2017 Cardiovascular Research Day Abstract Book
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<strong>Cardiovascular</strong><br />
<strong>Research</strong> <strong>Day</strong><br />
ABSTRACT BOOK<br />
November 3, <strong>2017</strong><br />
Lexington Convention Center<br />
HealthCare ®<br />
GILL HEART INSTITUTE<br />
Saha <strong>Cardiovascular</strong><br />
<strong>Research</strong> Center
TABLE OF CONTENTS<br />
Schedule for the <strong>Day</strong> ............................................................................................................................. 2<br />
<strong>2017</strong> Gill Heart Institute Young Investigator Award ................................................................. 6<br />
<strong>2017</strong> Distinguished Alumni Speaker .............................................................................................. 7<br />
Featured Speakers ................................................................................................................................. 8<br />
Event Supporters ................................................................................................................................. 11<br />
Poster Judges ........................................................................................................................................ 13<br />
Poster Participants ............................................................................................................................. 14<br />
<strong>Abstract</strong>s ................................................................................................................................................ 16<br />
Survey ................................................................................................................................................... 121<br />
1
SCHEDULE FOR THE DAY<br />
Friday, November 3<br />
University of Kentucky <strong>Cardiovascular</strong> <strong>Research</strong> <strong>Day</strong><br />
Lexington Convention Center | Bluegrass Ballrooms<br />
8:15 am<br />
Guest Check –In Begins | Continental Breakfast | Bluegrass Prefunction<br />
8:30 am<br />
Career Development Session for Trainees | Continental Breakfast | Thoroughbred<br />
Room 8<br />
9:30 am<br />
Scientific Session I | Bluegrass Ballroom I<br />
Chair: Gregory Graf, Ph.D. and Gia Mudd-Martin, Ph.D., MPH, R.N.<br />
9:30 am<br />
Alan Daugherty, Ph.D., D.Sc., Director, Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center<br />
Welcoming Comments<br />
9:35 am<br />
Trainee Presentations<br />
Ahmed Al-Darraji | Ahmed Abdel-Latif Lab | University of Kentucky<br />
Azithromycin Therapy Reduces Cardiac Inflammation and Mitigates<br />
Adverse Cardiac Remodeling after Myocardial Infarction: Potential<br />
Therapeutic Targets<br />
Kelsey Conrad | Phil Owens Lab | University of Cincinnati<br />
Increased Circulating Trimethylamine N-oxide (TMAO) Augments the<br />
Incidence of Abdominal Aortic Aneurysm in Low Penetrant C57BL/6J<br />
Mice<br />
Ryan Allen, Ph.D. | Kasey Vickers Lab | Vanderbilt University<br />
Circulating Bacterial Small RNA Bound to LDL Induce Inflammatory<br />
Activation of Macrophages<br />
2
10:35 am<br />
<strong>Research</strong> Blitz I<br />
David Henson | University of Kentucky<br />
Mohamed AboAly, MD | University of Kentucky<br />
Zheying “Jeff” Chen | University of Kentucky<br />
Dylan Colli | University of Kentucky<br />
Allison Cooke | University of Cincinnati<br />
Mihir Shah | University of Kentucky<br />
Jiansheng Huang, PhD | Vanderbilt University<br />
Aida Javidan, MS | University of Kentucky<br />
Michael Petriello, PhD | University of Kentucky<br />
Michael Kuefner | University of Tennessee Health Science Center<br />
Robin Shoemaker | University of Kentucky<br />
10:45 am<br />
Featured Speaker<br />
Calum MacRae, M.D., Ph.D. | Harvard Medical School<br />
Bridging the Gaps in <strong>Cardiovascular</strong> Translation<br />
11:30 am<br />
Lunch | Scientific Session II | Bluegrass Prefunction<br />
12:00 pm<br />
Welcoming Remarks<br />
Mark Newman, M.D. | University of Kentucky Executive Vice<br />
President for Health Affairs<br />
12:15 pm<br />
Distinguished Alumni Presentation<br />
John Charles, Ph.D. | NASA Human <strong>Research</strong> Program at Johnson<br />
Space Center<br />
From the Bluegrass to Beyond the Blue<br />
1:00 pm<br />
Poster Session | Bluegrass Ballroom II<br />
1:00 pm<br />
Odd Numbered Posters<br />
2:00 pm<br />
Even Numbered Posters<br />
3
SCHEDULE FOR THE DAY continued<br />
Friday, November 3<br />
University of Kentucky <strong>Cardiovascular</strong> <strong>Research</strong> <strong>Day</strong><br />
Lexington Convention Center | Bluegrass Ballrooms<br />
3:00 pm<br />
Scientific Session III | Bluegrass Ballroom I<br />
Chair: Lisa Cassis, Ph.D. and Dan Rader, M.D.<br />
3:00 pm<br />
Trainee Presentations<br />
Brooke Ahern | Jon Satin Lab | University of Kentucky<br />
Cardiac Specific Rad Deletion Enhances Cardiac Function through Safe,<br />
Stable Positive Inotropic Support<br />
Ibra Fancher, Ph.D. | Irena Levitan Lab | University of Illinois at<br />
Chicago<br />
Hypercholesterolemia-induced endothelial dysfunction is rescued by<br />
overexpression of endothelial Kir2.1 in resistance arteries<br />
3:30 pm<br />
<strong>Research</strong> Blitz II<br />
Shayan Mohammadmoradi, MS | University of Kentucky<br />
Feiming Ye | University of Kentucky<br />
Chia-Hua Wu | University of Kentucky<br />
Hannah Russell | University of Cincinnati<br />
Katelyn Ahern | University of Virginia<br />
Hisashi Sawada, MD, PhD | University of Kentucky<br />
Courtney Turpin, MS | University of Kentucky<br />
Zhihong Yang, PhD | National Institutes of Health<br />
Miao Liu, PhD | University of Kentucky<br />
Bradley Wright | University of Kentucky<br />
4
3:40 pm<br />
Featured Speaker<br />
Steven Houser, Ph.D. | Temple University<br />
Cardiac Repair and Injury<br />
4:25 pm<br />
Gill Heart Institute Translational Early Career Award Recipient<br />
Kiran Musunuru, M.D., Ph.D., MPH | University of Pennsylvania<br />
Discovery and Therapeutic Genome Editing of <strong>Cardiovascular</strong> Disease<br />
Genes<br />
5:15 pm<br />
Networking Reception | Bluegrass Prefunction<br />
6:00 pm<br />
Dinner and Awards Ceremony | Bluegrass Prefunction<br />
Special Presentation from Mark Stoops |University of Kentucky Head Football Coach<br />
5
<strong>2017</strong> GILL HEART INSTITUTE<br />
YOUNG INVESTIGATOR AWARD<br />
Kiran Musunuru, M.D., Ph.D., MPH<br />
Associate Professor<br />
<strong>Cardiovascular</strong> Medicine and Genetics<br />
Perelman School of Medicine<br />
University of Pennsylvania<br />
Dr. Musunuru received his medical degree from Weill<br />
Cornell Medical College, his Ph.D. from The Rockefeller<br />
University, and his Masters of Public Health from Johns<br />
Hopkins Bloomberg School of Public Health. He trained in<br />
Internal Medicine at Brigham and Women's Hospital and<br />
<strong>Cardiovascular</strong> Medicine at Johns Hopkins Hospital,<br />
followed by postdoctoral work at Massachusetts General<br />
Hospital and the Broad Institute of MIT and Harvard.<br />
Dr. Musunuru's research focuses on the genetics of cardiovascular and metabolic diseases<br />
and seeks to identify naturally occurring genetic variants that predispose to or protect<br />
against disease and can be used to develop therapies to protect the entire population. In<br />
2016, he received the Presidential Early Career Award for Scientists and Engineers from<br />
the White House, as well as the American Heart Association's Award for Meritorious<br />
Achievement.<br />
6
<strong>2017</strong> DISTINGUISHED ALUMNI<br />
SPEAKER<br />
John Charles, Ph.D.<br />
Chief Scientist, NASA Human <strong>Research</strong> Program at Johnson<br />
Space Center<br />
John B. Charles, Ph.D., is the Chief Scientist of NASA’s<br />
Human <strong>Research</strong> Program (HRP), responsible for the<br />
scientific direction of human research and technology<br />
development enabling astronauts to go beyond low Earth<br />
orbit and eventually to Mars. Previously he was HRP’s<br />
Associate Manager for International Science and led NASA’s<br />
space life sciences planning for the joint US/Russian oneyear<br />
mission on ISS and the Twins Study.<br />
Dr. Charles earned his B.S. in biophysics at The Ohio State<br />
University and his doctorate in physiology and biophysics at the University of Kentucky.<br />
He came to the Johnson Space Center in 1983 a postdoctoral fellow and became a civil<br />
servant in 1985. He is co-developer of the Shuttle-era fluid-loading countermeasure, and<br />
investigated the cardiovascular effects of space flight using ultrasound, re-entry data<br />
recording and in-flight lower body negative pressure on Space Shuttle astronauts and on<br />
crewmembers of the Russian space station Mir. He coordinated all of the NASA-sponsored<br />
biomedical, biological and microgravity science investigations as Mission Scientist for<br />
American astronaut missions on Mir, on STS-95, John Glenn’s Shuttle flight, and on STS-<br />
107, Columbia’s last mission in January 2003.<br />
He is a Fellow of the Aerospace Medical Association and has been a member since 1983. He<br />
is also a Full Member of the International Academy of Astronautics (IAA) and co-chaired<br />
the 18 th IAA “Humans in Space Symposium” in Houston in 2011.<br />
He has published 75 scientific papers and space history articles and has received several<br />
professional awards, including National Space Club and Foundation Eagle Manned Mission<br />
Award (<strong>2017</strong>), the NASA Exceptional Achievement Medal (2014), the ASMA “Joe Kerwin<br />
Award” (2011), the “Hubertus Strughold Award” of the Space Medicine Association (2001)<br />
and the NASA Exceptional Service Medal (2000).<br />
7
FEATURED SPEAKER<br />
Calum MacRae, M.D., Ph.D.<br />
Chief, <strong>Cardiovascular</strong> Medicine, Brigham and Women’s Hospital<br />
Associate Professor of Medicine, Harvard Medical School<br />
Calum MacRae is a cardiologist, geneticist and developmental<br />
biologist who trained in Edinburgh, London and Boston. He is<br />
Chief of <strong>Cardiovascular</strong> Medicine at Brigham and Women’s<br />
Hospital and Associate Professor of Medicine at Harvard Medical<br />
School. He is also an Associate Member at the Broad Institute<br />
and a Principal Faculty Member at the Harvard Stem Cell<br />
Institute. His research is focused on understanding the<br />
fundamental mechanisms of disease using human studies and<br />
complementary efforts combining systems level modeling with empiric high-throughput<br />
biology in the zebrafish. His lab uses automated screens in fish to define the genetic<br />
architecture of disease and to explore gene-drug (or environment) interactions through the<br />
interrogation of large-scale chemical libraries. His interests include heart failure,<br />
arrhythmias and coronary disease, genomic medicine, innovation in phenotyping and the<br />
redesign of clinical care.<br />
8
FEATURED SPEAKER<br />
Steve Houser, Ph.D., FAHA<br />
Senior Associate Dean, <strong>Research</strong><br />
Vera J. Goodfriend Endowed Chair, <strong>Cardiovascular</strong> <strong>Research</strong><br />
Chair and Professor, Physiology<br />
Director, <strong>Cardiovascular</strong> <strong>Research</strong> Center<br />
Professor, Medicine<br />
For forty years, Steven R. Houser, Ph.D. , FAHA has been a<br />
constant presence at the Lewis Katz School of Medicine at<br />
Temple University. A committed alumnus, beloved<br />
instructor, scientific-leader and an internationallyrecognized<br />
expert in cardiac biology and pathophysiology,<br />
A widely respected and prolific cardiovascular researcher,<br />
Dr. Houser first came to Temple as a Ph.D. student in physiology before earning a research<br />
fellowship in Cardiology at the School of Medicine. In 1979 he joined the faculty full time,<br />
was named director of the <strong>Cardiovascular</strong> <strong>Research</strong> Center in 2003, chair of the<br />
Department of Physiology in 2006 and Senior Associate Dean of <strong>Research</strong> in 2013 (having<br />
previously held the position from 2003-2007). In 2015 he was appointed to the Vera J.<br />
Goodfriend Endowed Chair in <strong>Cardiovascular</strong> <strong>Research</strong>.<br />
Outside of his Temple appointments, Dr. Houser has recently served as President of the<br />
American Heart Association. During Houser’s long association with the AHA, he has also<br />
served as a fellow, board member, chair of the research committee and president of the<br />
southeastern Pennsylvania affiliate. He has also worked on a number of national AHA<br />
committees.<br />
His research group has helped define many fundamental features of the normal cardiac<br />
myocyte (a muscle cell) and identified defective molecular and cellular processes that<br />
produce abnormal cardiac myocyte function in cardiovascular disease. In 2012, the group<br />
was awarded a five-year $11.6 million grant from the National Heart, Lung, and Blood<br />
Institute of the National Institutes of Health (NIH) to develop new approaches to prevent,<br />
slow or reverse damage to the heart after a heart attack. He is a much sought-after expert<br />
in his field, having been an invited speaker at conferences and symposiums more than 200<br />
times, a reviewer or editor for 13 peer-review journals, served as the chair or member of<br />
numerous NIH study sections and has published more than 210 manuscripts, papers or<br />
book chapters.<br />
9
SPECIAL PRESENTATION<br />
Mark Stoops<br />
Head Coach, University of Kentucky Football<br />
Mark Stoops is in the middle of his fifth year as the University of<br />
Kentucky head football coach. In his first season, Coach Stoops<br />
inherited a program that came off a 2-10 season, averaging 22.1<br />
points per loss. It took just four seasons to return the program to<br />
bowl eligibility and restore a winning culture. In addition to<br />
shaping the Wildcats’ performance on the field, Stoops has been<br />
instrumental in shaping the program off the field. Coach Stoops<br />
assisted in planning and fundraising for the $125 million<br />
renovation of Kroger Field, as well as the new $45 million football complex. These<br />
renovations and the winning culture Coach Stoops brought with him to Lexington have led<br />
to multiple top-25 recruiting classes and optimism for the future of this program.<br />
Coach Stoops grew up surrounded by a football family and is native of Youngstown, Ohio,<br />
one of the nation’s biggest football towns. After high school, he went on to play for Hall of<br />
Fame coach Hayden Fry at the University of Iowa, playing in four bowl games and winning<br />
25 games in three years. After graduation, Coach Stoops spent two seasons as a graduate<br />
assistant coach under Coach Fry. After the ’91 season, he spent four years coaching high<br />
school football before entering the collegiate ranks. Coach Stoops made stops at several<br />
major programs including Iowa, USF, Wyoming, Houston, Arizona, Miami and Florida State.<br />
Fast forward to <strong>2017</strong>, and Coach Mark Stoops is in his 28th season of coaching. He has<br />
compiled a 4-1 career BCS bowl record, won a BCS National Championship, and has<br />
developed 11 NFL 1st round draft picks.<br />
10
EVENT SUPPORTS<br />
Gill Foundation of Texas<br />
●<br />
The Saha Fund for <strong>Cardiovascular</strong> <strong>Research</strong><br />
and Education<br />
●<br />
The Estate and Family of Mrs. Hager Koostra<br />
●<br />
Mr. and Mrs. Bob Allen<br />
11
NOTES<br />
12
<strong>2017</strong> POSTER JUDGES<br />
Douglas Andres<br />
Jason Brandon<br />
J. Mark Brown<br />
William Brown<br />
Lisa Cassis<br />
John Charles<br />
Cherry Croft<br />
Alan Daugherty<br />
Brian Delisle<br />
Sanda Despa<br />
Joyce Evans<br />
Ming Gong<br />
Gregory Graf<br />
Kathryn Gunn<br />
Zhenheng Guo<br />
Rebecca Haeusler<br />
Steven Houser<br />
Wendy Katz<br />
Kevin Pearson<br />
Joe Layne<br />
Sangderk Lee<br />
Norbert Leitinger<br />
Irena Levitan<br />
Zhenyu Li<br />
Xiangan Li<br />
MacRae Linton<br />
Robert Lodder<br />
Analia Loria<br />
Hong Lu<br />
Calum MacRae<br />
Andrew Morris<br />
Kiran Musunuru<br />
Saskia Neher<br />
Fredrick Onono<br />
Phillip Owens<br />
Sabire Ozcan<br />
Romeo Papazyan<br />
Edwards Park<br />
Julie Pendergast<br />
Jonathan Satin<br />
Travis Sexton<br />
Mary Sheppard<br />
Sony Soman<br />
Venkateswaran Subramanian<br />
Lisa Tannock<br />
Elizabeth Tarling<br />
Ryan Temel<br />
Joel Thompson<br />
Jenelle Timmins-Persley<br />
Michael Tranter<br />
Vincent Venditto<br />
Kasey Vickers<br />
Nancy Webb<br />
Wally Whiteheart<br />
Jeremy Wood<br />
Changcheng Zhou<br />
Lin Zhu<br />
Zhihui Zhu<br />
Lin Zhu<br />
Zhihui Zhu<br />
13
<strong>2017</strong> POSTER PARTICIPANTS<br />
40 Mohamed AboAly<br />
11 Brooke Ahern<br />
85 Katelyn Ahern<br />
8 Ahmed Al-Darraji<br />
74 Ryan Allen<br />
67 Yasir Alsiraj<br />
86 Sarah Anthony<br />
26 Kavya Balaji<br />
49 Meenakshi Banerjee<br />
89 Nicolas Barbera<br />
32 Jazmyne Barney<br />
45 Esias Bedingar<br />
12 Dibyajyoti Biswal<br />
88 Yedida Bogachkov<br />
94 Lei Cai<br />
17 Zheying Chen<br />
13 Dylan Colli<br />
72 Kelsey Conrad<br />
73 Allison Cooke<br />
55 Robert Correll<br />
66 Marcielle de Beer<br />
43 Mohamed El-Helw<br />
56 Eman Elsawalhy<br />
70 Ibra Fancher<br />
44 Demetria Fischesser<br />
63 Christy Gliniak<br />
78 Aspen Gutgsell<br />
35 Allison Hall<br />
1 Dwight Harris<br />
15 Peter Hecker<br />
97 David Henson<br />
10 Joseph Herald<br />
31 Jessie Hoffman<br />
38 Amanda Hoskins<br />
41 Tianfei Hou<br />
81 Jiansheng Huang<br />
75 Aida Javidan<br />
80 Ailing Ji<br />
82 Shannon Jones<br />
57 Smita Joshi<br />
54 Tara Keenan<br />
30 Peter Kekenes-Huskey<br />
50 Morgan Kelly<br />
96 Seonwook Kim<br />
77 Alison Kohan<br />
61 Michael Kuefner<br />
92 Lindsey Lanzillotta<br />
18 Bryana Levitan<br />
53 Zhenyu Li<br />
90 Yanzhang Li<br />
93 Tong Li<br />
7 Miao Liu<br />
34 Shu Liu<br />
33 Guogen Mao<br />
51 Eric McGann<br />
46 Gregory Milburn<br />
84 Shayan Mohammadmoradi<br />
9 Mohammad Javad Mollakazemi<br />
60 Kathryn Moore<br />
4 Chase Neumann<br />
5 Laura Peterson<br />
79 Michael Petriello<br />
22 Joshua Preston<br />
20 Seema Rao<br />
62 Benjamin Roberts<br />
71 Hannah Russell<br />
21 Keegan Sammons<br />
39 Kaitlyn Samuels<br />
27 Hisashi Sawada<br />
76 Leslie Sedgeman<br />
48 Travis Sexton<br />
14 Mihir Shah<br />
68 Robin Shoemaker<br />
6 Preetha Shridas<br />
3 Tara Shrout<br />
65 Daniel Silver<br />
87 Loren Smith<br />
91 Wenliang Song<br />
14
83 Stefanka Spassieva<br />
47 Cody Sutphin<br />
37 Joel Thompson<br />
36 Himi Tripathi<br />
64 Courtney Turpin<br />
99 Clint Upchurch<br />
24 Patrick Van Hoose<br />
103 Sahar VarnoosfaderanI<br />
59 Arielle Waller<br />
25 Fang Wang<br />
52 Robert Wendroth<br />
98 Carrie Wiese<br />
29 Patricia Wilson<br />
58 Cassandra Woolley<br />
28 Bradley Wright<br />
19 Congqing Wu<br />
23 Chia-Hua Wu<br />
42 Liping Yang<br />
69 Zhihong Yang<br />
95 Lihua Yang<br />
2 Feiming Ye<br />
16 Xiang Ye<br />
102 Jinchao Zhang<br />
100 Lin Zhu<br />
101 Wanying Zhu<br />
15
<strong>2017</strong> ABSTRACTS<br />
16
1<br />
Extracorporeal Membrane Oxygenation as a Rescue Measure in Patients Experiencing<br />
Respiratory Distress After Non-cardiac Thoracic Surgery<br />
Dwight Harris 1 • Sibu Saha, MD 2<br />
1College of Medicine, University of Kentucky • 2 Cardiothoracic Surgery, University of Kentucky<br />
Medical Student<br />
Extracorporeal Membrane Oxygenation (ECMO) is a rescue measure used to treat patients in every<br />
age group from neonates to adults. ECMO is used to treat many different conditions including<br />
respiratory distress, cardiogenic shock, and trauma. In these patient groups ECMO has been<br />
extensively studied. Recently, physicians have begun using ECMO as a rescue measure in patients<br />
experiencing acute respiratory distress syndrome (ARDS) after non-cardiac thoracic surgery.<br />
Studies on the use of ECMO after non-cardiac thoracic surgery are lacking with only a hand full of<br />
reports published. The goal of this report is to present our experience with patients experiencing<br />
ARDS after non-cardiac thoracic surgery. We conducted a retrospective review of all patients that<br />
had received ECMO after thoracic surgery at the University of Kentucky from January 9, 2012 to<br />
January 9, <strong>2017</strong>. We identified 8 patients. The average time on ECMO was 9.125 days, and the<br />
average hospital stay was 65.125 days. Of the eight patients placed on ECMO three of them survived<br />
to discharge. Of the 3 that survived to discharge one died within six months and two are less than<br />
four months out to follow up. The average charge per patient was calculated to be 1,053,551<br />
dollars, and the average charge per day for ECMO was 16,177 dollars. The contribution margin per<br />
case was $109,200. ECMO is a tool that saves lives, but ECMO does not appear to have the same<br />
yield in patients experiencing ARDS after Thoracic surgery compared to other patient groups.<br />
ECMO after non-cardiac thoracic surgery is low yield and costly compared to other patient<br />
populations.<br />
17
2<br />
Inhibition of Megalin Reduces Atherosclerosis and Improves Hypercholesterolemia-induced<br />
Lysosomal Dysfunction in Kidney<br />
Feiming Ye 1 • Chia-Hua Wu 1 • Deborah Howatt 1 • Adam Mullick 2 • Mark Graham 2 •<br />
Anju Balakrishnan 1 • Craig Vander Kooi 3 • Jan Danser 4 • Jian-An Wang 5 • Alan Daugherty 1 •<br />
Hong Lu 1<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky • 2 Ionis Pharmaceuticals •<br />
3Molecular and Cellular Biochemistry, University of Kentucky • 4 Erasmus MC • 5 Department of<br />
Cardiology, Zhejiang University<br />
Graduate Student<br />
Objective: Our previous study has demonstrated that hepatocyte-specific deficiency of<br />
angiotensinogen (hepAGT-/-) reduces atherosclerosis. Megalin interacts with AGT and determines<br />
its presence in proximal convoluted tubules of kidney. In this study, we investigated effects of<br />
megalin inhibition on hypercholesterolemia-induced atherosclerosis and kidney pathologies.<br />
Methods and Results: Male LDL receptor -/- mice at the age of 8 - 10 weeks were injected<br />
subcutaneously with PBS, control antisense oligonucleotides (ASO), or megalin ASO for 13 weeks.<br />
One week after the initial injection, mice were fed a Western diet enriched with saturated fat for 12<br />
weeks. Inhibition of megalin profoundly reduced atherosclerotic lesion area, compared to the two<br />
control groups (Percent lesion area in PBS, control ASO and megalin ASO groups: 23 ± 2, 20 ± 2, 12<br />
± 1 %, respectively; P < 0.001). Histological analysis of kidney sections exhibited extensive vacuolar<br />
formation in proximal convoluted tubules of kidney in mice injected with PBS or control ASO, which<br />
were absent in mice injected with megalin ASO. Immunostaining of lysosomal-associated<br />
membrane protein 1 demonstrated that these vacuoles were derived from enlarged lysosomes.<br />
Same kidney pathology was also found in hepAGT+/+ mice in an LDL receptor -/- background fed<br />
the Western diet, but not observed in hepAGT-/- mice fed this diet.<br />
Conclusions: Megalin in proximal convoluted tubules of kidney and AGT derived from hepatocytes<br />
may interact to promote hypercholesterolemia-induced atherosclerosis and renal lysosomal<br />
dysfunction.<br />
18
3<br />
Improving Patient Care on Left Ventricular Assist Device Support: Novel Outcome Predictors<br />
& Preventative Therapies<br />
Tara Shrout 1 • Travis Sexton, PhD 2 • Susan Smyth, MD, PhD 3<br />
1College of Medicine, University of Kentucky • 2 Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of<br />
Kentucky • 3 Chief, Division of <strong>Cardiovascular</strong> Medicine, University of Kentucky<br />
Medical Student<br />
Background:<br />
Left ventricular assist device (LVAD) implantation is an increasingly common and important<br />
intervention for heart failure. Despite advancements, implantation introduces a foreign body across<br />
which blood experiences aberrant sheer stress. Clinical complications of bleeding and stroke<br />
develop secondary to an acquired coagulopathy, and patients’ quality of life is an important<br />
outcome to consider. This study uniquely correlates early platelet function and biomarkers<br />
alterations with clinical outcomes, as well as patient quality of life measures to help predict,<br />
address, and explore complications with LVAD support.<br />
Objectives:<br />
1. Evaluate correlations between predictive measures of platelet function, biomarkers, laboratory<br />
trends with clinical complications outcomes and LVAD types.<br />
2. Characterize the role of a novel hemostatic agent as a preventative therapy for bleeding.<br />
3. Understand patients’ quality of life on LVAD support.<br />
Design:<br />
March 2014-August 2016, 68 enrolled patients underwent implantation of Heartmate II (n= 48),<br />
Heartmate III (n=2), Heartware (n=18). Blood collections: 24-hours preoperatively and 1-, 24-, 72-,<br />
and 168-hours postoperatively. Follow-up collections: between 30-90-days postoperatively.<br />
Platelet function analyzed via impedance aggregometry, five agonists, and novel hemostatic agent.<br />
Biomarkers of coagulation, platelet activation, and inflammation analyzed. Daily clinical data<br />
collected.<br />
Participants:<br />
Consecutive-based sample. Sociodemographics: age, gender, ethnicity, co-existing conditions<br />
collected. Eligibility: First-time LVAD implant. Exclusion: Age
4<br />
Identification of a Lipid Signature in Clear Cell Renal Carcinoma (ccRCC) Suggest a Role of<br />
Phosphatidylinositol Remodeling in Disease Progression<br />
Chase Neumann 1 • Renliang Zhang, PhD 1 • Christopher Przybicin, MD 2 • Brian Rini, MD 3 •<br />
Mark Brown, PhD 1<br />
1Cellular and Molecular Medicine, Cleveland Clinic • 2 Robert J. Tomsich Pathology and Laboratory<br />
Medicine, Cleveland Clinic • 3 Hematology and Oncology Taussig Cancer Institute, Cleveland Clinic<br />
Graduate Student<br />
The most lethal urological cancer and third most common urological cancer in the United States is<br />
clear cell renal carcinoma (ccRCC). Approximately 64,000 individuals in the United States are<br />
diagnosed with ccRCC annually, and more than 140,000 individuals die worldwide of ccRCC every<br />
year. The “clear cell” characteristic of this deadly kidney cancer is due to abnormal deposition of<br />
lipids in a large unilocular lipid droplet in cancer cells, and urinary biomarker studies have<br />
identified lipid droplet-associated proteins as predictive markers of disease progression. However,<br />
the mechanisms by which lipid metabolism impacts ccRCC progression are not well understood. We<br />
hypothesized that a group of mitogenic lipids exist within the ccRCC microenvironment that<br />
promote proliferation and metastatic potential. A high throughput Triple Time-of-Flight (TOF) LC-<br />
MS/MS lipidomics screen of metastatic ccRCC tumor samples and matched non-tumor adjacent<br />
tissue identifies a highly reproducible lipid signature of ccRCC. In agreement with previous reports,<br />
cholesteryl esters are increased in the tumor vs non-tumor. We also found a striking increase in<br />
phosphatidylinositol (PI) 36:4 and 34:1 in tumors. Membrane bound O-acyltransferase domain<br />
containing 7 (MBOAT7) is a lysophosphatidylinositol (LPI) acyltransferase that preferentially<br />
utilizes arachidonyl-CoA and 16:0 LPI as substrates. To further investigate the formation of 36:4 PI,<br />
an MBOAT7 product, we used TCGA datasets to analyze stage-specific expression of MBOAT7.<br />
MBOAT7 expression was found to increase with staging. This potential increase in<br />
phosphatidylinositol production, coupled with decreases in known phosphatase activity like PTEN,<br />
SACM1L, and MTM1 may lead to increased activation of the PI3K/Akt signaling activity. The impact<br />
of this work may suggest mechanisms why some patients are refractory to PI3K/mTOR inhibitors,<br />
while a small percentage (
5<br />
Potential Role of Muscle Specific Ribosomal Protein L3-like in Cardiomyocyte Transverse<br />
Tubule Formation<br />
Laura Peterson 1 • Alexander Alimov, PhD 1 • Jon Satin, PhD 1 • Brian Delisle, PhD 1 •<br />
John McCarthy, PhD 1<br />
1Physiology, University of Kentucky<br />
Graduate Student<br />
Ribosomes have long been thought as serving in a constitutive housekeeping function and possess<br />
no real regulative capacity. Recently however, ribosomal proteins have been shown to provide the<br />
ribosome with a regulatory capacity across phyla. Ribosomal protein paralogs arose from a<br />
duplication event millions of years ago and have contributed to the development of ribosome<br />
specialization. By allowing a ribosome to function in its original capacity and acquire new roles,<br />
some ribosomal protein paralogs have acquired specialized roles. Ribosomal protein L3-like (RPL3-<br />
like) is expressed only in skeletal muscle and the heart but its paralog, RPL3, is found ubiquitously<br />
throughout the body. Strikingly, the expression in the heart is limited to the ventricles and is not<br />
expressed in the atria. One of the major anatomical differences in the atria and ventricle is the<br />
patterning of transverse tubules (t-tubules) with the ventricles exhibiting a highly symmetrical,<br />
regular transverse pattern and the atria having an irregular pattern with more prominent axial<br />
projections. RPL3-like knock out in the adult mouse disrupts ventricular t-tubule organization. We<br />
hypothesize that RPL3-like containing ribosomes preferentially associate with transcripts that<br />
encode for proteins involved in t-tubule formation thereby controlling morphology.<br />
21
6<br />
Serum Amyloid A Activates the NLRP3 Inflammasome in Macrophages<br />
Preetha Shridas, PhD 1 • Maria de Beer, PhD 2 • Nancy Webb, PhD 3<br />
1Internal Medicine, University of Kentucky • 2 Physiology, University of Kentucky • 3 Molecular and<br />
Biomedical Pharmacology, University of Kentucky<br />
Faculty<br />
Objectives: Interleukin-1beta (IL-1β) has been implicated in many chronic diseases including<br />
atherosclerosis and type 2 diabetes. Production of bioactive IL-1β is controlled by the<br />
inflammasome, a multi-protein complex that regulates caspase-1 activity. Serum Amyloid A (SAA) is<br />
an acute-phase protein whose levels in circulation can increase more than 1000-fold during severe<br />
infection and tissue damage. SAA is more modestly elevated in conditions associated with chronic<br />
inflammation, including obesity. SAA has been identified as the first known physiological mediator<br />
capable of activating the NRLP3 inflammasome. The objective of this study is to investigate the<br />
mechanisms involved in SAA-mediated inflammasome activation in macrophages.<br />
Methods/Results: J774 macrophage-like cells and mouse bone-marrow derived macrophages<br />
(BMM) were stimulated with 5-25 µg/ml purified lipid-free mouse SAA, concentrations<br />
corresponding to those typically observed in obese individuals. The analyses of culture media and<br />
cell lysates demonstrated that SAA dose-dependently induced both caspase-1 activation and IL-1β<br />
secretion. The ability of SAA to induce IL-1β secretion was significantly reduced in BMM deficient in<br />
NRLP3. There was significant suppression in IL-1β secretion by SAA when J774 cells were treated<br />
with SAA in the presence of caspase-1inhibitor, Z-YVAD-FMK. A P2X7-receptor antagonist,<br />
AA38079, did not have any effect on SAA-mediated IL-1β production. Inhibition of reactive oxygen<br />
species (ROS) and cathepsin-B activation by N-acetyl-L-cysteine and CA-074, respectively, inhibited<br />
inflammasome activation by SAA. Inhibiting cellular potassium efflux by glyburide also significantly<br />
reduced SAA-mediated IL-1β secretion. Pre-incubating SAA with HDL prior to cell treatments<br />
completely inhibited its ability to trigger inflammasome activation. HDL also abrogated SAAmediated<br />
ROS generation in J774 cells.<br />
Conclusions: SAA-mediated NRLP3 inflammasome activation in macrophages is dependent on ROS<br />
generation, release of cathepsin-B, and potassium efflux, and is independent of the P2X7 receptor.<br />
Ongoing studies are investigating the cellular receptor(s) involved and the mechanism by which<br />
lipoproteins mask SAA’s effects. Blocking SAA-mediated inflammasome activation may ameliorate<br />
increased risk for type 2 diabetes and atherosclerosis in obese individuals.<br />
22
7<br />
Diabetic Hfpef in a Rat Model of Systemic Amylin Dyshomeostasis<br />
Miao Liu, PhD 1 • Nirmal Verma, PhD 1 • Analia Loria, PhD 1 • Sanda Despa, PhD 1 •<br />
Florin Despa, PhD 1<br />
1Pharmacology and Nutritional Sciences, University of Kentucky<br />
Postdoc<br />
Diabetic heart failure with preserved ejection fraction (HFpEF) is a complex disease characterized<br />
by diastolic dysfunction, altered insulin sensitivity and multiple organ impairments. Amylin, a β-cell<br />
hormone co-secreted with insulin, participates in normal glucose regulation, but is also known to<br />
induce insulin resistance and to form pancreatic amyloid when oversecreted (hyperamylinemia).<br />
Accumulating data from several laboratories have confirmed that, in addition to pancreatic islets,<br />
the hearts, kidneys and brains of patients with type-2 diabetes contain also abnormally increased<br />
levels of aggregated amylin. Our overall hypothesis is that the interplay between insulin resistance<br />
and hyperamylinemia results in toxic accumulation of aggregated amylin in the microvasculature<br />
that adversely affects function of multiple organs, including, but not limited to, the heart. Here, we<br />
used rats overexpressing human amylin in the pancreas (HIP rats) to test whether a “human”<br />
hyperamylinemia predisposes to HFpEF. Wild-type (WT) littermates expressing non-amyloidogenic<br />
rat amylin served as control. Hearts and kidneys of HIP rats showed amylin deposition in<br />
capillaries, intravascular macrophage accumulation, microhemorrhages and loss of vascular<br />
endothelial cell coverage and tight junctions. These changes were associated with diastolic<br />
dysfunction, cardiac hypertrophy and mid-range cardiac ejection fraction (~50%). HIP rats also had<br />
increased blood pressure and renal dysfunction, including diuresis, natriuresis, creatinine clearance<br />
and microalbuminuria. Abundant amylin deposition was detected in HIP rat red blood cells (RBCs).<br />
Amylin-loaded RBCs have altered deformability and increased adherence to cultured endothelial<br />
cells. Intravenous infusion of RBCs from HIP rats in WT rats resulted in release of amylin in plasma<br />
and capillaries, attachment of RBCs to the vascular endothelium, intravascular macrophage<br />
accumulation and microhemorrhages. In conclusion, hyperamylinemia provokes systemic amylin<br />
dyshomeostasis which negatively affects cardiac and renal function via amylin-mediated<br />
microvascular injury. Circulating level of amylin may serve as a biomarker of diabetic HFpEF and<br />
therapeutic target to reduce the development/progression of HFpEF.<br />
23
8<br />
Azithromycin Therapy Reduces Cardiac Inflammation and Mitigates Adverse Cardiac<br />
Remodeling after Myocardial Infarction: Potential Therapeutic Targets<br />
Ahmed Al-Darraji, PharmD 1 • Dalia Haydar 2 • Lakshman Chelvarajan, PhD 3 • Himi Tripathi, PhD 3 •<br />
Bryana Levitan 4 • Shaojing Ye, PhD 3 • Vincent Venditto, PhD 5 • John Gensel, PhD 4 •<br />
David Feola, PhD 5 • Ahmed Abdel-Latif, MD, PhD 6<br />
1Pharmacology and Nutritional Sciences, University of Kentucky • 2 Pharmacy College, University of<br />
Kentucky • 3 SAHA cardiovascular center, University of Kentucky • 4 Physiology, University of<br />
Kentucky • 5 College of Pharmacy, University of Kentucky • 6 Cardiology, University of Kentucky<br />
Graduate Student<br />
<strong>Abstract</strong><br />
Introduction and Hypothesis:<br />
Acute myocardial infarction (MI) is a primary cause of worldwide morbidity and mortality.<br />
Macrophages are fundamental components of post-AMI inflammation. Pro-inflammatory<br />
macrophages (M1-like) can lead to adverse cardiac remodeling and heart failure while<br />
regulatory/reparative macrophages (M2-like) enhance tissue healing. Shifting the balance between<br />
M1 and M2 macrophages post-MI is a novel therapeutic strategy. Azithromycin (AZM), a commonly<br />
used macrolide antibiotic, polarizes macrophages towards an M2-like phenotype in animal and<br />
human studies. We hypothesized that using AZM can decrease adverse cardiac remodeling and<br />
improve heart function following MI.<br />
Methods and results:<br />
Male mice (C57BL/6, 6–8 weeks old) were treated with either oral AZM (160 mg/kg/day) or<br />
vehicle control starting 3 days prior to MI and continued to day 7 post-MI. We observed significant<br />
reduction in mortality with AZM therapy. AZM-treated mice showed reduction in M1-like<br />
(CD45+/Ly6G-/F4-80+/CD86+) and increase in M2-like (CD45+/Ly6G-/F4-80+/CD206+)<br />
macrophages leading to significant reduction in the M1/M2 ratio in the heart and peripheral blood<br />
as assessed by flow cytometry and immunohistochemistry. Macrophage changes were associated<br />
with significant reduction in pro-inflammatory and increase in anti-inflammatory cytokine<br />
production as assessed by real-time PCR. AZM treatment was associated with increased neutrophil<br />
apoptosis, a known signal for shifting macrophages towards an M2-like phenotype. Finally, AZM<br />
treatment was associated with enhanced cardiac recovery, smaller scar size and enhanced<br />
angiogenesis.<br />
Conclusion:<br />
Azithromycin plays a cardioprotective role post-MI through attenuating inflammation and<br />
enhancing cardiac recovery. Long term and human translational studies are planned to examine the<br />
therapeutic applications of AZM.<br />
24
9<br />
Cerebro-cardiac and Cerebro-respiratory Interactions while Listening to Songs<br />
Mohammad Javad Mollakazemi 1 • Dibyajyoti Biswal 1 • Sridevi Thyagarajan 1 • Joyce Evans 1 •<br />
Abhijit Patwardhan, PhD 1<br />
1 Biomedical Engineering, University of Kentucky<br />
Graduate Student<br />
Introduction: Since ancient times listening to music has been thought to affect mental as well as physical wellbeing.<br />
Previous studies have shown that autonomic responses change while listening to songs, but the interactions<br />
between respiratory, cardiovascular and cerebral rhythms while listening to songs are not as well understood. In<br />
this study, we investigated relations between cerebral, cardiac and respiratory rhythms while subjects listened to a<br />
variety of songs.<br />
Materials and Methods: All study procedures were approved by the Institutional Review Board at the University of<br />
Kentucky. Data were collected from 8 adult subjects (4 males and 4 females). A pair of circumaural headphones was<br />
used to listen to songs. A short audio acuity test was administered to each subject to rule out overt hearing loss. The<br />
following signals were recorded during the study: 6 channels of EEG (F3, T3, P3, F4, T4, P4), ECG (lead II,<br />
Spacelabs), Respiration (abdominal and thoracic, AMI Inductotrace) and non-invasive continuous finger blood<br />
pressure (Finapres). Some results from cardio-respiratory variables are reported in a companion abstract. All<br />
signals were digitized online at a rate of 1000 samples/second. In order to determine the contribution of cognition<br />
and memory recall in the evoked responses, we scrambled local phase spectra for a song of the subject’s choosing<br />
while preserving the magnitude spectra. In addition, subjects listened to two songs, one with a slow and the other<br />
with a fast tempo. Data were collected during 10 minutes of control at the beginning of the study when the subjects<br />
sat quietly in the chair without listening to any music. To find interactions between cerebro-cardiac and cerebrorespiratory<br />
rhythms, frequency spectra, magnitude squared coherencies and transfer functions were computed for<br />
all possible combinations of EEGs and cardiovascular variable (RR intervals, computed from the ECG) and between<br />
EEGs and respiratory time series. Welch’s method with 100 sec segments, 50% overlap and a Hanning window<br />
were used for these computations.<br />
Results and Discussion: Coherence, integrated over dc-0.5 Hz (the Nyquist frequency used for cardiac and<br />
respiratory variables) between RR intervals and most channels of EEG increased during listening to songs relative<br />
to control. An increase was seen regardless of the type of song that was listened to. A similar increase in integrated<br />
coherence was also observed between respiratory oscillations and EEGs, again, regardless of type of the song.<br />
Comparing slow and fast tempo songs: the fast tempo song increased the coherence between RR intervals and EEGs<br />
and between respiratory oscillations and EEGs more than the slow tempo song. When subjects listened to the song<br />
of their choosing, the average coherence between cerebro-cardiac and cerebro-respiratory rhythms was higher<br />
than control, the phase randomized version of the same song had a somewhat lower coherence but was still higher<br />
than control. However, differences in integrated cerebro-cardiac and cerebro-respiratory coherencies between the<br />
song of choice and the phase randomized version of it were not consistent. The autospectra of the envelopes of all<br />
channels of the EEG showed an overall decrease in spectral power within the 0-5 Hz range while listening to songs.<br />
Conclusions: Although we had anticipated that the differences between the responses for the song of choice and its<br />
phase scrambled version would be small, our results do not show a consistent difference. Many subjects could<br />
identify the phase scrambled song as being the same or similar to the one of their choosing, which may explain this<br />
finding. The increase in integrated coherencies between respiration and EEGs is indicative of entrainment of<br />
respiration to the music. The increase in coherencies between EEGs and RR intervals, during listening to songs, also<br />
indicates entrainment of RR intervals with music; however, whether this entrainment happens in parallel with<br />
entrainment with respiration or it is sequential remains unknown. Collectively, these results show that listening to<br />
songs increases coherent entrainment between cerebro-cardio and cerebro -respiratory variables.<br />
Acknowledgement: Supported by a grant from the National Science Foundation (EPSCoR RII Track-2).<br />
25
10<br />
Evidence of Angiotensin II-dependent Obesity-induced Hypertension in Female Mice<br />
Exposed to Postnatal Neglect<br />
Joseph Herald 1 • Jacqueline Leachman 1 • Analia Loria, PhD 1<br />
1Pharmacology and Nutritional Sciences, University of Kentucky<br />
Undergraduate<br />
Previously, we have shown that female mice subjected to maternal separation with early weaning<br />
(MSEW), a model of postnatal neglect, display exacerbated diet-induced obesity and high blood<br />
pressure (BP) compared with control mice. Female MSEW mice show activated renin-angiotensin<br />
system components, including increased plasma renin activity and adipose tissue-derived<br />
angiotensinogen secretion. The goal of this study was to test whether augmented obesity-induced<br />
hypertension in female MSEW mice is AngII-dependent. Mouse MSEW was achieved by repeated,<br />
daily separations from the dam and 4-day early weaning. Normally reared controls (C) were<br />
weaned at postnatal day 21. Each experimental group of female weanlings was comprised of 6 mice<br />
each and derived from 3 different litters, that were placed on high fat diet (HFD, 60% kcal from fat).<br />
After 18 weeks, mice were implanted with radiotelemetry devices for BP measurement. At week 20,<br />
average 24-hr systolic blood pressure (SBP) was 134±2 mmHg in MSEW mice and 126±2 in C<br />
(P
11<br />
Cardiac Specific Rad Deletion Enhances Cardiac Function through Safe, Stable Positive<br />
Inotropic Support<br />
Brooke Ahern 1 • Bryana Levitan 2 • Mihir Shah 1 • Andrea Sebastian 1 • Zach Bessinger 3 • Radu<br />
Paul Mihail 3 • Landon Simpson 4 • Nathan Jacobs, PhD 3 • Douglas Andres, PhD 4 •<br />
Jonathan Satin, PhD 1<br />
1Physiology, University of Kentucky • 2 Gill Heart and Vascular Institute, University of Kentucky •<br />
3Computer Science, University of Kentucky • 4 Biochemistry, University of Kentucky<br />
Graduate Student<br />
Background: The cardiac L-type Calcium Channel (LTCC) is a heteromultimeric protein complex.<br />
Rad interacts with the main pore forming subunit (CaV1.2) and auxiliary subunit CaVβ2. Rad<br />
modifies LTCC function and Rad protein levels are reduced by pathological stimuli. Global,<br />
constitutive deletion of Rad results in an increase in calcium handling and improved ejection<br />
fraction without progression to heart failure.<br />
Goals: It is unknown if the effects of global Rad deletion are also exhibited in cardiac-specific Rad<br />
deletion, and if the effects are preserved long-term. Our hypothesis is that cardiac-specific Rad<br />
ablation causes positive inotropic effects that are maintained over time.<br />
Methods and Results: We crossed Radfl/fl onto αMHC-MerCreMer mice to create a cardiacrestricted<br />
inducible Rad knockout (cRadKO). Mice were induced for gene deletion at ~3 months of<br />
age. We assessed cardiac structure and function through echocardiography from 7 days through 9<br />
months after cRadKO induction. Ejection fraction improved in females in comparison to wild type 7<br />
days post knock out (762% vs. 594%; cRadKO n=16 vs. WT n=7, respectively; p=0.001). Improved<br />
function was stable for 9 months in females (774% vs. 587%;cRadKO vs. WT, respectively; p=0.01).<br />
Similar results were found in males. The key mechanism for improved function includes elevated<br />
trigger Ca2+ in cardiomyocytes measured as a Ca2+-transient departure velocity (48.72.4 units/s<br />
vs. 38.13.4 units/s; cRadKO n=8 vs. WT n=10, respectively; p=.014). Cellular Ca2+ synchrony was<br />
preserved in cRadKO despite chronic elevated Ca2+ homeostasis.<br />
Conclusion: Cardiomyocyte Rad deletion preserves increased ejection fraction over time through a<br />
mechanism including increased calcium handling resulting in safe, stable positive inotropic support<br />
for heart function.<br />
27
12<br />
Auditory Entrainment of Respiration and RR Intervals<br />
Dibyajyoti Biswal, PhD 1 • Javad Mohammad Mollakazemi, PhD 1 • Sridevi Thyagarajan, PhD 1 •<br />
Joyce Evans, PhD 1 • Abhijit Patwardhan, PhD 1<br />
1 Biomedical Engineering, University of Kentucky<br />
Graduate Student<br />
Introduction:- It is known that music has palliative effect in a number of patients. Listening to music is also<br />
known to evoke autonomically regulated cardiovascular and respiratory responses. In adults, these responses<br />
are a combination of sensory input and cognition. While it is known that listening to music evokes<br />
measurable responses in cardiovascular and respiratory variables,<br />
much is unknown in terms of causal links between these responses and the mechanisms by which they get<br />
entrained. Our overall objective is to determine the causal links between auditory stimuli, respiration and<br />
cardiovascular regulatory variables.<br />
Materials and Methods:- The study was approved by the Institutional Review Board at the University of<br />
Kentucky. Subjects gave informed consent. Subjects (n=8, equal numbers of males and females, age 18 to 35)<br />
sat in a comfortable chair and listened to songs through a pair of circumaural headphones. An internet based<br />
audio acuity test was administered to each subject to rule out overt hearing loss. The following<br />
measurements were made: non-invasive continuous finger blood pressure (Portapres, Finapres), ECG (lead II,<br />
Spacelabs), Respiration (abdominal and thoracic, AMI Inductotrace) and EEG. Results of analyses using EEG is<br />
reported elsewhere. All signals were digitized on-line at a rate of 1000 samples/second. To align the<br />
physiological data with the music, a square wave pulse was inserted at the beginning of the songs. For each<br />
study, the subject was asked to provide a song of their choice which in their opinion “moves” them. Data were<br />
collected during 5 trials in each study: control (10 minutes), songs of slow and fast tempo, the song of the<br />
subject’s choice, and a phase scrambled version of the song of the subjects’ choice. Each song was 3-4 minutes<br />
long. To minimize cognitive response, we scrambled the phase of the song of the subject’s chosen song by<br />
segmenting the songs and scrambling the phase spectrum of that segment while preserving the magnitude<br />
spectrum. From the recorded data, time series of RR intervals were generated. These time series and the<br />
respiratory signals were low pass filtered (cut off 5Hz) and subsampled at a rate of 10 samples/second. From<br />
the subsampled data auto-spectra of RR intervals and respiration, coherencies and transfer functions<br />
between these signals were computed using Welch’s method.<br />
Results and Discussion:- The most pronounced effect during listening to songs was in respiration. In all but<br />
one subject, the mean respiratory rate increased relative to control while listening to songs. In addition to an<br />
increase in mean rate, the power distribution in the autospectra of respiration markedly narrowed during<br />
auditory stimulation. Similar changes were seen in RR interval spectra, the so called HF power, i.e. respiratory<br />
region power, was more narrowly distributed while listening to songs. Coherence between RR intervals and<br />
respiration exhibited a broad band increase during listening to songs as compared with control. Although<br />
there was a slight tendency during phase scrambled song for respiratory spectra to move closer to those<br />
during control, these differences were not very robust.<br />
Conclusion:- Our results show that auditory stimulation does entrain respiration as well as RR interval. Taken<br />
together, these results suggest that RR intervals were entrained by respiration while listening to both slow<br />
and fast tempo songs. The increase in average coherencies suggests entrainment of respiration to auditory<br />
stimulation which in turn entrains RR intervals. Further analyses using phase synchronization indexes would<br />
be useful to better reveal the causal links in these entrainment processes.<br />
Supported by a grant from the National Science Foundation (EPSCoR RII Track-2).<br />
28
13<br />
An Unbiased Classification Algorithm for Transverse Tubule Remodeling Within Murine<br />
Heart Failure Models.<br />
Dylan Colli 1 • Bradley Stewart 2 • Peter Kekenes-Huskey, PhD 2<br />
1Chemical and Materials Engineering, University of Kentucky • 2 Chemistry, University of Kentucky<br />
Undergraduate<br />
Transverse tubules (TTs) are the main method of delivery of extracellular calcium into the myocyte,<br />
responsible for the initiation of calcium induced calcium release (CICR) necessary to carry out<br />
excitation contraction coupling (EC coupling) within ventricular myocytes. Disruptions in the<br />
regularly striated TT network are correlated with various degrees of calcium mishandling and are<br />
generally observed within heart failure models. Currently, TT remodeling is often judged<br />
qualitatively, subject to bias of the experimenter. To address the need to eliminate this bias, we<br />
propose a technique that utilizes: 1. Computer vision libraries. 2. Signal processing techniques such<br />
as matched filtering that provide signal-to-noise ratios, a quantitative measure of observed<br />
remodeling. Combining these methods, an unbiased classification algorithm is presented that is<br />
able to quantify both regions of TT loss and longitudinal remodeling as well as characterize whole<br />
myocytes by the magnitude of their deviation from the prototypical healthy myocyte. This allows<br />
for the expedient, and more importantly, unbiased and reliable classification of TT remodeling<br />
observed within murine ventricular myocytes.<br />
29
14<br />
Cardiac specific Rad knockout Increases Calcium Release<br />
Mihir Shah 1 • Andrea Sebastian 1 • Landon Simpson 2 • Doug Andres, PhD 2 • Jonathan Satin, PhD 1 •<br />
Brooke Ahern 1<br />
1Physiology, University of Kentucky • 2 Biochemistry, University of Kentucky<br />
Undergraduate<br />
Background: The L-type calcium channel complex (LTCC) associates with regulatory proteins<br />
including Rad that govern calcium channel function. Overexpression of Rad inhibits LTCC inward<br />
calcium current. In contrast, whole body constitutive Rad knockout in mice have higher calcium<br />
influx in cardiac myocytes. It is unknown whether the effect of constitutive whole body Rad<br />
knockout arise from development, from the influence of non-cardiac myocytes, or is a primary<br />
result of Rad reduction in cardiac myocytes. Our recently engineered inducible, cardiac myocyte<br />
restricted Rad-knockout mouse model allows us to address these possibilities.<br />
Hypothesis: Cardiac specific Rad knockout will recapitulate the same effects of global Rad knockout.<br />
Acute induction (1-4 weeks) of Rad knock out produces an immediate effect on cytosolic calcium<br />
entry.<br />
Methods: Cytosolic Ca2+ in single, live cells were analyzed using Fura-2. Cells were paced at 1 Hz.<br />
Twitch calcium, calcium reuptake, and release kinetics were measured before and after the addition<br />
of isoproterenol in both wild-type (Radfl/fl abbreviated as WT) and induced, Rad-/-<br />
cardiomyocytes (cRadKO).<br />
Results: Twitch calcium levels were significantly higher in cRadKO compared to WT (1.99±0.09 and<br />
1.54±0.13 FU for WT and RadKO, respectively, p=0.008). cRadKO and WT twitch calcium<br />
significantly increased in response to acute isoproterenol (ISO; 2.63±0.13 and was 1.98±0.20 FU for<br />
WT and RadKO, respectively, p=0.0082). Calcium reuptake also increased with the knockout of Rad<br />
and in the presence of ISO. The onset velocity of calcium release increased in cRadKO (38.13±3.46<br />
FU/ms and 48.71±2.38 FU/ms, for WT and cRadKO, respectively, p=0.01). The ISO response for<br />
onset velocity was retained with cRadKO, in parallel with amplitude effects.<br />
Conclusion: Reduction of Rad protein elevated baseline cytosolic calcium levels approaching that of<br />
ISO-stimulated WT cardiomyocytes. Thus, Rad knockout provides positive inotropic support to<br />
heart function by elevating baseline cytosolic Ca2+. cRadKO maintains modulated myocardial<br />
function to sympathetic activity.<br />
30
15<br />
Atherosclerosis in the Cerebrovasculature of Nonhuman Primates Promotes Reactive<br />
Gliosis: A Potential Model for Vascular Dementia<br />
Peter Hecker 1 • Lei Cai, PhD 1 • Elizabeth Head, PhD 2 • Donna M. Wilcock, PhD 3 • Ryan Temel, PhD 1<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center and Department of Pharmacology and Nutritional Sciences,<br />
University of Kentucky • 2 Sanders-Brown Center of Aging and Department of Pharmacology and<br />
Nutritional Sciences, University of Kentucky • 3 Sanders-Brown Center of Aging and Department of<br />
Physiology, University of Kentucky<br />
Graduate Student<br />
Age- and lifestyle-dependent vascular insults drive and/or exacerbate cardio- and cerebro- vascular<br />
disease. While several animal models exist for studying vascular contributions to cardiovascular<br />
disease there are few models for studying the vascular contributions to cognitive impairment and<br />
dementia (VCID). Our lab is studying atherosclerosis progression and regression in male<br />
cynomolgus monkeys fed a high-fat/high-cholesterol diet. After 20 months on the atherogenic diet,<br />
a subset of monkeys are switched to standard nonhuman primate “chow” diet and are<br />
concomitantly treated with vehicle or a microRNA-33 antagonist (anti-miR-33). We hypothesize<br />
that anti-miR-33 treatment will create more stable atherosclerotic plaques by increasing<br />
cholesterol efflux from foam cells and dampening inflammation in the artery wall. While our initial<br />
interest was atherosclerosis in the coronary arteries, we have recently turned our attention to<br />
atherosclerosis in the main arterial network that supplies the brain. Evaluation of the circle of<br />
Willis (COW) revealed the presence of atherosclerotic plaques in major branch points of the large<br />
arteries. Immunohistochemistry (IHC) on arterial sections has begun to evaluate the size and<br />
molecular/cellular composition of these atherosclerotic plaques. Understanding that<br />
atherosclerosis could reduce cerebral blood flow (CBF) and/or promote endothelial dysfunction,<br />
we have started to examine brain sections from our monkeys for underlying neuropathology.<br />
Preliminary IHC data shows increased microglia and astrocyte reactivity around small cerebral<br />
vessels from a monkey with atherosclerosis versus a healthy, control animal. The marked increase<br />
in glial reactivity around small vessels may suggest extravasation or decreased CBF, both capable of<br />
driving neuroinflammation and downstream neurodegeneration. In the near future, we plan to pair<br />
MRI analysis of hypoperfusion and white matter lesions (WML) with histology and IHC assessments<br />
of WML, cerebral amyloid angiopathy, and astrocyte endfeet engagement with the vasculature.<br />
Although much work remains to be done, we are optimistic that we have created a unique and<br />
important translational model that mimics human cerebrovascular atherosclerosis and<br />
neuropathology associated with VCID.<br />
31
16<br />
Hypercholesterolemia Induces Acute Thymic Atrophy Through Blocking Thymocyte<br />
Differentiation and Expansion<br />
Xiang Ye, PhD 1 • XiangAn Li, PhD 1<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky<br />
Postdoc<br />
Background: Thymus is the primary site for T cell development, however its function is decreasing<br />
with age, defined as thymic involution. The age-related thymic atrophy reduces the ability to<br />
produce naïve T cells, which is one of the main factors contributes to lower immune-surveillance in<br />
the elderly. Other than aging, thymic atrophy also can be accelerated by various stressors, like<br />
chemotherapy, corticosteroids, disturbed lipid metabolism, infection, inflammation and<br />
psychological stress. However, our knowledge on thymic atrophy is very limited. Here we report<br />
that hypercholesterolemia induces acute thymic atrophy through inhibiting thymocyte expansion<br />
and differentiation.<br />
Methods and Results: To investigate the role of hypercholesterolemia, we use albumin driven cre to<br />
specifically knock out SRBI in the liver, causing defect in reverse cholesterol transport and thus<br />
hypercholesterolemia. One week of HCD induces acute thymic atrophy (thymus weight:<br />
SRBIflox/flox vs SRBIflox/flox-Alb-Cre, 28.22mg vs 9.28mg, p < 0.0001) in RCT defected mice, and a<br />
more dramatic decrease of thymocyte cellularity (5.9 X 10E7 vs 4.5 X 10E6). In order to know what<br />
happened with T cell development, we profiled development process using flowcytometry with<br />
CD4, CD8a, CD44 and CD25. We found that CD4+CD8a+ cell decreases to be only 18% of thymocyte<br />
from more than 80%, which indicates blocks in thymocyte differentiation. Besides, we also see an<br />
increase of DN1 (CD4-CD8a-CD25-CD44+) progenitor cells increased from 17% to 38% of double<br />
negatives (CD4-CD8a-). To exclude the possibility of accelerated cell apoptosis of certain cell type<br />
rather than ceased differentiation, we measured thymocyte apoptosis using annexin V staining. The<br />
results only show a very mild increase in cell apoptosis (annexin V+: 8% vs 11%) after 3 days of<br />
HCD treatment, which could not explain the dramatic change in thymocytes numbers alone. Then<br />
we performed gene expression profile on the thymus from mice after 3 days of HCD. Gene<br />
enrichment analysis using the differentially expressed genes reveals an enrichment of cell cycle<br />
genes, with many cell cycle-promoting genes decreased and cell cycle inhibiting genes increased.<br />
Consistently, BrdU incorporation combined with 7-AAD staining also indicates impaired thymocyte<br />
proliferation, the thymocytes reside in G2-M phase decrease from 2.8% to 1.7% of total thymocytes.<br />
The earlier developing cells are most impacted, with G2-M cells decrease from 5.3% to 1.4% of DN.<br />
Conclusions: Hypercholesterolemia induces acute thymic atrophy through inhibiting thymocyte<br />
expansion and differentiation. This study indicates high cholesterol diet could exert its impact on<br />
immune system through accelerating thymic atrophy.<br />
32
17<br />
Cardiac Cycle Affects Ultrasound Measurements of Ascending Aortic Diameter in a Marfan<br />
Mouse Model<br />
Zheying Chen 1 • Hisashi Sawada, MD, PhD 1 • Debra Rateri 1 • Alan Daugherty 1 •<br />
Mary Sheppard, MD 1<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky<br />
Graduate Student<br />
Objective:<br />
Ultrasound measurements of aortic diameter are a common endpoint in preclinical studies.<br />
However, there is a lack of standardization in both image capture and analysis. For our study, we<br />
developed a standardized protocol for measuring ascending aortic diameter and examined effects<br />
of cardiac cycle in wild type and fibrillin-1 hypomorphic (FBN1mgR/mgR) mice.<br />
Methods and Results:<br />
Twelve week old male and female FBN1mgR/mgR mice were anesthetized and maintained at a<br />
heart rate of 450-550 beats per minute. Ultrasound images were captured using a Vevo 2100<br />
system with a 40MHz tranducer. Images captured were standardized according to two anatomical<br />
landmarks: the innominate artery branchpoint and aortic valves. The largest luminal ascending<br />
aortic diameter between the sinotubular junction and the innominate artery were measured in<br />
mid-systole and end-diastole by two blinded, independent observers. Aortic diameters were<br />
significantly different (p
18<br />
A Novel Approach for Modifying Intrinsic Heart Rate: RRad knock-down<br />
Bryana Levitan 1 • Mihir Shah 2 • Joshua Rutland, MD 1 • Brooke Ahern 3 • Douglas Andres, PhD 4 •<br />
Jonathan Satin, PhD 3<br />
1Cardiology, University of Kentucky • 2 University of Kentucky • 3 Physiology, University of Kentucky<br />
• 4 Molecular and Cellular Biochemistry, University of Kentucky<br />
Staff<br />
Introduction: About 3 million people worldwide are dependent on pacemakers. Each year 600,000<br />
pacemakers are implanted worldwide, for the primary indication of symptomatic bradycardia. Biological<br />
pacemakers represent an emerging technology for bradyarrhythmias that require a pacemaker such as<br />
sinoatrial nodal failure due to congenital disease or aging. Leading edge biological pacemaker<br />
approaches include implantation of induced, pluripotent stem cell-derived cardiomyocytes (iPSC-CMs);<br />
however, intrinsic rates of implanted cells might be insufficient to meet physiologic demands, and cell<br />
implantation carries a variety of obstacles. In the present report, we introduce a novel molecular<br />
mechanism for increasing heart rate. Rad is a monomeric G-protein and constituent of the L-type<br />
calcium channel macromolecular complex (LTCC). Rad inhibits LTCC inward calcium current. LTCC<br />
activity contributes to pacemaker depolarization. Therefore, we tested the hypothesis that reduction of<br />
cardiac Rad will result in a stable increased heart rate (HR). We suggest that knockdown of cardiac Rad<br />
could facilitate the development of rate support alone or in combination with cells as a biologic<br />
pacemaker.<br />
Methods & Experimental Design: Cardiac-restricted, inducible Rad knockout (cRadKO) female mice were<br />
implanted with radio-telemeters (model F10A, DSI, Minneapolis MN). Baseline recordings (continuous<br />
72h) were followed by induction of Rad knockout (RadKO). Continuous recordings followed induction of<br />
RadKO at times specified in results. Intrinsic heart rate was measured after complete autonomic<br />
blockade with injection of propranolol (1 mg/kg) and atropine (1 mg/kg). Peak heart rate was achieved<br />
by injection of isoproterenol (ISO) at diurnal peak. Startle response was performed using a series of air<br />
jet challenges to provoke an increase in native adrenergic stimulation.<br />
Results: Baseline measures demonstrated normal diurnal rhythms of HR (daytime trough, night-time<br />
peak) without evidence of significant arrhythmias. Trough HR increased without evidence of significant<br />
arrhythmias 4 days post cRadKO, while peak HR remained unchanged. The increase in trough HR was<br />
sustained 1 month after cRadKO. During trough periods episodes of sinus bradycardia without SA node<br />
or AV node block were noted consistent with high vagal tone. Intrinsic HR was also significantly<br />
increased post-cRadKO over baseline. ISO challenge induced sinus tachycardia with a blunted response<br />
as compared to controls. During recovery 1-8 hours after ISO challenge both cRadKO and control mice<br />
exhibited transient episodes of bradyarrhythmias. All animals returned to pre-ISO rhythm without<br />
incident. The startle response did not elicit arrhythmias.<br />
Conclusion: Cardiac Rad deletion results in a stable increase in intrinsic heart rate with retained<br />
physiologic autonomic responsiveness. Rad depletion did not induce any significant arrhythmias under<br />
physiological stressors. We conclude that inhibition of Rad can be used to increase the intrinsic heart<br />
rate. This could represent a novel mechanism for tuning iPSC-CMs to more physiologic heart rates.<br />
34
19<br />
Inflammasome Activation Triggers Blood Coagulation through Pyroptosis<br />
Congqing Wu, PhD 1<br />
1CVRC, College of Medicine<br />
Postdoc<br />
Disseminated intravascular coagulation (DIC) is a fatal complication of systemic bacterial infection.<br />
Mechanisms for blood coagulation induced by bacterial infection are often attributed to host<br />
inflammatory response to bacterial virulence factors, yet the molecular events linking bacteria<br />
sensing to initiation of the coagulation cascade remain unclear. Here we identify inflammasome<br />
activation as a trigger for coagulation induced by gram-negative bacterial products. Specifically,<br />
canonical inflammasome activation elicited by the conserved type III secretion system (T3SS) rod<br />
protein EprJ from E. coli induces systemic coagulation through caspase-1 activation, whereas<br />
noncanonical inflammasome activation by lipopolysaccharide (LPS), a major outer membrane<br />
component of gram-negative bacteria, produces similar effects involving caspase-11. Deficiency of<br />
gasdermin D (Gsdmd) or depletion of monocytes and macrophages blocks inflammasome-mediated<br />
coagulation. Together, our data unveil inflammasome-dependent pyroptosis leads to blood<br />
coagulation.<br />
35
20<br />
Clinical Use of the Amplatzer Septal Occluder in Percutaneous Closure: A Single-Center<br />
Experience<br />
Seema Rao, MS 1 • Tyler Scaff 1 • John Gurley, MD 2 • Sibu Saha, MD 3<br />
1College of Medicine, University of Kentucky • 2 Cardiology, University of Kentucky • 3 Cardiothoracic<br />
Surgery, University of Kentucky<br />
Medical Student<br />
Device therapy is a viable alternative to open heart surgery in the management of intracardiac<br />
defects. The Amplatzer Septal Occluder (ASO) is one such device that has been adapted to close a<br />
wide variety of cardiac defects with few complications and a high success rate. This is a<br />
retrospective review of all the patients who received the ASO between 2012-2016 at our<br />
institution. In total, 169 patients underwent percutaneous closure of a septal defect with Amplatzer<br />
during the timeframe studied, and of those, 91% received the device for an atrial septal defect or<br />
patent foramen ovale. All Amplatzer devices achieved successful closure without requiring a second<br />
procedure. Four patients sustained complications of the procedure, with two experiencing<br />
tamponade, one with hematoma, and one with cellulitis. Three fatalities occurred within thirty days<br />
and were attributed to comorbidities not related to the device. The ASO has performed very well at<br />
our institution and we expect it to serve additional functions as the field of transcatheter cardiology<br />
develops.<br />
36
21<br />
Exposure to PCB126 during the Nursing Period Significantly Impairs Early-Life Glucose<br />
Tolerance<br />
Keegan Sammons 1 • Sara Tenlep, MS 1 • Leryn Reynolds, PhD 1 • Hollie Swanson, PhD 1 •<br />
Kevin Pearson, PhD 1<br />
1Pharmacology and Nutritional Sciences, University of Kentucky<br />
Undergraduate<br />
Polychlorinated Biphenyls (PCBs) are persistent environmental organic pollutants that are known<br />
to have detrimental health effects. In a mouse model in our lab, PCB126 exposure during pregnancy<br />
and nursing alters offspring body composition and glucose tolerance. The purpose of this study was<br />
to expose dams to PCB126 during the nursing period only. Female ICR mice were bred and half of<br />
the dams were exposed to either vehicle (safflower oil) or 1 µmole PCB126 per kg of body weight<br />
via oral gavage on postnatal days 3, 10, and 17 (n = 9/group). Offspring body weight, lean and fat<br />
mass, and glucose tolerance were measured. Both male and female offspring displayed normal body<br />
weights as well as body composition (p > 0.05). However, both male and female offspring that were<br />
exposed to PCBs during the nursing period had significantly impaired glucose tolerance at 3 weeks<br />
of age (p < 0.05). This persisted until 9 weeks of age in the female offspring (p < 0.05), but the<br />
difference disappeared as the male offspring aged (p > 0.05). Our earlier work suggests that in<br />
utero and postnatal PCB126 exposure predisposes offspring to having lower lean mass and<br />
impaired glucose tolerance later in life. However, our current study shows that exposure to PCB126<br />
through the mother's milk impairs glucose tolerance in the short-term and is likely caused by<br />
impairments in insulin receptor signaling in the periphery as others have shown with direct PCB<br />
exposures in adult mice. Future experiments will investigate the mechanisms of dysfunction caused<br />
by in utero PCB126 exposure, which may be driving the increased risk of obesity and insulin<br />
resistance in adult offspring.<br />
37
22<br />
Regulation of Akt Signaling by NQO1<br />
Joshua Preston 1 • Andrea Di Francesco, PhD 2 • Krystle Kalafut 2 • Tyler Rhinesmith 2 • Clara Di<br />
Germanio, PhD 2 • Michel Bernier, PhD 2 • Rafael de Cabo, PhD 2<br />
1Pharmacology and Nutritional Sciences, University of Kentucky • 2 Translational Gerontology<br />
Branch, National Institute on Aging, National Institutes of Health<br />
Undergraduate<br />
Background: NQO1 is an inducible quinone reductase that participates in the cellular defense<br />
system in response to chemical and oxidative stress. By using NAD(P)H as electron donor, NQO1<br />
enzymatic activity shifts the cellular redox state leading to a significant increase in the NAD+/NADH<br />
ratio. The propensity of NQO1 knockout mice to exhibit a diabetes-like phenotype and a null NQO1<br />
polymorphism associated with metabolic syndrome phenotypes in humans suggests a role for this<br />
enzyme in cellular energetics and metabolism. Here, we explored a possible link between NQO1, an<br />
intracellular generator of NAD+, and nutrient-sensitive NAD+-dependent deacetylase activity in the<br />
regulation of Akt, which plays a central role in regulating cellular signaling and energy pathways<br />
critically involved in type 2 diabetes and associated metabolic disorders.<br />
Results: Pharmacological inhibition of NQO1 with the mechanism-based inhibitor Mac609<br />
increased insulin-stimulated Akt phosphorylation in HepG2 and HeLa cell lines. Consistent with<br />
increased Akt activation, Mac609 promoted nuclear exclusion and inactivation of the downstream<br />
Akt targets, the gluconeogenic transcription factors forkhead box O1 (FOXO1) and O3 (FOXO3a) in<br />
liver-derived HepG2 cells treated with insulin. Conversely, siRNA-mediated NQO1 knockdown<br />
increased insulin- or serum-stimulated Akt phosphorylation. Similarly, MDA-MB-468 cells, which<br />
carry a polymorphic form of NQO1 that results in an absence of NQO1 protein and activity, showed<br />
markedly higher constitutive and insulin-stimulated Akt phosphorylation levels compared to an<br />
isogenic cell line stably expressing NQO1. We then explored the impact of pharmacological<br />
inhibition or genetic down-regulation of NQO1 on the global pattern of protein acetylation in HeLa<br />
cells. The reduction in NQO1 level and/or activity was associated with an increase in protein<br />
acetylation levels likely due to the inhibition of NAD+-dependent sirtuins. Lastly, cell treatment<br />
with the NAD+ precursor nicotinamide mononucleotide (NMN) inhibited Akt phosphorylation in a<br />
dose- and time-dependent manner, thus recapitulating the effects of NQO1 on Akt activity.<br />
Conclusions: We have identified a molecular mechanism whereby NQO1 functions as a modulator<br />
of insulin/Akt signaling pathway. These findings have implications for future basic and<br />
translational research on pathways that control energy homeostasis and diabetes.<br />
38
23<br />
Substitution of Leu11 and Tyr12 in Mouse Angiotensinogen Does Not Affect Angiotensin IImediated<br />
Functions in Mice<br />
Chia-Hua Wu 1 • Congqing Wu 1 • Feiming Ye 1 • Deborah Howatt 1 • Anju Balakrishnan 1 •<br />
Jessica Moorleghen 1 • Craig Vander Kooi 2 • Alan Daugherty 3 • Hong Lu 1<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center , University of Kentucky • 2 Molecular and Cellular<br />
Pharmacology, University of Kentucky • 3 Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of<br />
Kentucky<br />
Graduate Student<br />
Background and Objective: Angiotensinogen (AGT) is the unique precursor of angiotensin II<br />
(AngII), which is a critical contributor to atherogenesis. Renin cleavage of AGT exhibits species<br />
specificity. It has been determined in in vitro studies that Leu11-Tyr12 in mouse AGT and Val11-<br />
Ile12 in human AGT are essential for species-specific renin cleavage. In this study, we compared<br />
adeno-associated viral (AAV) vectors encoding human AGT or mouse AGT with Leu11Val and<br />
Tyr12Ile mutations to determine whether substitution of these residues regulated AngII-mediated<br />
functions.<br />
Methods and Results: Male hepatocyte-specific AGT deficient (hepAGT-/-) mice were injected<br />
intraperitoneally with AAV vector containing a null insert or encoding human AGT, while their wild<br />
type littermates (hepAGT+/+) were injected with AAV containing the null insert. All mice were in<br />
an LDLR -/- background. Two weeks after AAV injections, mice were fed a saturated fat-enriched<br />
diet for 12 weeks. Administration of AAV encoding human AGT led to high plasma human AGT<br />
concentrations, but had no effect on plasma renin concentrations and hypercholesterolemiainduced<br />
atherosclerosis. In a subsequent study, AAV encoding mutated mouse AGT with Leu11Val<br />
and Tyr12Ile were injected into hepAGT-/- mice. Mutated mouse AGT resulted in a significant<br />
increase of plasma AGT concentrations. Plasma renin concentrations in hepAGT-/- mice<br />
repopulated with mutated mouse AGT were decreased to a level comparable to their<br />
concentrations in hepAGT+/+ mice injected with a null AAV. AAV-driven expression of mutated<br />
mouse AGT also augmented atherosclerosis in hepAGT-/- mice.<br />
Conclusion: Human AGT does not repopulate AngII-mediated functions, whereas mutations of<br />
Leu11Val and Tyr12Ile in mouse AGT, to mimic the two amino acids in human AGT, does not affect<br />
AngII-mediated effects.<br />
39
24<br />
Vascular Inflammatory Regulation of Lipid Phosphate Phosphatase 3 Expression<br />
Patrick Van Hoose, PhD 1 • Andrew Morris, PhD 1 • Susan Smyth, MD, PhD 1<br />
1<strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky<br />
Postdoc<br />
Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3, is a cell surface enzyme that<br />
regulates lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) availability and<br />
signaling. Genome wide association studies in humans identified heritable single nucleotide<br />
polymorphisms (SNPs) in the final intron of PLPP3 that independently predicted coronary artery<br />
disease (odds ratio, 1.17; P=3.81×10–19). PLPP3 is dynamically regulated during vascular<br />
inflammation and the risk allele reduces gene expression by disrupting binding of CCAAT enhancer<br />
binding protein beta (CEBPβ). However, other mechanisms may control dynamic regulation of<br />
PLPP3. The USC Genome Browser identifies potential target sequences for NFκB responsive<br />
elements in the PLPP3 promoter, including three potential RelA (p65) binding sites. Previous work<br />
has established that smooth muscle expression of Plpp3 attenuates experimental atherosclerosis<br />
and development of intimal hyperplasia.These observations led to the hypothesis that dynamic<br />
regulation of PLPP3 is a crucial step in controlling vascular inflammation and disease progression.<br />
Coronary human smooth muscle cells (caHSMCs) were treated for 72hrs with 1µM angiotensin II<br />
(ATII) in the presence or absence of 1 µM parthenolide. Ldlr-/- mice were randomized to receive<br />
either vehicle saline or 1000ng/kg/min angiotensin II via osmotic mini-pump for 7 days. Following<br />
72hr exposure to angiotensin II (ATII) PLPP3 expression increases in caHSMCs and is accompanied<br />
by increases in p65 but not CEBPβ expression. Parthenolide, an inhibitor of IκBα degradation,<br />
blocks ATII induced PLPP3 expression. To determine whether Plpp3 is upregulated in response to<br />
ATII in vivo, Ldlr-/- mice were treated with ATII for 7 days. Plpp3 and p65 expression remained<br />
unchanged in the aortic arch, thoracic aorta, abdominal aorta and mesenteric arteries.These<br />
observations suggest potential novel regulation of PLPP3 expression governed by an ATII-NFκB<br />
pathway that could be important in the context of vascular inflammation and disease.<br />
40
25<br />
Myeloid β-catenin deficiency exacerbates atherosclerosis in low-density lipoprotein<br />
receptor-deficient mice<br />
Fang Wang, PhD 1 • Zun Liu, PhD 1 • Se-Hyung Park, PhD 1 • Taesik Gwag, PhD 1 • Weiwei Lu, PhD 1 •<br />
Yipeng Sui, PhD 1 • Changcheng Zhou, PhD 1<br />
1Pharmacology and Nutritional Science, University of Kentucky<br />
Postdoc<br />
Objective: The Wnt/β-catenin signaling pathway is an ancient and evolutionarily conserved<br />
pathway regulating essential aspects of cell fate determination, proliferation, migration and<br />
polarity. Canonical Wnt/β-catenin signaling has been implicated in atherosclerosis development<br />
but the cell-specific role of β-catenin in atherogenesis remains elusive. Macrophage is one of the<br />
major cell types involved in the initiation and progression of atherosclerosis, and this study aims to<br />
investigate the impact of β-catenin expression on macrophage functions and atherosclerosis<br />
development.<br />
Methods and Results: To investigate the role of macrophage Wnt/β-catenin signaling in<br />
atherogenesis, we generated myeloid-specific β-catenin-deficient low-density lipoprotein receptordeficient<br />
mice (β-catenin∆myeLDLR-/-). As expected, deletion of β-catenin decreased macrophage<br />
adhesion and migration in vitro. However, β-catenin∆myeLDLR-/- mice had significantly increased<br />
atherosclerosis as compared with control littermates. Mechanistic studies revealed that β-catenin<br />
can directly regulate signal transducer and activator of transcription (STAT) pathway in<br />
macrophages, and ablation of β-catenin resulted in STAT1 activation, leading to elevated<br />
macrophage inflammatory responses and increased atherosclerosis.<br />
Conclusions: This study demonstrates a critical role of myeloid β-catenin expression in<br />
atherosclerosis by modulating macrophage inflammatory responses.<br />
41
26<br />
Vascular inflammation induced expression of lipid phosphate phosphatase<br />
Kavya Balaji 1 • Patrick Van Hoose, PhD 1 • Andrew Morris, PhD 1 • Susan Smyth, MD, PhD 1<br />
1<strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky<br />
Undergraduate<br />
Lipid phosphate phosphatase 3 (PLPP3) is a polymorphic gene that is a member of the phosphatidic<br />
acid phosphatase family. It encodes for the cell surface enzyme, lipid phosphate phosphatase 3<br />
(LPP3) that regulates lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) availability<br />
and signaling. Genome wide association studies in humans identified heritable single nucleotide<br />
polymorphisms (SNPs) in PLPP3 that predicts coronary artery disease risk independently of<br />
traditional risk factors (odds ratio, 1.17; P=3.81×10–19). PLPP3 is dynamically regulated during<br />
vascular inflammation and the risk allele reduces gene expression by disrupting binding of CCAAT<br />
enhancer binding protein beta (CEBPβ). However, other mechanisms may control dynamic<br />
regulation of PLPP3. The USC Genome Browser identifies potential target sequences for NFκB<br />
responsive elements in the PLPP3 promoter, including three potential RelA (p65) binding sites.<br />
Hypothesis: These observations led to the hypothesis that angiotensin II, an inducer of vascular<br />
inflammation could regulate of PLPP3.<br />
Methods: Coronary human smooth muscle cells (caHSMCs) were treated for 12, 24, 48 and 72hrs<br />
hours with 1 M angiotensin II (ATII) in the presence or absence of parthenolide. Following<br />
treatment PLPP3 and p65 gene expression was examined.<br />
Results: The time course treatment of ATII revealed 72hr treatment upregulated PLPP3 gene<br />
expression in caHSMCs and this upregulation of PLPP3 was inhibited in the presence of<br />
parthenolide, an inhibitor of IκBα degradation.<br />
Conclusion: These observations suggest possible regulation of PLPP3 via an angiotensin II-NFkB<br />
dependent pathway, which may be important in the context of vascular disease.<br />
42
27<br />
LRP1 Deletion in Smooth Muscle Cells of the Outer Aortic Media Promotes Angiotensin IIinduced<br />
Thoracic Aortic Aneurysm<br />
Hisashi Sawada, MD, PhD 1 • Debra Rateri 1 • Mark Majesky, PhD 2 • Alan Daugherty, PhD 1<br />
1Saha CVRC, University of Kentucky • 2 Center for <strong>Cardiovascular</strong> Biology, University of Washington<br />
Postdoc<br />
Objective: Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional protein<br />
that is linked to several vascular pathologies. LRP1 deletion in smooth muscle cells (SMCs)<br />
accelerates angiotensin II (AngII)-induced thoracic aortic aneurysm (TAA). In association with TAA<br />
formation, there is medial thickening that is characterized by a transmural gradient in which<br />
pathology progressively increases from lumen to adventitial aspect. We hypothesized that deletion<br />
of LRP1 in the outer medial layers of the proximal thoracic aorta has a pivotal role in the<br />
pathogenesis of TAA. The aim of this study was to determine whether LRP1 deletion in the outer<br />
media accelerates AngII-induced TAA formation.<br />
Methods and Results: SMCs in the outer media of the ascending aorta are derived from the second<br />
heart field, as demonstrated by lineage tracing studies using Cre under the control of Mef2c.<br />
Therefore, we used Mef2c-driven Cre to delete LRP1 in SMCs of the outer medial layers. Female<br />
LRP1 flox/flox mice were bred to male Mef2c-Cre1/0 mice to generate study mice. We first<br />
confirmed LRP1 deletion in Cre1/0 mice by both immunostaining and Western blot. LRP1 was<br />
expressed ubiquitously across smooth muscle cells of all aortic medial layers in Cre 0/0 mice. In<br />
mice expressing Mef2c-Cre, aortic LRP1 protein was detected only in SMCs of the inner laminar<br />
medial layers. Western blotting demonstrated LRP1 protein abundance in Cre expressing mice was<br />
reduced by 43%. Saline or AngII (1,000 ng/kg/min) was infused by subcutaneous osmotic pumps<br />
for 28 days into 12 - 14 week-old male Cre0/0 and 1/0 mice. As expected, systolic blood pressure<br />
increased similarly in both AngII-infused Cre 0/0 and 1/0 mice compared to saline-infused mice.<br />
Aortic rupture occurred within 3 to 10 days after AngII infusion in 17% of AngII-infused Cre 0/0<br />
mice, while LRP1 deletion in Cre 1/0 mice increased aortic rupture to 27%. Aortic diameter in the<br />
survivors was significantly increased in Cre1/0 mice compared to Cre0/0 mice. Histologically,<br />
elastin fragmentation was detected in the aorta of AngII-infused Cre 0/0 mice and greater in Cre1/0<br />
mice.<br />
Conclusion: LRP1 in second heart field-derived SMCs of the outer media may play a critical role in<br />
the pathogenesis of TAA.<br />
43
28<br />
Sexual Dimorphism of Angiotensin II-induced Thoracic Aortic Rupture in Mice with LRP1<br />
Deficient Second Heart Field–derived Smooth Muscle Cells<br />
Bradley Wright 1 • Hisashi Sawada, MD, PhD 1 • Jessica Moorleghen 1 • Richard Charnigo, PhD 2 •<br />
Debra Rateri 1 • Mark Majesky, PhD 3 • Alan Daugherty, PhD 1<br />
1Saha CVRC, University of Kentucky • 2 Biostatistics, University of Kentucky • 3 Division of<br />
Cardiology, University of Washington<br />
Undergraduate<br />
Objective: Low-density lipoprotein receptor-related protein 1 (LRP1) maintains vascular<br />
homeostasis. Deletion of LRP1 in smooth muscle cells (SMCs) accelerates angiotensin II (AngII)-<br />
induced thoracic rupture and ascending aortic aneurysm. We demonstrated recently that SMCs in<br />
the outer medial layers of the ascending aorta were derived from second heart field (SHF). Deletion<br />
of LRP1 from SHF-SMCs significantly increased thoracic aortic rupture and aneurysms in male mice.<br />
The aim of this study was to determine if female sex influenced AngII-induced rupture and<br />
aneurysm in LRP1 deficient SHF-SMC mice.<br />
Methods and Results: Female LRP1 flox/flox mice were bred to male Mef2c-Cre 1/0 mice to<br />
generate study mice. Either saline or AngII (1,000 ng/kg/min) was infused by subcutaneous<br />
osmotic pumps for 28 days into 12-14 week old Cre 0/0 and Cre 1/0 mice of both sexes (N=12-31).<br />
As expected, AngII infusion increased systolic blood pressure in both sexes. During AngII infusion,<br />
aortic rupture occurred within 3 to10 days in 17% of Cre 0/0 mice, while LRP1 deletion in Cre 1/0<br />
mice increased significantly to 27% in males. Aortic rupture rate in AngII-infused female mice was<br />
decreased significantly compared to male mice, as there were none in Cre 0/0 and within 6-14 days<br />
only 9% in Cre 1/0 female mice. Ultrasonography was used to measure ascending aortic dilation as<br />
an index of thoracic aneurysm. Ascending aortic diameter in the survivors was significantly<br />
increased in AngII-infused Cre 1/0 vs Cre 0/0 and saline-infused controls in both sexes.<br />
Conclusion: Although male and female Cre1/0 mice experienced similar dilation of the ascending<br />
aorta under AngII infusion, female Cre1/0 mice experienced a significantly increased rate of<br />
survival compared to male litter mates. Future studies will determine the mechanism of sexual<br />
dimorphism that reduces AngII-induced thoracic aortic rupture in females.<br />
44
29<br />
SAA is not Just an HDL-Associated Lipoprotein<br />
Patricia Wilson, MS 1 • Joel Thompson, PhD 1 • Frederick de Beer, MD, PhD 1 • Nancy Webb, PhD 1 •<br />
Lisa Tannock, MD, PhD 2<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky • 2 Endocrinology/Saha<br />
<strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky<br />
Staff<br />
<strong>Cardiovascular</strong> disease (CVD) is the leading cause of death in developed nations despite widespread<br />
use of pharmacological interventions. Individuals with obesity and/or diabetes are at particularly<br />
high risk for CVD and research suggests this may be due to elevated levels of serum amyloid A<br />
(SAA). Brief and chronic elevations of SAA have been indeed found to be atherogenic in mouse<br />
models. The current dogma is that SAA is exclusively an HDL associated lipoprotein. However, we<br />
and others have reported SAA on apoB-containing lipoproteins in both humans and mice and<br />
several studies have recently suggested that SAA-LDL is a risk factor for CVD. The goal of this study<br />
was to determine if SAA can shift between HDL and apoB-containing particles, and if the presence<br />
of SAA on apoB particles affects their atherogenicity. To determine if SAA could be exchanged<br />
between lipoproteins in vitro, we first incubated SAA with each lipoprotein separately, then mixed<br />
the lipoprotein containing SAA with each of the other two lipoproteins. To determine is this<br />
exchange could also occur in vivo, we injected apoE-/- x SAA1.1/2.1-DKO mice with murine SAA<br />
complexed with individual murine lipoproteins. To elucidate the role of CETP in SAA shifting, some<br />
mice were preinjected with an adenovirus expressing CETP 72 hours prior to injection with SAA-<br />
HDL. We now have evidence that SAA does indeed shift between lipoproteins both in vitro and in<br />
vivo, facilitated by CETP, and that delipidated SAA will completely associate with any available<br />
lipoproteins although preferentially on HDL. The presence of SAA on apoB-containing lipoproteins<br />
has physiological relevance, as we have demonstrated that this augments the proteoglycan binding<br />
affinity of these particles thus increasing their atherogencity. Collectively our data shows that SAA<br />
can be exchanged from HDL to apoB-containing lipoproteins and that this exchange is proatherogenic.<br />
45
30<br />
Computational modeling of amylin-induced calcium dysregulation in rat ventricular<br />
cardiomyocytes<br />
Peter Kekenes-Huskey 1<br />
1Chemistry, University of Kentucky<br />
Faculty<br />
Hyperamylinemia is a condition that accompanies obesity and precedes type II diabetes, and it is<br />
characterized by above-normal blood levels of amylin, the pancreas-derived peptide. Human amylin<br />
oligomerizes easily and can deposit in the pancreas [1], brain [2], and heart [3], where they have<br />
been associated with calcium dysregulation. In the heart, accumulating evidence<br />
suggests that human amylin oligomers form moderately cation-selective [4, 5] channels that embed<br />
in the cell sarcolemma (SL). The oligomers increase membrane conductance in a concentrationdependent<br />
manner [5], which is correlated with elevated cytosolic Ca 2+ . These findings motivate<br />
our core hypothesis that non-selective inward Ca 2+ conduction afforded by human amylin<br />
oligomers increase cytosolic and SR Ca 2+ load, which thereby magnifies intracellular Ca 2+<br />
transients. Questions remain however regarding the mechanism of amylin-induced Ca 2+<br />
dysregulation, including whether enhanced SL Ca 2+ influx is sufficient to elevate cytosolic Ca 2+<br />
load [6], and if so, how might amplified Ca 2+ transients perturb Ca 2+ -dependent cardiac<br />
pathways. To investigate these questions, we modified a computational model of cardiomyocytes Ca<br />
2+ signaling to reflect experimentally-measured changes in SL membrane permeation and<br />
decreased sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) function stemming from<br />
acute and transgenic human amylin peptide exposure. With this model, we confirmed the<br />
hypothesis that increasing SL permeation alone was sufficient to enhance Ca 2+ transient<br />
amplitudes. Our model indicated that amplified cytosolic transients are driven by increased Ca 2+<br />
loading of the sarcoplasmic reticulum (SR) and the greater fractional release may contribute to the<br />
Ca 2+ -dependent activation of calmodulin. Importantly, elevated Ca 2+ in the SR and dyadic space<br />
collectively drive greater fractional SR Ca 2+ release for human amylin expressing rats (HIP) and<br />
acute amylin-exposed rats (+Amylin) mice, which contributes to the inotropic rise in cytosolic Ca 2+<br />
transients. These findings suggest that increased membrane permeation induced by<br />
oligomeratization of amylin peptide in cell sarcolemma contributes to Ca 2+ dysregulation in prediabetes.<br />
46
31<br />
PCB 126 Disrupts Gut Microbiota and Increases Intestinal Inflammation in a Mouse Model of<br />
Atherosclerosis<br />
Jessie Hoffman, MS 1 • Michael Petriello, PhD 2 • Olga Vsevolozhskaya, PhD 3 •<br />
Richard Charningo, PhD 3 • Andrew Morris, PhD 2 • Bernhard Hennig, PhD 4<br />
1Pharmacology and Nutritional Sciences, University of Kentucky • 2 Division of <strong>Cardiovascular</strong><br />
Medicine, University of Kentucky • 3 Biostatistics, University of Kentucky • 4 Animal and Food<br />
Sciences, University of Kentucky<br />
Graduate Student<br />
The gut microbiome is sensitive to diet and environmental factors and is involved in the regulation<br />
of many host metabolic responses. Dioxin-like pollutants contaminate many food sources, and thus<br />
human exposure begins within the gut, which may play a role in pollutant-induced systemic<br />
toxicity. Additionally, gut dysbiosis and inflammation pose risk factors for the development of<br />
cardiovascular diseases, specifically atherosclerosis. We hypothesize that the dioxin-like pollutant<br />
PCB 126 will perturb gut microbial populations and impair gut health, which may contribute to<br />
pollutant-induced systemic toxicity in an atherosclerotic mouse model. LDLr -/- mice were fed a<br />
low fat atherogenic diet (10% fat, 0.15% cholesterol) for 14 weeks and exposed to PCB 126 at week<br />
2 and 4. Exposure to PCB 126 reduced gut microbial diversity and shifted populations at the<br />
phylum and genus levels in ways that mimic observations in chronic inflammatory diseases.<br />
Furthermore, PCB exposed mice exhibited increased markers of inflammation in intestinal and<br />
plasma samples. Interestingly, Cyp1a1 gene expression was increased in intestinal samples even 10<br />
weeks after PCB exposure, indicating a slow continual passage of pollutants through the<br />
enterohepatic circulation. These data imply that PCB toxicity is already initiated in the gut through<br />
disruption of healthy microbiota, and increases in gut inflammation. These observations highlight a<br />
unique opportunity for dietary interventions that are beneficial for both gut and overall health.<br />
Further research should examine how nutritional components can combat pollutant induced<br />
toxicity initiated at the gut level.<br />
47
32<br />
PCB 126 Exposure Increases Peripheral Vascular Disease Risk in Mice with Fatty Liver<br />
Jazmyne Barney 1 • Banrida Wahlang, PhD 2 • Brendan Thompson 1 • Chunyan Wang, PhD 3 •<br />
Omer Hamad 3 • Jessie Hoffman, MS 4 • Michael Petriello, PhD 2 • Andrew Morris, PhD 2 •<br />
Bernhard Hennig, PhD 3<br />
1Toxicology and Cancer Biology, University of Kentucky • 2 UK Superfund <strong>Research</strong> Center,<br />
University of Kentucky • 3 Animal and Food Sciences, University of Kentucky • 4 Pharmacology and<br />
Nutritional Sciences, University of Kentucky<br />
Graduate Student<br />
Xenobiotic and energy metabolism is dependent on a functional liver. Exposure to environmental<br />
pollutants like polychlorinated biphenyls (PCBs) is associated with inflammatory diseases,<br />
including non-alcoholic fatty liver disease and atherosclerosis. A compromised liver may play a<br />
critical role in modifying the induction of PCB toxicity and inflammation of the peripheral<br />
vasculature. Over a 14 week study, male C57Bl/6 mice (n=10) were fed an amino acid control diet<br />
or a methionine-choline deficient diet (MCD) with or without oral gavage of PCB 126 (0.5mg/kg).<br />
Post euthanasia, tissue and blood were collected for histological, toxicological, and inflammatory<br />
evaluation. Regardless of diet, PCB 126 induced hepatic steatosis. The MCD+PCB126 group<br />
exhibited fibrosis and increased liver to body weight ratio, suggesting liver injury and toxicity. Mice<br />
fed MCD-diet and exposed to PCB 126 demonstrated altered expression of hepatic genes involved in<br />
carbohydrate and lipid metabolism, indicating metabolic dysfunction. With regard to effects of PCB<br />
126 on extra-hepatic organs, all mice fed MCD diet had decreased expression of plasma leptin and<br />
resistin, and PCB 126 exposed groups appeared to have crown like structures in their epididymal<br />
adipose tissue, indicating the presence of inflammatory cells. In addition, MCD+PCB 126 mice<br />
displayed increased plasma inflammatory markers including Icam-1, Mcp-1, and Tnf-α.<br />
Interestingly, in the MCD+PCB 126 group, plasma ALT and AST levels were increased as well as proatherogenic<br />
trimethylamine-N-oxide (TMAO), implying simultaneous liver damage and increased<br />
peripheral vasculature disease risk. Together these results provide a novel linkage of a<br />
compromised liver to PCB-induced hepatic and vascular inflammation. These finding also a have<br />
translational component, suggesting environmental pollutants can cause inflammatory disease<br />
pathologies by stimulating cross-talk between individual organ systems. (Supported in part by<br />
NIEHS/NIH grant P42ES007380).<br />
48
33<br />
Promoter Enhancer Interactions Regulating PLPP3 Gene Expression<br />
Guogen Mao, PhD 1 • Shaojing Ye, PhD 1 • Susan S. Smyth, MD, PhD 2 • Andrew J. Morris, PhD 2<br />
1Gill Heart and Vascular Institute, University of Kentucky • 2 Gill Heart and Vascular Institute,<br />
Veterans Affairs Medical Center,, University of Kentucky<br />
Staff<br />
Common heritable intronic variants within the final fifth intron of the PLPP3 gene associate with<br />
significant inter individual differences in human coronary artery disease risk. PLPP3 is one of three<br />
closely related genes encoding lipid phosphate phosphatases 1-3 which are integral membrane<br />
enzymes that dephosphorylate lipid phosphate mono esters and some lipid polyphosphates. PLPP3<br />
expression in vascular endothelial cells is required for mouse development and while individuals<br />
with heterozygosity for loss of function PLPP3 alleles have been identified homozygotes have not so<br />
PLPP3 is likely also essential for human development. Because the three PLPP genes have<br />
overlapping expression patterns and encode enzymes with very similar activities it is reasonable to<br />
hypothesize that differences in regulation of expression may underlie these unique functions of<br />
PLPP3 gene in atherosclerosis and development. We used informatic approaches to identify<br />
regulatory elements within the PLPP3 promoter and coronary artery disease risk haplotype block.<br />
We then cloned the relevant sequences into reporter constructs and used transfection assays and<br />
site directed mutagenesis to examine sequence and allele specific effects on promoter and enhancer<br />
activity and on how these interact to control gene expression. The ~1.5 KB PLPP3 promoter<br />
comprises a ~0.5 KB enhancer indicated by H3K4ms1/H3K27Ac markers and a ~1KB core<br />
promoter containing three NF-kB (p50/RelA and p42/RelB) binding sites. Chromatin<br />
immunoprecipitation and reporter assays indicate that all three of these are functional regulators of<br />
basal activity of the PLPP3 promoter with very strong upregulation of promoter activity observed<br />
in transfection assays with these transcription factors. Similar informatic approaches identified<br />
cFOS and C/EBP target sequences in accessible chromatin within the coronary artery disease risk<br />
associated haplotype block in the final intron of the PLPP3 gene. These sequences are conserved in<br />
mice and humans and interact with their cognate transcription factors in immunoprecipitation and<br />
gel shift assays and regulate expression of both generic promoters and the PLPP3 promoter in<br />
reporter gene/transfection assays. The cFOS and C/EBP target sequences exhibit allelic variation in<br />
humans with coronary artery disease risk variants decreasing transcription factor binding and<br />
transcriptional enhancer activity. Taken together these results identify an NF-kB driven pathway<br />
that likely explains the widely observed strong upregulation of PLPP3 expression during<br />
inflammation and atherosclerosis and suggest a mechanism by which coronary artery disease risk<br />
associated variants of intronic enhancer sequences could decrease PLPP3 expression and reduce<br />
the normally protective functions of this gene observed in experimental models of atherosclerosis.<br />
49
34<br />
Endothelial Mineralocorticoid Receptor and Neutrophils Mediate Aldo plus Salt-Induced<br />
Abdominal Aortic Aneurysm<br />
Shu Liu 1 • Yu Zhong 1 • Zhenheng Guo 2 • Ming Gong 1<br />
1Physiology, University of Kentucky • 2 Pharmacology and Nutritional Sciences, University of<br />
Kentucky<br />
Staff<br />
<strong>Abstract</strong><br />
Objective—We recently reported that administration of mice with aldosterone (Aldo) or<br />
deoxycorticosterone acetate (DOCA) plus salt induces AAA via mineralocorticoid receptor (MR).<br />
The current study defines the specific roles of endothelial and myeloid cell MR in DOCA-salt<br />
induced AAA.<br />
Approach and Results—A tamoxifen inducible endothelial cell (EC)-specific MR knockout mouse<br />
model (iECMRKO) and a myeloid cell-specific MR knockout mouse model (MyMRKO) were<br />
developed. The iECMRKO mice, but not the MyMRKO mice were protected from Aldo- or DOCA-saltinduced<br />
AAA. Mechanistically, EC-specific MR deletion had little effect on Aldo-salt-induced salt<br />
retention, hypertension, and renal fibrosis, but largely suppressed aortic elastin degradation,<br />
matrix metalloproteinase-2 (MMP-2) and MMP-9 upregulation, macrophage and neutrophil<br />
infiltration. Surprisingly, neutrophils, but not macrophages, were observed in the aorta 1 week<br />
after Aldo-salt administration in control mice, but not in iECMRKO mice. Treatment of C57BL/6<br />
mice with an anti-PMN antibody selectively suppressed Aldo-salt-induced circulating Ly6G-postive<br />
neutrophils, but not CD4-postive leukocytes, and protected mice from Aldo-salt-induced AAA. In<br />
cell cultures, Aldo-induced endothelial adhesion molecules (E-selectin, P-selectin, and ICAM-1, but<br />
not VCAM-1) and proinflammatory cytokine (IL-6 and MCP-1) mRNA expressions were abolished in<br />
MR-deficient ECs. Importantly, Aldo-salt-induced ICAM-1 but not VCAM-1 protein upregulation was<br />
abolished in aortas from iECMRKO mice.<br />
Conclusions—Endothelial MR, but not myeloid cell MR, plays an important role in Aldo-salt-induced<br />
AAA. Moreover, ICAM-1, but not VCAM-1; and neutrophils, but not macrophages, mediate the early<br />
processes of Aldo-salt-induced and endothelial MR-mediated AAA development.<br />
50
35<br />
Identification of Candidate Long QT Syndrome Type 2 Patients Starting from Exome<br />
Sequences Identified in a Biobank Cohort<br />
Allison Hall, MS 1 • Don Burgess, PhD 2 • Pierre Fwelo 1 • Jennifer Smith 3 • Corey Anderson, PhD 4 •<br />
Craig T. January, MD, PhD 4 • Ann Stepanchick, PhD 4 • Uyenlinh Mirshah, PhD 4 • Jonathan Luo, PhD 4<br />
• Dustin Hartzel, PhD 4 • Michael Murray, MD 5 • Tooraj Mirshahi, PhD 5 • Brian Delisle, PhD 1<br />
1Physiology, University of Kentucky • 2 Physics, Asbury • 3 University of Kentucky • 4 University of<br />
Wisconsin • 5 Geisinger Health System<br />
Staff<br />
Introduction: Every year congenital long QT syndrome (LQTS) is thought to cause sudden cardiac<br />
death in hundreds of individuals in the US. Genetic screening potentially could identify LQTS<br />
patients before it strikes. However, genetic analyses often find novel rare sequence variants of<br />
uncertain physiological significance, and little is known about genetic screening in unaffected<br />
populations.<br />
Hypothesis: LQTS type 2 (LQT2) is caused by loss-of-function mutations in the rapidly activating<br />
delayed rectifier K+ channel gene KCNH2 (Kv11.1). Screening for KCNH2 variants using an<br />
approach similar to the Comprehensive In Vitro Proarrhythmia Assay for drug testing will allow the<br />
identification of candidate LQT2 patients starting from exome sequences.<br />
Methods: Ten KCNH2 mutations from the NCBI ClinVar database listed as “pathogenic”, “suspectedpathogenic”,<br />
or “conflicting interpretations” were identified in 10,000 Whole Exome Sequences<br />
(WES) from the Geisinger MyCode® cohort. The KCNH2 mutations were screened using Western<br />
blot to quantify terminally glycosylated mature Kv11.1 protein (a proxy for Kv11.1 channel<br />
trafficking); patch-clamp to measure Kv11.1 channel current (IKv11.1); and computational<br />
simulations with a human ventricular action potential (AP) model to predict AP duration (a<br />
correlate for the QT interval).<br />
Results: Two of the KCNH2 mutations were trafficking-deficient to decrease mature Kv11.1 protein<br />
and peak IKv11.1, and five mutations altered normal Kv11.1 channel activation or deactivation.<br />
Simulating the decrease in IKv11.1 caused by the trafficking-deficient KCNH2 mutations increased<br />
AP duration by >30%, whereas the mutations that disrupted Kv11.1 channel gating did not predict<br />
significant changes in AP duration. De-identified Electronic Health Records (EHR) from the<br />
Geisinger MyCode® subjects showed that the corrected QT interval (QTc Bazette) for the patients<br />
that harbored the trafficking-deficient KCNH2 mutations was ≥480 ms, whereas the average QTc<br />
for all EHR database subjects was
36<br />
Optimization of immunomagnetic separation for cryopreserved cord blood and apheresis<br />
mononuclear cell fractions derived endothelial progenitor cells<br />
Himi Tripathi, PhD 1 • Lakshman Chelvarajan, PhD 1 • Brad J Berron, PhD 2 • Ahmed Abdel Latif, MD,<br />
PhD 1<br />
1Gill Heart Institute and Division of <strong>Cardiovascular</strong> Medicine, University of Kentucky • 2 Department<br />
of Chemical and Materials Engineering, University of Kentucky<br />
Staff<br />
Introduction: <strong>Cardiovascular</strong> tissue damage in acute myocardial infarction mainly arises from the<br />
loss of blood flow, leading to changes in the composition and function of the ischemic region and<br />
heat failure. Multiple studies have examined the utility of CD34+ endothelial progenitor cells (EPCs)<br />
for limiting tissue damage and promoting tissue repair after an ischemic event. However, isolation<br />
of EPCs using traditional fluorescent activated cell sorting is cumbersome, expensive and time<br />
consuming. In this study, we examined the efficacy of novel antigen based methods for EPC<br />
isolation from human cord blood.<br />
Methods and Results: Immunomagnetic cell sorting protocol to purify CD34+ cells from<br />
cryopreserved cord blood and apheresis samples was optimized using MACS ultrapure CD 34<br />
microbead kit. Cell viability, CD 34 purity and endothelial progenitor cells phenotypic expression<br />
was assessed using flow cytometer and trypan blue assay. After thawing cryopreserved cord blood,<br />
we observed >80 ± 10% viable cells and 95±5% viable apheresis cells. CD34+ purity of 74 ± 16%<br />
and 94 ± 1.7% was achieved in cryopreserved cord blood and apheresis samples, respectively.<br />
Recovery of CD34+ cells was higher from apheresis (64.6%) in comparison to cord blood (31.3%).<br />
Post selection, the expression of EPCs markers was 65.6± 23.2 for CD31 and 90.3 ± 6.6 for CD133 in<br />
cord blood and 79.7± 5.5 and 69.6 ± 5.4 in apheresis samples. Additionally, we observed<br />
significantly higher recovery and system efficiency when using small input number of CD34+ cells<br />
(3.02 ± 2.7 X 106 vs. 31.3% ±54.1 X 106; p
37<br />
Elevated circulating TGF-beta is not the cause of increased atherosclerosis development in<br />
biglycan deficient mice<br />
Joel Thompson, PhD 1 • Patricia Wilson, PhD 1 • Alex Wyllie 1 • Adrian Wyllie 1 • Lisa Tannock, MD 1<br />
1Internal Medicine, University of Kentucky<br />
Staff<br />
Background: Vascular biglycan contributes to atherosclerosis development and increased biglycan<br />
expression correlates with increased atherosclerosis. However, mice deficient in biglycan have<br />
either no reduction in atherosclerosis or an unexpected increase in atherosclerosis. Biglycan<br />
deficient mice have systemically elevated TGF-beta, likely due to lack of sequestration of TGF-beta<br />
in extracellular matrix The purpose of this study was to determine if prevention of TGF-beta<br />
elevations in biglycan deficient mice affected atherosclerosis development.<br />
Methods: Biglycan deficient mice were crossed to Ldlr deficient mice. Diabetes was induced via<br />
streptozotocin and all mice were fed a high cholesterol diet. Diabetic biglycan wildtype and biglycan<br />
deficient Ldlr deficient mice were injected with the TGF-beta neutralizing antibody 1D11 or<br />
irrelevant control antibody 13C4.<br />
Results: Biglycan deficient mice had significantly elevated plasma TGF-beta levels, which was<br />
further increased by diabetes, and significantly increased atherosclerosis. There was a significant<br />
correlation between TGF-beta concentrations and atherosclerosis. However, despite nearly<br />
complete suppression of plasma TGF-beta levels in mice treated with the TGF-beta neutralizing<br />
antibody 1D11, there was no significant difference in atherosclerosis between mice with elevated<br />
TGF-beta levels and mice with suppressed TGF-beta levels.<br />
Conclusions: The increased atherosclerosis in biglycan deficient mice does not appear to be due to<br />
elevations in TGF-beta.<br />
53
38<br />
Gestational Diabetes Provokes Postpartum Cardiac Hypertrophy via Activation of Ca2+-<br />
Dependent Signaling<br />
Amanda Hoskins 2 • Nirmal Verma, PhD 1 • Florin Despa, PhD 1 • Sanda Despa, PhD 1<br />
1Pharmacology & Nutritional Sciences, University of Kentucky • 2 University of Kentucky<br />
Undergraduate<br />
Introduction: Gestational diabetes mellitus (GDM) complicates 9% of pregnancies in the US and its<br />
incidence is growing. Women with prior GDM have higher risk of developing cardiac hypertrophy<br />
and dysfunction, but the underlying mechanisms are largely unknown.<br />
Hypothesis: GDM promotes pathological growth of the heart through Ca2+-dependent hypertrophy<br />
signaling.<br />
Methods and Results: Female rats that express the human isoform of amylin, an amyloidogenic<br />
peptide co-secreted with insulin, in the pancreatic β-cells (HIP rats) were used as a GDM model. WT<br />
littermates served as controls. In both groups, glucose tolerance decreased during pregnancy and<br />
recovered after giving birth, with HIP females remaining glucose intolerant compared to the WT<br />
throughout the study. Cardiac hypertrophy, assessed from heart weight-to-body weight ratio, heart<br />
weight-to-tibia length ratio and echocardiographic measurements of the left-ventricular wall,<br />
occurred in both HIP and WT females during pregnancy. By two months postpartum, heart size<br />
returned to the pre-pregnancy level in the WT but remained significantly larger in HIP females. To<br />
uncover the cause of hypertrophy in postpartum HIP females, we investigated the activation status<br />
of calcineurin/NFAT and CaMKII/HDAC hypertrophy pathways. Calcineurin/NFAT signaling,<br />
assessed from the nuclear-to-cytosolic localization of NFATc4, was reduced during late pregnancy<br />
in both groups. In WT females, this pathway returned to its baseline activation level within two<br />
months postpartum. However, in postpartum HIP females the ratio of nuclear-to-cytosolic NFATc4<br />
was significantly larger than at baseline, indicating re-activation of this hypertrophy pathway. In<br />
contrast, the CaMKII/HDAC hypertrophy signaling was strongly activated in late pregnancy and<br />
returned to baseline postpartum in both HIP and WT females. Since calcineurin and CaMKII are<br />
activated by higher cytosolic Ca2+, we also analyzed myocyte Ca2+ cycling. Ca2+ transient decay<br />
was slower in myocytes from postpartum HIP females vs. baseline, while no differences occurred in<br />
the WT.<br />
Conclusion: Two months after a GDM-complicated pregnancy, female rats show cardiac<br />
hypertrophy that is likely caused by activation of calcineurin/NFAT hypertrophy pathway.<br />
54
39<br />
Effects of Molecular Clock Component Deletion in Mouse Cardiomyocytes on Long QT<br />
Syndrome Phenotype<br />
Kaitlyn Samuels 1 • Elizabeth Schroder 1 • Tanya Seward 1 • Brian Delisle, PhD 1<br />
1Physiology, University of Kentucky<br />
Undergraduate<br />
Cardiac excitability is affected by various genetic and environmental perturbations, which is often<br />
the cause of a multitude of human cardiac diseases. Specifically, this study explored the effects of<br />
the genetic knockout of the Bmal1 circadian transcription factor in mouse heart cells superimposed<br />
upon a congenital Scn5a sodium ion channel genetic mutation representative of long QT syndrome<br />
type 3 (LQT3) in humans. Changes in electrocardiographic (ECG) properties were evaluated to<br />
investigate the changes in cardiac excitability in these transgenic mice. There were a significantly<br />
higher number of cardiac arrhythmias after the Bmal1 gene knockout than before the knockout,<br />
specifically an increased number of RR-interval pauses. There was also a consistently slower heart<br />
rate after the Bmal1 gene knockout. Finally, there was increased slope factor between the RRinterval<br />
and QT-interval after the Bmal1 knockout than before the knockout. These findings are<br />
consistent with previous similar investigations and serve to help provide a better understanding of<br />
the pathophysiology associated with cardiac genetic mutations.<br />
55
40<br />
The Prognostic Role of Elevated Myeloperoxidase in Patients with Acute Coronary<br />
Syndrome: A Systematic Review and Meta-Analysis<br />
Mohamed Abo-Aly, MD 1 • Andrew kolodziej, MD 1 • Raphael Twerenbold, MD 1 •<br />
Christian Mueller, MD 1 • Ahmed Abdel-Latif, MD, PhD 1<br />
1Division of cardiovascular medicine, Gill heart institution., University of Kentucky<br />
Postdoc<br />
Background:<br />
Many reports have shown the correlation between myeloperoxidase (MPO) and the pathogenesis of<br />
acute coronary syndrome (ACS). However, the prognostic role of MPO for major cardiac events or<br />
mortality in ACS patients has not been well studied. We sought to perform a systematic review and<br />
meta-analysis to examine the prognostic value of inpatient MPO level in patients presenting with<br />
ACS.<br />
Methods:<br />
PubMed and Cochrane databases were searched from 1975 to September <strong>2017</strong> for studies that<br />
investigated the prognostic value of serum MPO in patients with ACS. Studies should have<br />
dichotomized patients into a high MPO and a low MPO group, reported clinical outcomes according<br />
to the same cutoff value of MPO and followed up patients for at least 30 days to be eligible for<br />
enrollment. Data were analyzed using random-effects model to control heterogeneous reporting.<br />
Results:<br />
We retrieved 3896 studies for initial screening which yielded 14 studies including 9268 subjects<br />
with a median follow up of 8.7 ± 21.96 months for the final analysis. High serum MPO level<br />
significantly predicts the future risk of mortality (odds ratio [OR] 2.039; 95% confidence interval<br />
[CI]: 1.405-2.959; P=0.0176) and MACE (OR 1.42; 95% CI: 1.00-1.99; p=0.044). we also observed a<br />
string trend towards higher incidence of recurrent myocardial infarction (MI) in patients with high<br />
MPO level (OR 1.24; 95% CI: 0.99-1.54; p=0.054) in comparison to patients with low serum MPO.<br />
Conclusion:<br />
In this meta-analysis examining the long outcomes in ACS patients, high MPO levels were associated<br />
with worse clinical outcomes. These observations support the use of MPO as a novel clinical<br />
prognostic marker in patients with acute coronary syndrome.<br />
56
41<br />
Desynchrony of Tissue Oscillators and Compromised Blood Pressure Circadian Rhythm in a<br />
Novel Diabetic Db/Db-Mper2luc Mouse Model<br />
Tianfei Hou, MS 1 • Wen Su, MD 1 • Ming Gong, MD, PhD 2 • Zhenheng Guo, PhD 1<br />
1Department of Pharmacology and Nutritional Sciences, University of Kentucky • 2 Department of<br />
Physiology, University of Kentucky<br />
Graduate Student<br />
Diabetic patients have a high prevalence of blood pressure (BP) circadian rhythm disruption mostly<br />
manifested as reduced BP dipping during the inactive phase (non-dipping profile). Accumulating<br />
evidence demonstrates a pathophysiological link between end-organ damage and non-dipping BP.<br />
Clock gene dysregulation may link diabetes with non-dipping BP but comprehensive study of clock<br />
gene oscillation dysregulation in diabetes has been hampered by the impractical requirement of<br />
intensive timed sampling. To address this issue, we generated a novel diabetic model, db/dbmPer2Luc<br />
mouse, by crossing the extensively used type 2 diabetic db/db mice with the clock gene<br />
Period 2 (mPer2) luciferase knock-in mPer2Luc mice. The db/db-mPer2Luc mice were obese,<br />
diabetic and had non-dipping BP. Real-time measurement of bioluminescence of the many tissues<br />
explanted from the db/db-mPer2Luc mice revealed that the phases of the mPer2 circadian<br />
oscillations shifted to different extents in aorta, mesenteric artery, kidney, liver, white adipose<br />
tissue and thymus, but no phase shift was detected in the central SCN tissue, or the adrenal gland<br />
and lung. The desynchrony of the tissue oscillators in the db/db-mPer2Luc mice was associated<br />
with compromised circadian rhythms in respiratory exchange ratio. Moreover, the time-of-day<br />
variations in vascular contractile responses and in baroreflex sensitivity were abolished. Taken<br />
together, we generated a novel diabetic db/db-mPer2Luc mouse model. Using this model, we<br />
revealed a desynchrony among tissue oscillators, a loss of the time-of-day variations in vascular<br />
contractile responses and baroreflex sensitivities, all of which likely contribute to the dampening of<br />
BP circadian rhythm in diabetes.<br />
57
42<br />
Lysophosphatidic Acid Receptor 4 Influence The development of Atherosclerotic Vascular<br />
Disease in Mouse Model<br />
Liping Yang, MD 1 • Andrew Morris, PhD 1 • Susan Smyth, MD, PhD 1<br />
1Internal Medicine - Cardiology, University of Kentucky<br />
Staff<br />
Background: Lysophosphatidic acid (LPA) is one of the simplest phospholipid signaling molecules.<br />
LPA elicits diverse biological functions including cell proliferation, differentiation, migration,<br />
survival and apoptosis through binding and activating specific cell surface G-protein coupled<br />
receptors (LPA1-6). LPA1/Edg2, LPA2/Edg4 and LPA3/Edg7 receptors are the endothelial<br />
differentiation gene (Edg) family. LPA4/p2y9/GPR23 of the purinergic receptor family and the<br />
related LPA5/GPR92 and LPA6/p2y5 have been identified as novel LPA receptors. LPA is generated<br />
by the secreted lysophospholipase D autotaxin (ATX) and inactivated by dephosphorylation by<br />
membrane lipid phosphate phosphatases (LPPs). Our previous work established that ATX and LPP3<br />
contribute to atherosclerotic vascular disease, but little is known about the role of specific LPA4<br />
receptors in atherosclerosis. We recently demonstrated upregulation of LPA4 in SMC undergoing a<br />
switch to assume a foam-cell like phenotype. The present work therefore aims to elucidate an<br />
association between LPA4 and experimental atherosclerosis in mice.<br />
Methods and Results: We generated mouse globally lacking LPA4 (LPA4-/-) on C57/BL background.<br />
Hyperlipidemia was elicited in the mice by PCSK9 virus injection at 5 weeks of age and then<br />
Western Diet feeding for 20 weeks. Although plasma cholesterol levels were similar, en face<br />
analysis of aortic atherosclerosis indicated a significant reduction in plaque area in LPA4-/-<br />
compared to LPA4-WT mice. Oil Red-O staining of aortic roots confirmed reduced neutral fat<br />
accumulation in the aortic roots of LPA4-/-mice. In sections taken at the level of the aortic root,<br />
CD68 and α-SMA immunoreactivity was lower in the absence of LPA4. SMCs isolated from mouse<br />
thoracic aorta were treated with ox-LDL to promote a foam cell phenotype, characterized by<br />
upregulation of CD68 expression. SMC isolated from LPA4-/- mice had lower CD68 mRNA after ox-<br />
LDL exposure than LPA4-WT cells.<br />
Conclusion: These findings revealed a role for LPA4 in vascular inflammation and the development<br />
of atherosclerotic vascular disease, potentially through effects of SMC phenotype and function.<br />
58
43<br />
Intracoronary Versus Intravenous Adenosine-Induced Maximal Hyperemia for Fractional<br />
Flow Reserve Measurement: A Systematic Review and Meta-Analysis<br />
Mohamed El-Helw, MD 1 • Mohamed Abo-Aly, MD 1 • Georges Lolay, MD 1 • Christopher Adams, MD 1 •<br />
Ahmed El-Sharaawy, MD 1 • Ahmed Abdel-Latif, MD, PhD 1 • Khaled Ziada, MD 1<br />
1Division of <strong>Cardiovascular</strong> Medicine, Gill Heart Institute, University of Kentucky<br />
Postdoc<br />
Background: Measurement of fractional flow reserve (FFR) is considered the gold standard<br />
technique for the invasive hemodynamic assessment of borderline coronary artery stenosis.<br />
Currently, intravenous (IV) adenosine is the recommended approach; however intracoronary (IC)<br />
administration is widely used due to its convenience and lower cost. The correlation between IV<br />
and IC administration to assess coronary blood flow is not well studied.<br />
Objective: This systematic review and meta-analysis is conducted to review the available literature<br />
that compared FFR measurements using bolus IC vs. standard continuous IV of adenosine infusion<br />
for detection of significant coronary artery stenosis.<br />
Methods: We systematically searched MEDLINE, EMBASE, Google scholar and the Cochrane Central<br />
Register of Controlled Trials databases. We reviewed data pertaining to the used adenosine doses,<br />
side effects of each method of administration and FFR values. We performed statistical analyses<br />
examining the sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and odds<br />
ratio in studies comparing bolus IC adenosine and continues IV adenosine infusion using random<br />
effects modelling.<br />
Results: We identified 8 studies addressing the primary review question. Compared to standard IV<br />
adenosine infusion, the overall sensitivity of IC adenosine is 0.843 (95% C.I. 0.701-0.925, P
44<br />
Recapitulating In Vivo Fibroblast Differentiation Using a Hydrogel Cell Culture System<br />
Demetria Fischesser 1 • Onur Kanisicak, PhD 1 • Mario Perera 2 • Neil Ayres, PhD 2 • Jeff<br />
D Molkentin, PhD 1<br />
1Molecular <strong>Cardiovascular</strong> Biology, Cincinnati Children's Hospital • 2 Chemistry, University of<br />
Cincinnati<br />
Graduate Student<br />
Cardiac fibroblasts are resident interstitial cells known to maintain the extracellular matrix (ECM)<br />
under normal physiological conditions through the secretion of collagens and other adherent<br />
proteins. During injury, these fibroblasts take on an activated, contractile state and deposit<br />
additional ECM proteins to contribute to scar formation. While this response is initially beneficial to<br />
prevent rupture and further injury, continuous scar development leads to fibrosis, pathological<br />
ventricular remodeling and dysfunction, and eventual organ failure. Though much has already been<br />
discovered about the importance of fibroblasts during development and injury response, there are<br />
still many aspects of their functions that remain unclear. In order to decipher these fibroblast<br />
functions, many in vitro systems have been used to easily manipulate fibroblasts while attempting<br />
to mimic in vivo conditions. However, most of these systems have used plastic or gelatin-coated<br />
plastic as the surface medium. These have proven to be much stiffer than physiological tissue and<br />
provide a static system inhospitable to the dynamic changes of the activating fibroblast. In order to<br />
mimic an adaptable in vivo environment for fibroblast culture, we have developed a new hydrogel<br />
technology to understand fibroblast behavior and differentiation. This technology utilizes gelatin<br />
norbornene and a thiol conjugated to a Reversible Addition-Fragmentation Chain Transfer (RAFT)<br />
polymer which can dynamically mimic the stiffness of both healthy and fibrotic tissue, thus allowing<br />
for seamless modeling of injury and disease in various tissues. Through morphology and<br />
differentiation studies, we have found that there is a significant difference between fibroblasts<br />
plated on plastic versus softer hydrogels which mimic in vivo conditions. Fibroblasts plated on<br />
plastic and stiffer surfaces become very large and flat, and express markers of differentiation, such<br />
as alpha-Smooth Muscle Actin (αSMA), within days of plating. On softer hydrogels, however, the<br />
fibroblasts remain small and eventually develop long stellate appendages. They produce αSMA<br />
much more slowly and at a much less robust level than on stiffer surfaces. The morphology and<br />
differentiation pattern seen on softer hydrogels is highly comparable to fibroblasts observed in<br />
vivo. We will use these initial findings to better understand the activation and differentiation of<br />
fibroblasts and how, in injury, they first inhibit tissue damage and later enhance it. In the future, we<br />
plan to manipulate this hydrogel system to intentionally change the stiffness of the surface on<br />
which fibroblasts have already been plated to determine if we can mimic disease states as well as<br />
reverse the fibroblast activation phenotype seen previously.<br />
60
45<br />
Determination of The Effects Of High Fat Feeding On Autotaxin Activity<br />
Esias Bedingar 1 • Frederick Onono, PhD 2<br />
1University of Kentucky • 2 Internal Medicine , College of Medicine<br />
Undergraduate<br />
Obesity is a major public health issue in the United States and is risk factor for several diseases such<br />
as coronary artery disease and certain types of cancers. Although the association between obesity<br />
and cardiovascular diseases is well known, the underlying molecular mechanisms are still unclear.<br />
One of the hypotheses proposed to explain the link between obesity and disease risk is that<br />
consumption of high fat diets or increased synthesis and storage of fats is associated with the<br />
production of bioactive lipids or lipid-derived molecules that promote disease. Lysophosphatidic<br />
acid (LPA) is a family of bioactive lysophospholipids well-recognized as an important signaling<br />
molecule acting primarily through actions at cell-surface G-protein coupled receptors. There are<br />
two major ways of LPA production. However, the enzyme responsible for making majority of the<br />
circulating LPA is autotaxin (ATX), a secreted phospholipase D. Autotaxin is strongly expressed in<br />
adipose tissue, thus synthesis of circulating LPA from dietary lipids could be further enhanced in<br />
obese individuals.<br />
In this study, we investigated the effects of fasting and high-fat feeding on the activity levels of ATX.<br />
Healthy volunteer subjects (n=11) were asked to fast overnight. In the morning, their blood was<br />
collected at t=0 for plasma preparation. Then, they were given a drink composed of boost protein<br />
shake and triglycerides and blood drawn at hourly intervals for up to 8 hours. Autotaxin activity<br />
was determined in the plasma using synthetic substrates. Preliminary results indicated that ATX<br />
activity is acutely sensitive to fasting and feeding, with the lowest activity observed following<br />
fasting and increasing with feeding of a high fat diet. To confirm the specificity of the ATX activity a<br />
novel inhibitor was used to inhibit ATX activity. Our findings indicate that ATX is dependent on the<br />
feeding status and is increased following feeding with a high fat diet. This study suggests the<br />
efficacy of ATX pharmacological inhibition could be enhanced if timed with the onset of feeding.<br />
61
46<br />
Sex-Specific Differences in Cardiac Fibrosis<br />
Gregory Milburn 1 • Autumn Conger 1 • Cheavar Blair, PhD 1 • Maya Guglin, MD 2 • Gretchen Wells, MD,<br />
PhD 2 • Rebekah Waikel, PhD 3 • Kenneth Campbell, PhD 1<br />
1Physiology, University of Kentucky • 2 <strong>Cardiovascular</strong> Medicine, University of Kentucky •<br />
3Biological Sciences, Eastern Kentucky University<br />
Undergraduate<br />
<strong>Cardiovascular</strong> disease is the leading causes of death in America, which prompted a concerted effort<br />
to better understand the causes and to develop innovative therapies. The etiology of heart disease<br />
is complex and depends on several factors such as age, race, and sex, but previous research<br />
conducted contains gaps, as it often did not look for difference between these groups. This study<br />
aims to fill one aspect of this research gap by examining sex-specific differences in cardiac fibrosis<br />
in failing and non-failing human hearts. Cardiac fibrosis is a result of cardiac remodeling and causes<br />
decreased heart function post cardiac damage. A previous study, conducted in our lab, used<br />
Nanostring analysis to examine sex-specific differences in the expression of genes related to<br />
fibrosis. The results of this study showed certain gene expressions were specific to sex or had an<br />
interaction between the sex and heart failure status. Several of these genes were regulators of<br />
collagen, a common type of cardiac fibrosis. We continued this study by quantifying collagen in<br />
human heart samples, collected from heart transplants and non-viable organ donors. These<br />
samples were stained using an established picrosirius red staining technique, turning the collagen<br />
tissue red and normal cardiac tissue yellow. These samples were then quantified using a k-means<br />
cluster, via a program written in lab, to eliminate any bias in determining red and yellow tissue. The<br />
results are pending as there are still samples left to be completed. We hope to continue pursuing<br />
this line of inquiry into sex-specific differences in heart disease by examining passive stiffness, of<br />
which collagen is a major component.<br />
62
47<br />
Dietary Effects on Lipoprotein-Associated Bioactive Mediators of Atherosclerosis<br />
Cody Sutphin 1 • Maria Kraemer, PhD 2 • Pan Deng, PhD 2 • Courtney Hammill, PhD 2 •<br />
Susan Smyth, MD, PhD 2 • Andrew Morris, PhD 2<br />
1Eastern Kentucky University • 2 <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky<br />
Undergraduate<br />
Elevated circulating levels of atherogenic lipoproteins associate with increased risk of<br />
cardiovascular disease. Multiple lines of evidence implicate bioactive components of lipoprotein<br />
particles (such as oxidized phospholipids) as signaling molecules that provoke responses in blood<br />
and vascular cells that cause the development and progression of atherosclerosis. Studies from<br />
ourselves and others identify a role for the bioactive lipid mediator lysophosphatidic acid (LPA) as<br />
a proinflammatory atherogenic signal in preclinical models and clinical settings. LPA accumulates<br />
in atheromas, and mice lacking certain LPA receptors are protected from experimentally induced<br />
atherosclerosis while mice lacking the LPA degrading enzyme lipid phosphate phosphatase 3<br />
(LPP3) exhibit accelerated atherosclerosis. These findings may explain why coronary artery disease<br />
risk is elevated in individuals with common genetic polymorphisms that decrease LPP3 expression.<br />
We hypothesized that LPA associated with low-density lipoprotein particles is sensitive to diet and<br />
enhanced by genetic hyperlipidemia. Plasma LDL-associated lipid species were analyzed in WT and<br />
LDLr-/- mice that were on either control or western diet. Mouse plasma was fractionated by sizeexclusion<br />
chromatography using a calibrated Superose 6 column to separate lipoprotein species<br />
and then analyzed by targeted and untargeted HPLC coupled mass spectrometry to identify and<br />
quantitate LPA species, as well as other lipid-class species, that were altered in LDL-containing<br />
plasma fractions. We found that circulating levels of LPA are highly sensitive to high fat feeding in<br />
mice and that genetic manipulations that reduce lipoprotein clearance (Ldlr-/-) dramatically<br />
increase circulating levels of LDL-associated LPA while reducing albumin-associated LPA.<br />
Additionally, we identified unexpected increases in a broad range of atherogenic lipoprotein<br />
particle- associated lipid species when hyperlipidemic mice are put onto a western diet. These<br />
studies provide new insights into how diet could influence coronary artery disease risk by<br />
promoting increase in atherogenic lipoprotein associated LPA.<br />
63
48<br />
Ticagrelor Reduces Inflammation and Mortality in a Murine Model of Sepsis and Reduces<br />
Platelet-Leukocyte Aggregates and Inflammation In Pneumonia<br />
Travis Sexton, PhD 1 • Guoying Zhang, MD 1 • Tracy Macaulay, PharmD 2 • Leigh Ann Callahan, MD 3 •<br />
Richard Charnigo, PhD 4 • Olga Vsevolozhskaya, PhD 4 • Zhenyu Li, PhD 1 • Susan Smyth, MD, PhD 2<br />
1<strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky • 2 Gill Heart and Vascular Institute,<br />
University of Kentucky • 3 Pulmonary, Critical Care & Sleep Medicine, University of Kentucky •<br />
4Statistics, University of Kentucky<br />
Staff<br />
Background: Sepsis is a life-threatening and dysregulated response to infection that leads to<br />
numerous complications and carries substantial risk of mortality. Pneumonia is one of the most<br />
common precipitators of sepsis. Despite advances in treatment for sepsis and pneumonia,<br />
significant improvements have not been realized and high rates of cardiovascular events remain an<br />
issue. Retrospective analysis of clinical studies suggest anti-platelet therapy may improve outcomes<br />
in patients with pneumonia and sepsis.<br />
Methods: We conducted a human study with pneumonia patients (XANTHIPPE) and a murine study<br />
using a sepsis model to determine the effect of ticagrelor on inflammation, thrombosis, and lung<br />
function.<br />
Results: Among subjects with pneumonia not taking a P2Y12 antagonist at baseline, ticagrelor<br />
lowered the percent of leukocytes with attached platelets 11.75% at 24 hours compared to a<br />
10.90% increase in placebo patients. Furthermore, ticagrelor lowered plasma IL-6 levels 83% at 24<br />
hours compared to minimal change with placebo. Ticagrelor had a transient effect on markers for<br />
NETosis showing a significant 60% spike in MPO-NE complexes at 24 hours followed by a return<br />
toward baseline at 48 hours while placebo had no significant effect. Lung function tests also<br />
numerically improved with ticagrelor, although statistical significance was not achieved. In the<br />
murine sepsis model, disruption of the P2Y12 receptor protected against inflammatory response,<br />
lung permeability, and mortality.<br />
Conclusions: Our findings indicate a mechanistic link between platelets, leukocytes, and lung injury<br />
in settings of pneumonia and sepsis and suggest possible therapeutic approaches to reduce<br />
complications of pneumonia.<br />
64
49<br />
Endocytosis Mediates Platelets' Responses to Viruses In The Vasculature<br />
Meenakshi Banerjee, PhD 1 • Sidney Whiteheart, PhD 1<br />
1Molecular and Cellular Biochemistry, University of Kentucky<br />
Postdoc<br />
Platelet endocytosis is essential for Fibrinogen (Fg) uptake, receptor trafficking of integrins<br />
(αIIbβ3, αvβ3) and other surface receptors. However, the mechanistic underpinnings of<br />
endocytosis, its importance in platelets, and the molecular machinery required and possible<br />
trafficking routes are understudied, in part due to a lack of viable experimental tools. Previously we<br />
showed the importance of ADP-ribosylation factor 6 (Arf6), which regulates αIIbβ3-mediated Fg<br />
uptake/storage and affects acute platelet functions e.g., clot retraction and spreading. To further<br />
identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing<br />
Soluble N-ethylmaleimide Factor Attachment Protein Receptor (v-SNARE) called<br />
Cellubrevin/Vesicle Associated Membrane Protein-3 (VAMP-3) in platelet function. VAMP-3 KO<br />
platelets have defective uptake and accumulation of Fg, which led to enhanced platelet spreading<br />
on Fg and faster clot retraction. Using both Arf6 KO and VAMP-3 KO mutants as tools to probe the<br />
importance of endocytosis in platelets, we posited that platelet endocytosis could potentially be<br />
critical for actively sensing pathogenic damage in the vascular microenvironments and allowing<br />
platelets to act as immune cells. Previous reports show that platelets do endocytose viruses such as<br />
HIV-1 but the molecular machinery is ill-defined. In nucleated cells, responses to HIV-1 are<br />
mediated by virus phagocytosis/endocytosis, degradation to release Toll-like Receptor ligands, and<br />
subsequent receptor activation. Is this process recapitulated in platelets? Here we show that<br />
platelets indeed use VAMP-3 and Arf6-dependent pathways to endocytose HIV-1 virions, degrade<br />
retroviral particles to release TLR ligands, which initiate platelet activation and secretion.<br />
Consequently, HIV-1 uptake and subsequent activation is abolished in VAMP-3 and Arf6 KO mice.<br />
Collectively, our studies shed light on how platelets act at the early stage of pathogen recognition<br />
and are able to process them to initiate an immune response.<br />
65
50<br />
Association between Plasma Cholesterol Levels and the Development of Xanthomas, Malaise,<br />
and Inappetance in Cynomlgus Monkeys<br />
Morgan Kelly 1 • Courtney Burkett 1 • Sierra Paxton 1 • Lei Cai 1 • Ryan Temel, PhD 1<br />
1<strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky<br />
Undergraduate<br />
<strong>Cardiovascular</strong> disease is the leading cause of death in the United States. There is a positive<br />
correlation between coronary heart disease (CHD) and LDL cholesterol (LDL-C) levels. Statins are<br />
used to lower LDL-C and CHD risk but do not completely eliminate CHD events caused by<br />
atherosclerotic lesion rupture. Therefore, finding a therapy capable of regressing or stabilizing<br />
atherosclerotic lesions is a priority for academic and pharmaceutical researchers. By stimulating<br />
macrophage cholesterol efflux and promoting anti-inflammatory macrophage polarization,<br />
antagonism of microRNA-33a (miR-33a) in mice causes aortic atherosclerotic lesions to acquire a<br />
more stable composition. However, the preclinical mouse studies have limited translational value<br />
since mice do not develop coronary artery atherosclerosis and express only one of the two miR-33<br />
family members found in humans. In contrast, nonhuman primates (NHPs) have miR-33a and miR-<br />
33b and develop coronary artery atherosclerosis and thus are a good model to determine the<br />
therapeutic potential of miR-33 antagonism in humans. In order to determine whether miR-33a/b<br />
antagonism can regress or stabilize atherosclerotic lesions, 36 male cynomolgus monkeys were fed<br />
for 20 months a diet high in fat and cholesterol, which increased LDL-C and drove atherosclerosis<br />
formation. During this period, the animals developed xanthomas and callosities due to cholesterol<br />
deposition in the skin. In addition, some of the monkeys went through prolonged periods of malaise<br />
and inappetance. The objectives of this project were to review the veterinary records and<br />
determine 1) the number of days on atherogenic diet needed to first observe xanthomas and<br />
callosities and 2) the number of incidences of malaise and prolonged inappetance per monkey. This<br />
data was then correlated to the average total plasma cholesterol (TPC) concentration during the 20<br />
months on atherogenic diet. A statistically significant, negative correlation was observed between<br />
the number of days to initial callosity formation and TPC (p= 0.049 and = 0.19). There were no<br />
statistically significant correlations between TPC and the other clinical observations. We are in the<br />
process of data collection but hypothesize that coronary artery atherosclerotic lesion size will be<br />
greater in animals that went through periods of malaise and inappetance. In addition, we<br />
hypothesize that the days for xanthoma/callosity formation will be inversely associated with<br />
plaque size. This information may allow our lab to predict atherosclerosis severity in monkeys<br />
based upon clinical observations.<br />
66
51<br />
Circadian disruption and atherosclerosis in ApolipoproteinE-deficient mice<br />
Eric McGann 1 • Jeffery Chalfant, MS 1 • Deborah Howatt 2 • Julie Pendergast, PhD 1<br />
1Biology, University of Kentucky • 2 Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky<br />
Staff<br />
The circadian system is a network of molecular clocks located throughout the body. These clocks<br />
coordinate daily rhythms of behavior and physiology with environmental cycles. Shift work, which<br />
chronically disrupts circadian rhythms, increases the risk of developing cardiovascular diseases.<br />
The mechanisms by which this happens, however, are largely unknown. Our long-term goal is to<br />
determine how disruption of circadian rhythms causes cardiovascular disease. Atherosclerosis is<br />
the build-up of plaques in arteries and can lead to myocardial infarction and stroke. In this study,<br />
we sought to investigate whether circadian disruption accelerates atherosclerosis in mice. Wildtype<br />
mice do not develop atherosclerotic lesions. Therefore, we studied C57BL/6J ApolipoproteinEdeficient<br />
(ApoE-/-) mice because they spontaneously develop atherosclerotic lesions. We first<br />
characterized circadian rhythms in ApoE-/- mice. We found that circadian behavioral rhythms,<br />
including free-running periods of activity in constant darkness and constant light, phase angles of<br />
entrainment, and phase shifts to light pulses, were similar between wild-type and ApoE-/- mice.<br />
These data show that ApoE-/- mice had no deficits in their circadian behavior rhythms or light<br />
responsiveness. Next, we determined if molecular circadian rhythms in tissues were altered in<br />
ApoE-/- mice. We analyzed the expression of the circadian timekeeping protein, PERIOD2, in<br />
central and peripheral tissues using a luciferase reporter. We found that PERIOD2::LUCIFERASE<br />
rhythms in tissues were similar in wild-type and ApoE-/- mice. We next determined the effect of<br />
constant light on the development of atherosclerosis in ApoE-/- mice. After 3 months in constant<br />
light, locomotor activity was arrhythmic or the rhythm was severely disrupted. In addition,<br />
atherosclerotic lesion area was increased in ApoE-/- mice in constant light compared to those in<br />
control 12h light-12h dark condition. Together, these data demonstrate that ApoE-/- mice have<br />
normal circadian rhythms and chronic circadian disruption accelerates atherosclerosis in ApoE-/-<br />
mice.<br />
This study was funded by National Institutes of Health grant P20GM103527, the Gertude F. Ribble<br />
Trust, and the University of Kentucky.<br />
67
52<br />
Does Light Pollution Affect the Development of Atherosclerosis?<br />
Robert Wendroth 1 • Eric McGann 1 • Deborah Howatt 2 • Julie Pendergast, PhD 1<br />
1Department of Biology, University of Kentucky • 2 Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University<br />
of Kentucky<br />
Undergraduate<br />
Exposure to light at night has been steadily increasing since industrialization. Light at night comes<br />
from light bulbs, light pollution produced by cities, and more recently from the widespread use of<br />
personal electronics (smart phones, tablets, laptops) at night. Previous studies in outbred mice<br />
have shown that dim light at night (DLAN) disrupts circadian rhythms and increases body weight,<br />
but it is unknown whether DLAN affects the development of cardiovascular disease. In this project,<br />
we are testing the hypothesis that DLAN accelerates the development of atherosclerotic lesions in<br />
C57BL/6J Apolipoprotein E-deficient (ApoE-/-) mice. We single-housed male ApoE-/- mice in lighttight<br />
boxes at 7 weeks of age for 1 week in 12h light:12h dark (12L:12D) and then in DLAN (12h<br />
light: 12h dim 5 lux light) for 12 weeks. Control mice were maintained in 12L:12D for 13 weeks.<br />
Body weights did not differ between ApoE-/- mice in 12L:12D and DLAN. In 12L:12D, ApoE-/- mice<br />
had a robust rhythm of eating behavior that peaked during the night. However, after a few days in<br />
DLAN, ApoE-/- mice had a low-amplitude eating behavior rhythm. Preliminary data also suggest<br />
that ApoE-/- mice in DLAN have increased atherosclerotic lesion area compared to ApoE-/- mice in<br />
12L:12D. These data suggest that DLAN disrupts eating behavior rhythms and may also increase<br />
atherosclerosis.<br />
68
53<br />
TRAF3 negatively regulates platelet activation and thrombosis<br />
Rui Zhang, PhD 1 • Guoying Zhang, MD 1 • Binggang Xiang, MD, PhD 1 • Xiaofeng Cheng, MD, PhD 2 •<br />
Lijang Tang, MD 3 • Shaojun Shi, MD 4 • Yani Liu, MD 4 • Xun Ai, PhD 5 • Ping Xie, PhD 6 •<br />
Zhenyu Li, MD, PhD 1<br />
1Division of <strong>Cardiovascular</strong> Medicine, University of Kentucky • 2 Taizhou Hospital, Wenzhou Medical<br />
University • 3 Department of Cardiology, Zhejiang Hospital • 4 Department of Pharmacy, Huazhong<br />
University of Science and Technology • 5 Department of Physiology and Biophysics, Rush University<br />
• 6 6Department of Cell Biology and Neuroscience, Rutgers University<br />
Faculty<br />
CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily, binds to CD40,<br />
leading to many effects depending on target cell type. Platelets express CD40L and are a major<br />
source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus<br />
formation, involving both CD40-dependent and -independent mechanisms. A family of proteins<br />
called TNF receptor associated factors (TRAFs) plays key roles in mediating CD40L-CD40 signaling.<br />
Platelets express several TRAFs. It has been shown that TRAF2 plays a role in CD40L-mediated<br />
platelet activation. Here we show that platelets also express TRAF3, which plays a negative role in<br />
regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are<br />
increased in TRAF3 knockout mice. The expression levels of collagen receptor GPVI and integrin<br />
αIIbβ3 in platelets were not affected by deletion of TRAF3, suggesting that increased platelet<br />
activation in the TRAF3 knockout mice was not due to increased expression platelet receptors.<br />
Time to formation of thrombi in a FeCl3-induced thrombosis model was significantly shortened in<br />
the TRAF3 knockout mice. However, mouse tail-bleeding times were not affected by deletion of<br />
TRAF3. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.<br />
69
54<br />
Impact of miR-33 antagonism on metabolic parameters in nonhuman primates<br />
Tara E. Keenan 1 • Tong Li, MD 1 • Lei Cai, PhD 1 • Sierra M. Paxton 1 • Courtney R. Burkett 1 • Peter<br />
I. Hecker 1 • Ryan E. Temel, PhD 1<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center and Department of Pharmacology and Nutritional Sciences,<br />
University of Kentucky<br />
Undergraduate<br />
Introduction: MicroRNA-33a (miR-33a) and miR-33b regulate lipid homeostasis by inducing<br />
degradation or blocking translation of mRNAs encoding proteins that control cholesterol efflux and<br />
fatty acid oxidation. By stimulating macrophage cholesterol efflux and promoting antiinflammatory<br />
macrophage polarization, antagonism of miR-33a in mice reduces atherosclerotic.<br />
However, some mouse studies have shown that disruption of miR-33 function has negative<br />
metabolic effects such as increased plasma and hepatic triglycerides. Mouse studies have limited<br />
translational value since mice express only one of the two miR-33 family members found in<br />
humans. Since nonhuman primates (NHPs) have miR-33a and miR-33b, NHPs are the best<br />
preclinical model for determining the therapeutic potential of miR-33 antagonism. The objective of<br />
this project was to determine whether miR-33 antagonism has adverse metabolic effects on NHPs.<br />
Methods: Liver was collected via laparotomy from 12 chow fed male cynomolgus monkeys. Male<br />
cynomolgus monkeys (n=36) were fed for 20 months a high fat/high cholesterol diet, which caused<br />
hypercholesterolemia and atherosclerosis development. At the end of the 20-month progression<br />
phase, a subset of monkeys (n=12) were euthanized to collect tissues. The remaining monkeys were<br />
switched to a cholesterol-lowering “chow” diet and treated with either vehicle (n=12) or anti-miR-<br />
33 (n=12) for 6 months. Blood was collected at 20 months of progression and 2, 4, and 6 months of<br />
regression and the serum was sent to ANTECH Diagnostics for chemical analysis. Liver lipid content<br />
was biochemically determined.<br />
Results: Feeding the monkeys a chow diet for 6 months caused serum triglycerides (TG) to<br />
significantly increase and total cholesterol (TC) to significantly decrease. However, serum TG and<br />
TC were similar for the vehicle and anti-miR-33 treated monkeys. The progression group had<br />
significantly greater hepatic TC and TG compared to the regression groups. Hepatic TC and TG<br />
levels of the vehicle and anti-miR-33 groups were similar and had returned to the baseline levels<br />
observed in monkeys fed only chow. In line with the liver lipids changes, serum ALT and AST levels<br />
were reduced to a similar extent in the regression NHPs. In addition, body weights and serum<br />
glucose concentrations were similar for the vehicle and anti-miR-33 treated NHPs during the 6-<br />
month regression phase.<br />
Conclusion: Metabolic parameters are normal in chow-fed NHPs treated with miR-33 antagonist.<br />
70
55<br />
ATF6 proteins regulate cardiac hypertrophy during pressure overload in the mouse heart<br />
Robert Correll, PhD 1 • Jeffrey Lynch, PhD 1 • Michelle Sargent 1 • Allen York 1 • Jeffery Molkentin, PhD 1<br />
1Molecular <strong>Cardiovascular</strong> Biology, Cincinnati Children's Hospital<br />
Postdoc<br />
Objective: The activating transcription factor 6 (ATF6) branch of the endoplasmic reticulum (ER)<br />
unfolded protein response (UPR) plays a critical protective role in the heart’s response to acute<br />
injury or long-term hemodynamic stress that results in cardiac hypertrophy. Here we seek to<br />
elucidate the molecular mechanisms by which engagement of ATF6 proteins results in protective<br />
signaling in the context of pressure overload in the mouse heart.<br />
Methods and Results: Overexpression of thrombospondin-4 (Thbs4) results in a protective ER<br />
stress response and mice gene-deleted for Thbs4 demonstrated compromised ER stress signaling,<br />
inhibition of ATF6α processing and nuclear localization, and decreased survival after transverse<br />
aortic constriction (TAC) or myocardial infarction (MI) surgery. Using gene-deleted mice lacking<br />
ATF6α we demonstrate that Thbs4-mediated upregulation of ER protein chaperone expression and<br />
expansion of the ER compartment requires ATF6α expression. Furthermore, mice lacking ATF6α or<br />
the related protein ATF6β show defects in the hypertrophic response after pressure overload,<br />
leading to accelerated decompensation and failure after long-term TAC surgery, accompanied by<br />
reduced expression of ER protein chaperones.<br />
Conclusion: We find that both ATF6α and ATF6β proteins are required for compensatory<br />
hypertrophy and ER chaperone mobilization following pressure overload in the mouse heart. We<br />
hypothesize that the inability to upregulate chaperone expression during pressure overload results<br />
in diminished ER protein folding capacity that impairs the hypertrophic response. These results<br />
position ATF6 proteins as essential regulators of compensatory cardiac hypertrophy following<br />
chronic hemodynamic stress.<br />
71
56<br />
Isolation and characterization of primary bone marrow mesenchymal stem cells.<br />
Eman El-sawalhy, MD 1 • Lakshman Chelvarajan, PhD 2 • Ahmed Abdel-Latif, MD, PhD 1<br />
1Division of <strong>Cardiovascular</strong> Medicine, Gill Heart Institute, University of Kentucky • 2 Saha<br />
cardiovascular research center, University of Kentucky<br />
Postdoc<br />
•Introduction: Myocardial infarction remains a major clinical problem and the leading cause of<br />
mortality in the world. Bone marrow derived stem cells have the capacity to participate in cardiac<br />
repair and regeneration of compromised heart muscle. The aim of this study is to generate highly<br />
enriched mesenchymal stem cells to inject 3x106 into the hearts of mice recovering from a<br />
myocardial infarction.<br />
•Methods: We isolated bone marrow (BM) from crushed long bones and hip bones of 6-8 weeks old<br />
GFP positive mice (C57BL/6). The stem cells were cultured in MesenCult media supplemented with<br />
MesenPure. Cells were cultured under either normoxic or hypoxic conditions (4% O2) for about<br />
two weeks. The cultures were then passaged when they reached 80% confluence. The cells were<br />
analyzed by flow cytometry using mesenchymal stem cell (CD90.2 and SCA-1) and hematopoietic<br />
(CD45) markers.<br />
•Results: As expected, the baseline bone marrow cells were negative for both stem cell markers,<br />
CD90 and Sca-1, while expressing the hematopoietic marker, CD45 (80%). During early expansion<br />
all the CD45– cells were Sca1+CD90–. Following subsequent passaging of the enriched cells, they<br />
began to express CD90. By the end of the second passage, the proportion of Sca1+CD90+<br />
mesenchymal stem cells was 65%, while cells expressing Sca-1 alone was down to 32%. Most of the<br />
live healthy cells remained GFP+ throughout the expansion and passaging.<br />
Conclusion: Mesenchymal stem cells can be isolated and enriched from murine bone marrow in<br />
sufficient amounts to be used in our in vivo study to enhance regeneration of cardiomyocytes and<br />
restore cardiac function after myocardial infarction.<br />
72
57<br />
Fine Tuning Platelet Secretion to Modulate Hemostasis<br />
Smita Joshi, MS 1 • Irina Pokrovskaya, MS 2 • Brian Storrie, PhD 2 • Sidney W. Whiteheart, PhD 1<br />
1Molecular and Cellular Biochemistry, University of Kentucky • 2 Department of Physiology and<br />
Biophysics, University of Arkansas for Medical Sciences<br />
Graduate Student<br />
Globally, occlusive thrombotic events: e.g., heart attacks and cerebral strokes, cause > 50% of total<br />
deaths attributed to the noninfectious disease. However, aggressive attempts to limit thrombosis<br />
cause bleeding, which can be equally catastrophic. What is needed is a strategy to limit clot<br />
formation, but not prevent it. Platelets play a critical role in controlling bleeding by sensing vascular<br />
damage and releasing a host of components to seal breaches. This secretion process is mediated by<br />
Soluble N-ethylmaleimide Sensitive Factor Attachment Protein Receptors (SNAREs) and their<br />
regulators. To drive secretion, vesicle (v)-SNARE on granules and target (t)-SNARE on the plasma<br />
membrane (PM) form a trans-bilayer complex that mediates membrane fusion. Syntaxin 11 and<br />
SNAP-23 form the functionally relevant t-SNARE heterodimer. For v-SNAREs, platelets contain<br />
Vesicle-Associated Membrane Protein (VAMP)-2, -3, -4, -5, -7, and -8. We focused on how the<br />
platelet VAMPs influence secretion and whether modulating secretion can modulate clot formation.<br />
To address this goal, we genetically titrated the different VAMPs to define their roles in exocytosis<br />
and hemostasis.<br />
We gathered global VAMP-3-/- and VAMP-8-/- animals. To overcome embryonic lethality of global<br />
VAMP-2 deletion, we generated platelet-specific VAMP-2/3-/- mice by using a tissue-specific<br />
promoter that facilitates expression of tetanus toxin that cleaves VAMP-2 and 3. We crossed these<br />
with VAMP-8-/- mice to create platelet-specific VAMP-2/3/8-/- mice. Structural analysis of wildtype<br />
and VAMP-deficient platelets showed that the α granule cargo solubilization/decondensation<br />
follows granule fusion. To define the structure of secretion, activation intermediates were fixed at<br />
various time points, post stimulation, and electron microscopy was performed. The data indicate<br />
that granule decondensation is time- and agonist concentration-dependent. Moreover,<br />
decondensation of granule cargo was VAMP dependent. Three dimensional EM analyses indicate<br />
that VAMP-8 plays a major role in compound, intra-granule fusion and also contributes to single,<br />
granule-PM fusion. Our structural data elucidate how platelet secretion occurs at the cellular level<br />
and explains the complex secretion kinetics previously reported in activated platelets.<br />
To further measure the functional importance of the VAMPs, ex vivo secretion assays were used to<br />
monitor the kinetics and the extent of release from all three platelet granules (dense, α, and<br />
lysosomes). Only VAMP-2/3/8-/- platelets showed a robust defect in secretion (~70% decrease),<br />
more than observed for VAMP-8-/- platelets (~50%). When we studied the effects of secretion on<br />
hemostasis, only VAMP-2/3/8-/- mice showed significantly increased tail-bleeding times and<br />
delayed arterial thrombosis. VAMP-8-/- animals did show a delay in thrombus formation but no<br />
overt bleeding diathesis. Our data show that small differences in secretion kinetics alter hemostasis,<br />
thus by modulating platelet secretion, we can control thrombus formation without inducing<br />
pathological bleeding. These data identify the secretory machinery as a viable target to control<br />
occlusive cardiovascular diseases.<br />
Our work is the first comprehensive study showing how by targeting secretion we can achieve the<br />
long-sought balance between occlusive thrombosis and spurious hemostasis. Additionally, by<br />
titrating amounts and types of VAMPs in platelets we have created a valuable set of animals to<br />
precisely analyze the role of platelet secretion in other vascular processes.<br />
73
58<br />
The XY sex chromosome complement augments Ang-II induced aortic arch aneurysms in<br />
female LDLr-/- mice<br />
Cassandra Woolley 1 • Yasir Al-Siraj 1 • Sean Thatcher, PhD 1 • Lisa Cassis, PhD 1<br />
1Department of Pharmacology and Nutritional Sciences, University of Kentucky<br />
Undergraduate<br />
Objective: Sex difference in cardiovascular disease has been a subject of research efforts aiming to<br />
improve the efficacy and approach of therapeutics available. In humans, abdominal aortic<br />
aneurysms (AAAs) display sexual dimorphism, with higher risk for development in males but with<br />
females exhibiting more rapid AAA growth rates and AAA ruptures at smaller aneurysm sizes.<br />
Previous studies performed in our laboratory have demonstrated that testosterone increases<br />
incidence and rupture rate of angiotensin II (AngII)-induced AAA in hyperlipidemic mice. After<br />
establishing this relationship, the four-core mouse model was used to analyze the role of sex<br />
chromosomes separate from sex hormones in aneurysm pathology. Female XY LDLr-/- mice are<br />
more susceptible to AngII-induced AAA than female XX LDLr-/- mice. The XY chromosome<br />
complement demonstrated more severe pathology and higher rupture rate. However, studies<br />
examining the role of sex chromosomes in the aortic arch between XX and XY female mice have not<br />
been performed. The purpose of this preliminary study was to observe whether differences were<br />
present between female XX and XY mice in formation of the aortic arch aneurysm.<br />
Methods and Results: Female XX and XY LDLr-/- mice were placed on a Western diet (TD.88137)<br />
one week prior to implantation of a 28-day AngII osmotic pump. The pumps administered AngII<br />
(1,000 ng/kg/min) for 28 days to induce aneurysm formation. The body weight of the mice was<br />
recorded weekly. Baseline measurements of the aortic arch diameter were made at D0 using<br />
ultrasound and found to be significantly different between XX and XY females (XX, 1.4 ± 0.03; XY,<br />
1.5 ± 0.05 mm; p
59<br />
Multilevel Mechanical Testing of Cardiac Valves<br />
Arielle Waller 1 • Daniel Perry 1 • Luke Knudson 1 • RS Baker 2 • David Morales, MD 3 •<br />
Farhan Zafar, MD 2 • Daria Narmoneva, PhD 1<br />
1Biomedical Engineering, University of Cincinnati • 2 Division of Pediatric Cardiothoracic Surgery,<br />
The Heart Institute, Cincinnati Children's Hospital • 3 Division of Pediatric Cardiothoracic Surgery,<br />
The Heart Institute, Cincinnati Children's Hospital<br />
Graduate Student<br />
The lack of adequate heart valve replacement options represents a major problem in pediatric care,<br />
where multiple operations are required to accommodate patient growth. Bioengineered valves may<br />
provide a viable alternative to existing strategies. Knowledge of the relationship between macroand<br />
micro-biomechanical properties and functional behavior of native or engineered valve tissue is<br />
critical for success of bioengineered valves, yet it is still poorly understood. Here, a tubular<br />
tricuspid valve bioprosthesis made of small intestinal submucosa-derived extracellular matrix (SIS-<br />
ECM) was used as a valve replacement in the in vivo sheep model. Current study developed distinct<br />
multilevel testing approaches to determine valve tissue biomechanical properties and quantify the<br />
structure-function and behaviors. The following analyses were performed: (i) tissue/organ level<br />
uniaxial testing of valve samples; (ii) microanalyses (cellular/subcellular level) of adjacent fullthickness<br />
valve sections using atomic force microscopy (AFM) to determine valve mechanical<br />
properties. The AFM approach was used in combination with MATLAB-based quantitative 2D<br />
histological mapping of valve matrix content (collagen & proteoglycans), for functional analyses of<br />
biomechanical behavior. Results demonstrated that valve tensile elastic modulus was higher for the<br />
valve annulus vs. the cusp, consistent with proteoglycan rich regions on the distal end of the cusp,<br />
and a collagen rich region on the proximal end of the cusp and fibrosa. Compression stiffness from<br />
AFM testing demonstrated an increased Young’s modulus for the fibrosa vs. spongiosa, with<br />
intermediate values for atrialis (mitral and tricuspid) and ventricularis (aortic and pulmonary),<br />
consistent with a collagen-rich fibrosa, proteoglycan-rich spongiosa, and elastin-rich<br />
atrialis/ventricularis. In summary, this demonstrates feasibility of this multilevel testing approach<br />
in functional assessment of bioengineered valve and its ability to provide necessary mechanical<br />
function immediately upon implantation, and to attain structural and biomechanical properties of<br />
the native tissue during growth and remodeling.<br />
75
60<br />
CHROME: a Long Non-coding RNA that Regulates Cholesterol Homeostasis<br />
Kathryn Moore, PhD 1<br />
1Medicine, New York University<br />
Faculty<br />
Thousands of long non-coding RNAs (lncRNAs) have been identified in the human genome, many of<br />
which are not conserved in lower mammals. The majority of these lncRNAs remain functionally<br />
uncharacterized and may have important implications in human physiology and disease. We<br />
identified a primate-specific lncRNA, CHROME, which is increased in the plasma and atherosclerotic<br />
plaques of individuals with coronary artery disease compared to healthy controls. Using gain- and<br />
loss-of-function approaches, we show that CHROME functions as a competing endogenous RNA of<br />
microRNAs (miRNAs) that repress cellular cholesterol efflux and plasma high density lipoproteins<br />
(HDL) levels, and regulates the concentration and biological functions of these miRNAs. CHROME<br />
knockdown in primary hepatocytes increases levels of its miRNA binding partners, thereby<br />
reducing expression of their target gene networks, hepatic cholesterol and phospholipid efflux, and<br />
the formation of nascent HDL particles. Consistent with these findings, hepatic levels of CHROME<br />
are positively correlated with plasma levels of HDL cholesterol in healthy individuals. Collectively,<br />
our findings identify CHROME as a central component of the non-coding RNA circuitry controlling<br />
cholesterol homeostasis in humans.<br />
76
61<br />
Secretory phospholipase A2 group IIA (PLA2G2A) enhances metabolism and insulin<br />
sensitivity<br />
Michael Kuefner 1 • Kevin Pham 1 • JeAnna Redd 2 • Erin Stephenson, PhD 3 • Innocence Harvey 2 •<br />
Xiong Deng, PhD 1 • Dave Bridges, PhD 2 • Eric Boilard, PhD 4 • Marshal Elam, MD, PhD 1 •<br />
Edwards Park, PhD 1<br />
1Pharmacology, University of Tennessee Health Science Center • 2 Nutritional Sciences, University of<br />
Michigan School of Public Health • 3 Physiology, University of Tennessee Health Science Center •<br />
4Infectious Diseases and Immunity, CHUQ <strong>Research</strong> Center and Division of Rheumatology<br />
Graduate Student<br />
Secretory phospholipase A2 group IIA (PLA2G2A) is a member of a family of secretory<br />
phospholipases that have been implicated in inflammation, atherogenesis, and antibacterial actions.<br />
Here, we evaluated the role of PLA2G2A in the metabolic response to a high fat diet. C57BL/6<br />
(BL/6) mice do not express PLA2g2a due to a frameshift mutation. We fed BL/6 mice expressing<br />
the human PLA2G2A gene (IIA+ mice) a fat diet and assessed the physiologic response. After 10<br />
weeks on the high fat diet, the BL/6 mice were obese, but the IIA+ mice did not gain weight or<br />
accumulate lipid. The lean mass in chow- and high fat-fed IIA+ mice was constant and similar to the<br />
BL/6 mice on a chow diet. Surprisingly, the IIA+ mice had an elevated metabolic rate, which was not<br />
due to differences in physical activity. The IIA+ mice were more insulin sensitive and glucose<br />
tolerant than the BL/6 mice, even when the IIA+ mice were provided the high fat diet. The IIA+<br />
mice had increased expression of uncoupling protein 1 (UCP1), sirtuin 1 (SIRT1), and PPARγ<br />
coactivator 1α (PGC-1α) in brown adipose tissue (BAT), suggesting that PLA2G2A activates<br />
mitochondrial uncoupling in BAT. Our data indicate that PLA2G2A has a previously undiscovered<br />
impact on insulin sensitivity and metabolism.<br />
77
62<br />
Making Good: Secretory Quality Control in Lipoprotein Lipase Trafficking<br />
Benjamin Roberts 1 • Saskia Neher, PhD 1<br />
1Biochemistry and Biophysics, UNC Chapel Hill<br />
Graduate Studnet<br />
Lipoprotein lipase (LPL) is an essential vascular regulator of serum triglycerides. Patients without<br />
LPL activity suffer from hypertriglyceridemia. Chronic elevated blood triglycerides increase the<br />
risks of acute pancreatitis, atherosclerosis, and cardiac disease. There is no cure for LPL deficiency<br />
available in the United States. Of the over 70 clinical LPL clinical mutations, some affect intracellular<br />
LPL trafficking. It is clear that misfolded LPL is subject to post-Endoplasmic Reticulum (ER) quality<br />
control, which results in its degradation. The regulation of this process and the LPL partners that<br />
promote LPL degradation are not well understood. To study atypical LPL trafficking we<br />
fluorescently tagged two clinically-identified LPL variants to follow intracellular LPL transport. We<br />
also characterized the trafficking of WT LPL produced in cells lacking its folding chaperone Lipase<br />
Maturation Factor 1. Finally, we used mass spectrometry to search for intracellular binding<br />
partners unique to misfolded LPL. Using these techniques, we quantified post-ER quality control of<br />
folded and misfolded LPL. These studies enhance our understanding of the regulation in LPL<br />
trafficking and help to explain the consequences of some LPL mutations resulting in LPL deficiency.<br />
78
63<br />
Gut Microbial Trimethylamine (TMA) Lyase Activity Coordinates Circadian Rhythms in Host<br />
Hepatic Lipid and Bile Acid Metabolism<br />
Christy Gliniak, PhD 1 • Rebecca Schugar, PhD 1 • Robert Helsley, PhD 1 • Anthony Gromovsky 1 •<br />
Chase Neumann 1 • Zeneng Wang, PhD 1 • Stanley Hazen, MD, PhD 2 • J. Mark Brown, PhD 2<br />
1Department of Cellular and Molecular Medicine, Cleveland Clinic • 2 Department of Cellular and<br />
Molecular Medicine, Center for Microbiome & Human Health, Cleveland Clinic<br />
Faculty<br />
From cyanobacteria to humans, circadian rhythms evolved to allow an organism to adapt and<br />
anticipate environmental cues, particularly events that regulate energy metabolism. Misalignment<br />
of circadian rhythms are associated with increased incidence of obesity, diabetes, cardiovascular<br />
disease, cancer, and other inflammatory disorders. Intestinal microbial composition and structure<br />
displays circadian rhythmicity, and the gut microbiome itself can regulate host endogenous<br />
circadian rhythms. However, it is not well understood how gut microbes regulate host metabolism<br />
and circadian rhythms. Gut microbes contribute to the production of the circulating metabolite<br />
trimethlyamine-N-oxide (TMAO), which is associated with cardiovascular disease in humans, and<br />
has been shown to enhance atherosclerosis and thrombosis potential in mice. Microbes produce<br />
trimethlyamine (TMA) from dietary choline or carnitine, which is later converted by the host to<br />
TMAO, primarily by host liver flavin monooxygenase 3 (FMO3). Previous studies have shown that<br />
pharmacologic inhibition of microbial TMA lyase activity or inhibition of FMO3 results in decreased<br />
atherosclerosis in mice, and the expression of the TMAO producing enzyme FMO3 exhibits<br />
circadian rhythmic patterns in the liver. Here we hypothesized that the co-metabolites TMA and<br />
TMAO may serve as gut microbe-derived signals that entrain host metabolic circadian rhythms,<br />
thereby impacting cardiometabolic disease. To test this we treated C57BL/6 mice with a highly<br />
potent and selective second-generation TMA lyase inhibitor CC08, and examined effects on<br />
circadian rhythms in host metabolism. As expected, mice treated with CC08 displayed reduced<br />
plasma TMA/TMAO levels across an entire 24-hour period. Unexpectedly, TMA lyase inhibition<br />
resulted in increased expression of the key nuclear receptors that regulate the circadian clock<br />
including Bmal1 (Arntl1) and Rev-erbα (Nr1d1) during the light cycle in the liver. The well known<br />
circadian cycling of transcription factors orchestrating hepatic fatty acid metabolism such as<br />
Srebp1c and Pparα were markedly altered in CC08-treated mice. TMA lyase inhibition resulted in<br />
elevated hepatic expression of Srebp1c during the light cycle, yet suppressed hepatic Pparα<br />
expression during the dark cycle. Furthermore, the expression of the bile acid-sensing nuclear<br />
receptor Fxr was lower in TMA lyase-inhibited animals during the dark cycle, which is associated<br />
with alterations in hepatic Fxr target gene expression and circulating bile acid levels. Collectively,<br />
these data suggest that pharmacologic inhibition of gut microbial TMA lyase activity can alter host<br />
transcriptional programs that dictate hepatic circadian rhythms in lipid and bile acid metabolism.<br />
These data provide the first clues into mechanisms by which TMA lyase inhibitors may protect mice<br />
against cardiometabolic disease.<br />
79
64<br />
Thrombospondin 1 (TSP1) Deficiency Protects Against Diet-Induced Fatty Liver Disease<br />
Courtney Turpin, MS 1 • Heather Norman-Burgdolf, PhD 2 • Ling Yao, MS 1 • Yanzhang Li, PhD 3 •<br />
Shuxia Wang, MD, PhD 1<br />
1Department of Pharmacology and Nutritional Sciences, University of Kentucky • 2 Department of<br />
Dietetics and Human Nutrition, University of Kentucky • 3 Department of Internal Medicine,<br />
University of Kentucky<br />
Graduate Student<br />
Background: Non-alcoholic fatty liver disease (NAFLD) is a widespread disease, affecting around<br />
20-30% of American adults with prevalence on the rise in many age groups. Obesity is thought to<br />
play a contributing role in the occurrence of this disease. NAFLD progression includes an initial<br />
state lipid accumulation, which is worsened by additional stresses. Thrombospondin 1 (TSP1) is a<br />
secreted matricellular protein that has been implicated in playing a role in causing obesityassociated<br />
inflammation, lipid accumulation, and fibrosis in various tissues, but TSP1 expression in<br />
the liver and its possible role in pathogenesis are not known.<br />
Methods: Eight-week-old control and TSP1 deficient mice were fed at either a low-fat (10% kcal<br />
from fat) or high-fat (60%kcal from fat) for 16 weeks. Liver histology, gene expression, and<br />
immunohistochemistry were performed to observe the effect of TSP1 deficiency on the liver<br />
physiology.<br />
Results: TSP1 expression was found to be significantly increased in control high-fat diet fed mice<br />
liver tissue. Liver histology showed less lipid accumulation in the TSP1 deficient mice fed a high-fat<br />
diet, which was further confirmed by reduced triglyceride levels. Although there was no significant<br />
change fatty acid uptake in the whole liver, there was a significant decrease in CD36 expression in<br />
the hepatocytes of the deficient mice, which also had reduced fatty acid uptake. There was also a<br />
significant decrease in de novo lipogenesis genes PPARγ and SREBP1c, but with normal feeding,<br />
there was no significant change in triglyceride production or secretion. In addition to the changes in<br />
lipid content, there was a reduction in inflammatory markers and the fibrosis marker Collagen1α1,<br />
but there were no observed changes in fibrosis histology staining.<br />
Discussion and implications: The findings of this study suggest that TSP1 expression could play a<br />
role in the development of the hallmark signs of NAFLD. Further studies using a NAFLD animal<br />
model and cell culture could help elucidate the mechanism of its involvement. In summary, the data<br />
suggests that TSP1 has involvement in lipid accumulation and the resulting inflammation and<br />
fibrosis that are seen in progressed disease states of NAFLD.<br />
80
65<br />
An obesity-generating diet drives the cancer stem cell phenotype and glioblastoma<br />
progression.<br />
Daniel J Silver, PhD 1 • Gustavo A Roversi 1 • Anthony Gromovsky 1 • J Mark Brown, PhD 1 • Justin<br />
D Lathia, PhD 1<br />
1Cellular and Molecular Medicine, Cleveland Clinic<br />
Staff<br />
Glioblastoma (GBM) is the most prevalent and lethal brain cancer. The disease occurs in two to<br />
three per 100,000 adults annually and accounts for approximately half of all brain cancers. While<br />
there are no known causes for GBM, obesity is an established risk factor for cancer in general.<br />
Recent work confirmed that overweight and obese women are at greater risk of developing glioma<br />
compared to women of healthy body weight. This finding represents a major change in the<br />
consideration of body mass for brain cancer patients, a factor that has been largely ignored in the<br />
clinic. Furthermore, it is well-established that consumption of a Western-pattern diet leads to<br />
increased rates of obesity. For these reasons, we hypothesized that obesity accelerates GBM<br />
progression by driving tumor cell proliferation and/or cancer stem cell enrichment. We have tested<br />
this hypothesis in vivo using three syngeneic glioma models transplanted into the brains of<br />
immune-competent C57BL/6 mice. Mice were maintained on either an obesity-generating, high-fat<br />
diet (HFD) or a standard rodent chow diet. Tumor-bearing mice fed the HFD succumbed to disease<br />
nearly two-fold faster than those fed the chow diet. For example, mice transplanted with the GL261<br />
syngeneic glioma model and maintained on HFD were culled, on average, 16 days earlier than those<br />
maintained on chow. Furthermore, we noted a nearly three-fold increase in the frequency of tumor<br />
generation in obese animals compared to lean, suggesting that systemic obesity enriches for a<br />
tumor-initiating cancer stem cell population in the brain. Tumor cells treated directly with oleic<br />
acid or linoleic acid in vitro demonstrated increased viability and enhanced sphere formation<br />
compared to vehicle controls. These data indicated that certain lipid components of the obesitygenerating<br />
diet were sufficient to drive tumor cell proliferation and the emergence of the cancer<br />
stem cell state. In order to identify the set of lipid species contributing to accelerated disease<br />
progression, we profiled the major lipids present in tumors of obese mice compared to tumors of<br />
lean mice using untargeted lipidomics. This analysis revealed four putative oncogenic lipids. These<br />
species were robustly expressed in tumors resected from obese mice compared to tumors resected<br />
from lean mice. We have additionally identified two potentially tumor-suppressive lipids that were<br />
expressed strongly in the contralateral hemisphere of lean mice compared to tumors resected from<br />
either lean or obese mice. This work confirms a shift in the lipid profile of tumors developing in the<br />
brains of the obese mice and suggests that select lipid species may directly drive the increased<br />
tumor cell proliferation and enhancement in the cancer stem cell compartment that we observe in<br />
vivo.<br />
81
66<br />
Serum Amyloid A3 is a High Density Lipoprotein-associated Acute Phase Protein<br />
Maria De Beer, PhD 1 • Ailing Ji, PhD 2 • Victoria Noffsinger 2 • Preetha Shridas, PhD 2 • Frederick De<br />
Beer, MD 2 • Lisa Tannock, MD 2 • Nancy Webb, PhD 2<br />
1Physiology, University of Kentucky • 2 Internal Medicine, University of Kentucky<br />
Faculty<br />
Acute phase serum amyloid (SAA) is a family of evolutionarily conserved, secreted proteins that<br />
exerts innate functions relevant to obesity and diabetes. In humans, two SAA isoforms (SAA1 and<br />
SAA2) are highly induced in the liver and extrahepatic tissues under the regulation of inflammatory<br />
cytokines. During severe inflammation, SAA1/2 levels can increase >1000-fold in plasma, where it<br />
is found associated with HDL. Mice produce an additional acute phase SAA, SAA3, which is thought<br />
to be produced mainly by adipocytes and macrophages and has not previously been found<br />
circulating on HDL.<br />
Objectives: The goal of this study was to investigate whether SAA3 serves as a third liver-derived,<br />
HDL-associated acute phase SAA in mice.<br />
Methods: Using isoform-specific oligonucleotide primers for qRT-PCR, we determined that SAA3 is<br />
transcriptionally induced to a similar extent (~2500-fold) compared to SAA1.1/2.1 (~6000-fold) in<br />
livers of C57BL/6 mice 19 hr after lipopolysaccharide (LPS) injection (100 µg/mouse). SAAs were<br />
also robustly induced in fat tissue (SAA1/2~100-fold; SAA3~400-fold). The analysis of primary<br />
mouse hepatocytes and in situ hybridization of mouse liver sections indicated that liver-derived<br />
SAAs are produced by hepatocytes and not other stromal cells, including Kupffer cells. All 3 SAA<br />
isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ~20% of<br />
SAA1/2. After separation by FPLC, virtually all of plasma SAA1/2 eluted with the HDL fraction,<br />
whereas ~15% of plasma SAA3 appeared to be lipid poor/free. HDL isolated from acute phase<br />
mouse plasma by density gradient ultracentrifugation was subjected to isoelectric focusing to<br />
determine the relative recovery of the various SAA isoforms. Whereas the bulk of plasma SAA1.1<br />
was found in the d=1.063-1.21 fraction, only ~50% of SAA2.1 and ~10% of SAA3 was recovered<br />
after ultracentrifugation. These findings suggest that SAA3 may be more loosely associated with<br />
HDL compared to SAA1.1/2.1, which may give rise to lipid poor/free SAA3 that is susceptible to<br />
more rapid clearance in vivo.<br />
Conclusions: We conclude that SAA3 is a major hepatic acute phase SAA in mice that may produce<br />
systemic effects during inflammation. Future studies investigating SAA pathobiology in mice must<br />
take into account the previously under-studied SAA3.<br />
82
67<br />
An XX Sex Chromosome Complement Promotes Hypercholesterolemia and Atherosclerosis<br />
in Ldlr-/- Mice Fed Western Diet<br />
Yasir Alsiraj, MS 1 • Sean Thatcher, PhD 1 • Lei Cai, PhD 2 • Ryan Temel, PhD 2 • Patrick Tso, PhD 3 •<br />
Lisa Cassis, PhD 1<br />
1Pharmacology and Nutritional Sciences, University of Kentucky • 2 Saha <strong>Cardiovascular</strong> <strong>Research</strong><br />
Center, University of Kentucky • 3 Department of Pathology and Laboratory Medicine, University of<br />
Cincinnati<br />
Graduate Student<br />
Objectives: Sex hormones are primary contributors to sexual dimorphism of cardiovascular<br />
diseases, but little is known about the influence of genes on sex chromosomes as mediators of<br />
cardiovascular sex differences. In this study, we hypothesized that genes on sex chromosomes<br />
influence the development of hypercholesterolemia and atherosclerosis.<br />
Methods and Results: Transgenic male mice with deletion of Sry from the Y-chromosome<br />
expressing Sry on autosomes (8-12 weeks of age) were bred to female Ldlr-/- mice to generate<br />
female and male mice with an XX or an XY sex chromosome complement. Male (M) and female (F)<br />
mice were fed a Western diet (Teklad TD88137) for 3 months. XX mice exhibited increased body<br />
weights compared to XY mice, regardless of gonadal sex (FXX, 38 ± 1.8; FXY, 30.6 ± 1.3 g; P
68<br />
Adipocyte deficiency of ACE2 increases systolic blood pressures of obese female C57BL/6<br />
mice<br />
Robin Shoemaker, PhD 1 • Wen Su, MD 2 • Ming Gong, PhD 2 • Lisa Cassis, PhD 1<br />
1Pharmacology and Nutritional Sciences, University of Kentucky • 2 Physiology, University of<br />
Kentucky<br />
Postdoc<br />
Background: We demonstrated that sexual dimorphism of obesity-hypertension was associated<br />
with differential activity of adipocyte angiotensin-converting enzyme 2 (ACE2) in male versus (vs)<br />
female mice. These data suggest that adipocyte ACE2 regulates blood pressure by influencing the<br />
balance of angiotensin II (AngII, a substrate of ACE2) vs angiotensin-(1-7) (Ang-(1-7)), which<br />
differs in male and female mice. We hypothesized that deficiency of ACE2 in adipocytes increases<br />
blood pressure of HF-fed female mice.<br />
Methods/Results: Mice with adipocyte ACE2 deficiency were developed from breeding Ace2fl/fl<br />
mice to transgenic C57BL/6 mice with heterozygous transgenic expression of Cre recombinase<br />
driven by the adiponectin promoter (Ace2Adipo). Female or male Ace2fl/fl and Ace2Adipo mice (8<br />
weeks old) were fed a HF (60% kcal as fat) or low fat (LF; 10% kcal from fat) diet for 16 weeks,<br />
after which blood pressure was quantified by radiotelemetry. Systolic blood pressures (SBP) were<br />
not different in LF-fed female mice of either genotype (24 hr average SBP [mmHg]: Ace2fl./fl:<br />
121+/-1; Ace2Adipo: 121+/-1; p>0.05). However, SBP was significantly increased in HF-fed female<br />
Ace2Adipo vs Ace2fl/fl mice (Ace2fl/fl: 127+/-2; Ace2Adipo: 133+/-3; p
69<br />
Fish oil-derived long-chain monounsaturated fatty acid (LCMUFA) for cardiovascular<br />
diseases : a novel approach for cardioprotection<br />
Zhihong Yang, PhD 1 • Scott Gordon, PhD 1 • Milton Pryor 1 • Shuibang Wang, PhD 2 •<br />
Robert Danner, MD, PhD 2 • Alan Remaley, MD, PhD 1<br />
1Lipoprotein Metabolism Section, CPB/NHLBI, National Institutes of Health • 2 Critical Care<br />
Medicine Department, Clinical Center, National Institutes of Health<br />
Postdoc<br />
<strong>Cardiovascular</strong> disease (CVD) remains the leading cause of death, disability, and healthcare expense<br />
in the United States and is also a major healthcare problem worldwide. Numerous studies have<br />
shown cardiovascular benefits of fish oil, and most of these favorable effects have been attributed<br />
to omega-3 fatty acids. Fish oils, however, also contain varying amounts of other unusual types of<br />
fatty acids not commonly found in other food sources. For example, oils derived from saury, pollock<br />
and herring are all enriched in long-chain monounsaturated fatty acids (LCMUFA) with aliphatic<br />
tails longer than 18 C atoms (i.e., C20:1 and C22:1 isomers combined). Compared with well-studied<br />
omega-3, limited information is available on the role of LCMUFA in cardiovascular health.<br />
In the current study, we first examined the effect of saury fish oil-derived LCMUFA concentrate on<br />
the pathogenesis of atherosclerosis in atherosclerosis animal model. In LDLR-deficient female<br />
mice, we observed that 12-week supplementation of 2% LCMUFA on a western diet significantly<br />
decreased atherosclerosis lesion areas and accumulation of macrophages, compared with western<br />
diet (control) or western diet supplemented with 2% olive oil enriched in shorter-chain MUFA oleic<br />
acid (C18:1 n-9), although there were no differences in plasma lipoprotein profiles between the<br />
three groups. LCMUFA diet also decreased plasma inflammatory cytokine levels, and improved<br />
cholesterol efflux capacity to apoB-depleted plasma in BHK cells overexpressing ABCA1. RNA<br />
sequencing and subsequent qPCR analyses revealed that LCMUFA upregulated hepatic PPAR<br />
signaling pathway. To clarify the individual effect of LCMUFA isomers, we further produced<br />
concentrated C20:1 and C22:1 fractions, and fed LDLR-deficient mice a western diet supplemented<br />
with 5% C20:1, C22:1, or not (control) for 12 weeks. In good agreement with the first study, both<br />
LCMUFA isomers showed beneficial effects on atherosclerosis development, systematic<br />
inflammation, and cholesterol efflux capacity, without lowering plasma lipids. It is suggested that<br />
the atheroprotective effect of LCMUFA is likely due to both isomer fractions. Alterations in<br />
lipoprotein proteome revealed by LC-MS/MS proteomic analysis were favorably correlated with<br />
reduction in atherosclerotic plaque areas. For mechanistic study, we hypothesized that some of the<br />
beneficial effect of LCMUFA were derived from their metabolite, and we synthesized LCMUFAderived<br />
ethanolamides to estimate their effect on PPAR activation. In vitro PPAR transactivation<br />
assay revealed a beneficial role of LCMUFA-derived ethanolamides in PPAR transcriptional activity.<br />
Based on these positive findings, we are now conducting a double-blind crossover clinical trial of<br />
LCMUFA-rich saury oil to estimate the effect of LCMUFA-rich diet on lipoprotein metabolism in<br />
adults (ClinicalTrials.gov Identifier: NCT03043365).<br />
In conclusion, our research showed for the first time that fish oil-derived LCMUFA-rich diet<br />
attenuates atherosclerosis, possibly by regulating PPAR signaling pathway and modulating<br />
lipoprotein proteome, other than lipid-lowering effects. Although omega-3 fatty acids are generally<br />
considered the major active components in fish lipids, our findings provide novel insights into<br />
potential cardioprotective effect of LCMUFA-rich fish oil, and build on past efforts to understand the<br />
impacts of MUFA on health outcomes.<br />
85
70<br />
Hypercholesterolemia-induced endothelial dysfunction is rescued by overexpression of<br />
endothelial Kir2.1 in resistance arteries<br />
Ibra Fancher, PhD 1 • Sang Joon Ahn, PhD 2 • Crystal Adamos 3 • Catherine Osborn 3 •<br />
Catherine Reardon, PhD 4 • Godfrey Getz, MD, PhD 4 • Shane Phillips, PhD 3 • Irena Levitan, PhD 3<br />
1Medicine, University of Illinois at Chicago • 2 Yale University • 3 University of Illinois at Chicago •<br />
4University of Chicago<br />
Postdoc<br />
Rationale: Hypercholesterolemia induces endothelial dysfunction, a key stage in the development of<br />
cardiovascular disease. Impairment of nitric oxide (NO) dependent vasomotor function is<br />
established as an important factor. We recently identified endothelial Kir2.1 as upstream mediators<br />
of flow-induced NO production. Our earlier studies established that endothelial Kir is suppressed by<br />
cholesterol.<br />
Objective: We tested the hypothesis that hypercholesterolemia induces microvascular endothelial<br />
dysfunction through suppression of Kir2.1, which we propose to be responsible for the inhibition of<br />
flow-induced NO production and the loss of NO-dependent component of flow-induced vasodilation<br />
(FIV).<br />
Methods and Results: Kir2.1 currents and their sensitivity to flow are significantly suppressed in<br />
microvascular endothelial cells exposed to acetylated-LDL without any change in Kir2.1 expression.<br />
Genetic deficiency of Kir2.1 on the background of hypercholesterolemic Apoe-/- mice, Kir2.1+/-<br />
/Apoe-/- exhibit the same blunted FIV and flow-induced NO response as Apoe-/- and Kir2.1+/-<br />
alone, as we showed earlier Kir2.1+/- arteries show significant impairment of (FIV) and NO<br />
production. Endothelial-specific overexpression of Kir2.1 via adenoviral transduction of arteries<br />
from Apoe-/- and Kir2.1+/-/Apoe-/- mice results in full rescue of FIV and NO production in both<br />
hypercholesterolemic models. Conversely, endothelial-specific expression of dominant-negative<br />
Kir2.1 results in impairment of FIV in WT arteries but has no further effect on the blunted FIV in<br />
Apoe-/- arteries. No differences were detected in Kir2.1 or eNOS expression between WT and Apoe-<br />
/- mice. Notably, a full rescue of the FIV response by endothelial-specific Kir2.1 was observed in<br />
Apoe-/- mice with and without the addition of high fat diet.<br />
Conclusions: We conclude that hypercholesterolemia-induced reduction in FIV is largely<br />
attributable to cholesterol suppression of Kir2.1 function via the loss of flow-induced NO<br />
production. The stages downstream of flow-induced Kir2.1 activation appear to be intact because<br />
overexpression of endothelial Kir2.1 rescues FIV and flow-induced NO production in arteries from<br />
hypercholesterolemic mice.<br />
86
71<br />
Fibrinogen Depletion Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm<br />
Hannah Russell 1 • Keith Saum 1 • Alexandra Sundermann 2 • Shannon Jones 1 •<br />
Anders Wanhainen, MD, PhD 3 • Todd Edwards, PhD 2 • Lori Holle 4 • Alisa Wolberg, PhD 4 • A.<br />
Phillip Owens III, PhD 1<br />
1Internal Medicine, University of Cincinnati • 2 Medicine and Public Health, Vanderbilt University<br />
Medical Center • 3 Surgery, Uppsala University • 4 Pathology, UNC Chapel Hill<br />
Graduate Student<br />
Background: Fibrinogen and fibrin [collectively fibrin(ogen)] provides physical and biochemical<br />
support to a developing clot and is one of the most crucial independent risk factors for<br />
cardiovascular diseases (CVDs). In addition to clot formation, fibrin(ogen) also promotes wound<br />
healing and powerful inflammatory and immune responses via engagement of leukocytes.<br />
Increased levels of circulating fibrin(ogen) degradation products are correlated with increased<br />
diameter and progression of the inflammatory disease abdominal aortic aneurysm (AAA). However,<br />
a causal link between fibrinogen and AAA has not yet been established. The objective of this study<br />
was to determine the role of fibrinogen depletion in a mouse model of AAA.<br />
Methods and results: We first determine whether AAA resulted in elevated procoagulant activity by<br />
quantifying plasma levels of thrombin anti-thrombin (TAT) and thrombin generation via calibrated<br />
automated thrombography (CAT). Compared to controls, both mice and humans with AAA had<br />
significantly elevated TAT as well as CAT parameters velocity index, peak height, and endogenous<br />
thrombin potential. To identify a role of fibrinogen in AAA, low density lipoprotein receptor<br />
deficient (Ldlr-/-) mice were injected intraperitoneally with scrambled anti-sense oligonucleotide<br />
(ASO) or β-fibrinogen ASO (30 mg/kg) 3 weeks prior to experimentation and throughout the study.<br />
Administration of fibrinogen ASO successfully achieved > 90% depletion of fibrinogen. After 3<br />
weeks of ASO, mice were fed a high fat/cholesterol ‘Western’ diet for 1 week prior to and<br />
throughout infusion with angiotensin II (AngII; 1,000 ng/kg/day) for 28 days. Compared to<br />
controls, fibrinogen depletion decreased abdominal diameter (33% decrease; P = 0.001),<br />
atherosclerotic lesion area (~63% decrease; P = 0.001), and inflammatory cytokines (>75%<br />
decreased IL-1 and IL-6; P = 0.001). Fibrinogen depletion also decreased aneurysm incidence (P <<br />
0.05) and rupture-induced death (P = 0.078).<br />
Conclusions: Our results show that AAAs are associated with elevated TAT and thrombin<br />
generation in both patients and mice, and that fibrinogen depletion attenuates AAA incidence,<br />
diameter, rupture-induced death, atherosclerosis, and inflammation. We propose that increased<br />
procoagulant activity accelerates the rate of fibrinogen to fibrin conversion, which promotes<br />
inflammation and increases cardiovascular disease. Further studies will define whether reducing<br />
plasma fibrinogen may be a novel treatment strategy.<br />
87
72<br />
Increased Circulating Trimethylamine N-oxide (TMAO) Augments the Incidence of<br />
Abdominal Aortic Aneurysm in Low Penetrant C57BL/6J Mice<br />
Kelsey Conrad, MS 1 • Shannon Jones 1 • Robert Helsley, PhD 2 • Rebecca Schugar, PhD 2 •<br />
Zeneng Wang, PhD 2 • Stanley Hazen, MD, PhD 2 • J. Mark Brown, PhD 2 • A. Phillip Owens III, PhD 1<br />
1Internal Medicine, University of Cincinnati • 2 Cellular and Molecular Medicine, Cleveland Clinic<br />
Graduate Student<br />
Background: The gut microbiota is a metabolically active endocrine organ critical to the<br />
maintenance of cardiovascular health. Nutrients common in high fat foods (phosphatidylcholine,<br />
choline, L-carnitine) are metabolized by microbial enzymes to form the gut microbial metabolite<br />
trimethylamine (TMA). Metabolism by the host enzyme flavin-containing monooxygenase 3 (FMO3)<br />
converts TMA to the pro-inflammatory molecule trimethylamine N-oxide (TMAO). Human clinical<br />
trials have correlated high levels of circulating TMAO to an increased risk of cardiovascular<br />
diseases. However, this meta-organismal pathway has not been evaluated in the context of<br />
abdominal aortic aneurysm (AAA). The objective of this study was to determine the effects of a high<br />
choline diet on the development of AAA.<br />
Methods: C57BL/6J male (n=20) and female (n=20) mice were fed either a standard chow control<br />
diet (n = 10 each sex) or a choline-rich diet (1%; n = 10 each sex) for 5 weeks. After 1 week of diet,<br />
basal abdominal ultrasounds were performed and angiotensin II (AngII; 1,000 ng/kg/min) was<br />
infused for 28 days via implantation of osmotic minipumps. Termination ultrasounds were<br />
performed on day 27 and mice were sacrificed on day 28. Aortas were harvested for evaluation of<br />
aneurysm progression and plasma was analyzed for the metabolites TMA, TMAO, and choline. To<br />
determine whether TMAO was elevated in human patients with AAA, plasma samples from<br />
participants with fast growing AAAs (n = 85), slow growing AAAs (n = 84), and normal (nonaneurysmal)<br />
aortas (n = 115) were analyzed for plasma TMAO levels via liquid chromatography<br />
tandem mass spectrometry (LC-MS/MS).<br />
Results: Administration of a choline-rich diet augmented the incidence (P < 0.02) and aortic<br />
diameter (P < 0.001) of AAAs in both male and female mice versus placebo-fed mice. Plasma levels<br />
of TMA, TMAO, and choline were significantly elevated in choline-fed mice versus normal chow (P <<br />
0.05). Importantly, circulating levels of plasma TMAO were significantly elevated in a step-wise<br />
fashion with the rate of aneurysm growth versus non-aneurysmal control patients (fast growing ><br />
slow growing > normal patients; P < 0.001).<br />
Conclusions: Our results indicate increases in circulating TMAO augments the growth status of<br />
aneurysms in human patients and the incidence of AAA in a low penetrant C57BL/6J mouse model.<br />
88
73<br />
A specific rotamer of apolipoprotein A-I enables Lecithin-cholesterol acyl transferase<br />
activation by discoidal HDL<br />
Allison L. Cooke 1 • Jamie C. Morris 1 • John T. Melchior, PhD 1 • Rong Huang, PhD 1 • W.<br />
Gray Jerome, PhD 2 • Scott E Street 1 • W. Sean Davidson, PhD 1<br />
1Pathology, University of Cincinnati • 2 Pathology, Microbiology, and Immunology, Vanderbilt<br />
University Medical Center<br />
Graduate Student<br />
Investigating the structure of apolipoprotein (apo)A-I, the primary protein component of high<br />
density lipoprotein (HDL), is important for understanding how HDL interacts with lipid-modifying<br />
proteins to mediate its cardioprotective functions in plasma. HDL lipid discs contain two molecules<br />
of apoA-I arranged in an antiparallel, stacked ring-structure. ApoA-I is composed of 10 tandem,<br />
amphipathic, alpha-helical repeats that encapsulate lipid for plasma transport on the hydrophobic<br />
face of the ring-structure, and allows HDL to interact with modifying proteins on the hydrophilic<br />
face. For example, lecithin-cholesterol acyl transferase (LCAT) is a critical lipoprotein-associated<br />
enzyme that converts cholesterol to cholesterol ester for transport out of peripheral cells. It was<br />
hypothesized that changing the orientation of apoA-I on HDL discs can modulate LCAT reactivity.<br />
To test this, site-directed cysteine-point mutagenesis was used to generate different orientations<br />
(rotamers) of apoA-I on HDL discs of similar size and composition: K133C- helix 5 of one apoA-I<br />
molecule adjacent to helix 5 of its antiparallel partner (5/5 rotamer), K206C- helix 5 adjacent to<br />
helix 2 (5/2 rotamer), and K195C-helix 5 adjacent to helix 1 (5/1 rotamer). Initially, an LCAT<br />
activation assay revealed LCAT had significantly increased reactivity to discs with the 5/5 rotamer<br />
relative to wild-type or the other rotamers (p
74<br />
Circulating Bacterial Small RNA Bound to LDL Induce Inflammatory Activation of<br />
Macrophages<br />
Ryan Allen, PhD 1 • Shilin Zhao, PhD 2 • Marisol Ramirez, MS 1 • Bradley Richmond, MD, PhD 3 •<br />
Timothy Blackwell, MD 3 • Quanhu Sheng, PhD 2 • Kasey Vickers, PhD 1<br />
1<strong>Cardiovascular</strong> Medicine, Vanderbilt University Medical Center • 2 Center for Quantitative Sciences,<br />
Vanderbilt University Medical Center • 3 Allergy and Inflammation, Vanderbilt University Medical<br />
Center<br />
Postdoc<br />
Chronic, sub-acute inflammation is an active component of many human diseases of diverse<br />
etiology, characterized by sustained recruitment and activation of macrophages. Macrophages are<br />
phagocytic, myeloid-derived cells that are resident in nearly every tissue to provide innate defense<br />
against invasive pathogens. Upon injury, macrophages respond to remove damaged cells and cell<br />
debris and facilitate tissue repair. However, in disease, prolonged activation of macrophages<br />
promotes tissue remodeling, often to the detriment of tissue function. This is classically observed in<br />
the development of atherosclerosis, in which monocytes infiltrate the artery wall, differentiate to<br />
macrophages, and phagocytize LDL to become cholesterol-engorged “foam cells”, which rapidly<br />
develop a pro-inflammatory phenotype that contributes to further cardiovascular disease. Our lab<br />
previously reported that high-density lipoproteins (HDL) traffic miRNAs between cells as part of<br />
intercellular gene-regulation networks that we proposed could mediate anti-inflammatory<br />
properties of HDL. Although low-density lipoproteins (LDL) have also been reported to traffic<br />
miRNA, more recent data from small-RNA sequencing (sRNA-SEQ) indicates that LDL-associated<br />
sRNA are primarily exogenous, with most sRNA fragments originating from bacteria. Most<br />
interestingly, taxonomic analysis of bacteria contributing sRNA to the LDL pool revealed a profile<br />
distinct from any characterized profile of the comprehensive human microbiome. Additionally,<br />
antibiotic mediated ablation of the gut microbiome failed to disrupt lipoprotein-sRNA pools of mice,<br />
suggesting that bacterial sRNA fragments are not linked to the gut microbiome. However,<br />
manipulation of housing conditions was capable of inducing stark changes in the landscape of<br />
bacterial sRNA bound to lipoproteins of mice, indicative of a potential role for the environment in<br />
shaping the lipoprotein-sRNA fingerprint. In support of this, we characterized the lipoprotein-sRNA<br />
profiles of wild-type and polymeric IgA receptor deficient (Pigr-/-) mice, a model of compromised<br />
airway mucosal immunity, and found reduced abundance of bacterial sRNA fragments, most<br />
notably of Proteobacteria and Firmicutes. Although many potential functions remain to be explored,<br />
we hypothesized that exogenous sRNA associated with LDL may influence the inflammatory<br />
potential of LDL upon uptake by artery-wall macrophages by activation of endosomal, nucleic acidsensing<br />
toll-like receptors (TLR), which are highly expressed in macrophages and facilitate innate<br />
immunity. To test this hypothesis, we treated primary mouse macrophages and PMA-stimulated<br />
THP1 cells with physiologically relevant concentrations of fresh, native LDL (nLDL) and observed<br />
induction of pro-inflammatory cytokine expression. Strikingly, partial silencing of nucleic acidsensing<br />
TLRs blunted this nLDL induced activation of pro-inflammatory gene expression.<br />
Conversely, treatment of human hepatoma cells, which express low levels of exogenous RNAsensing<br />
TLRs, with nLDL failed to induce pro-inflammatory cytokine expression. Taken together,<br />
our data identify a novel, non-lipid cargo of LDL that is capable of modulating inflammatory gene<br />
expression in macrophages and may contribute to the pathogenesis of atherosclerosis, and many<br />
other metabolic diseases.<br />
90
75<br />
Inducible Depletion of Calpain-2 Attenuates Obesity-accelerated Abdominal Aortic<br />
Aneurysms in mice<br />
Aida Javidan, MS 1 • Weihua Jiang, MS 1 • Jessica Moorleghen, MS 1 •<br />
Venkateswaran Subramanian, PhD 2<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky • 2 Physiology, University of Kentucky<br />
Graduate Student<br />
Background and Objective: Recent clinical studies demonstrated that abdominal adiposity is<br />
associated with increased risk of abdominal aortic aneurysm (AAA) development. Calpains are nonlysosomal<br />
calcium dependent cysteine proteases that are highly expressed in human and<br />
experimental AAAs. Using a pharmacological inhibitor and genetically deficient mice, we identified<br />
that calpain-2 (a major ubiquitous isoform) plays a critical role in Angiotensin II (AngII)-induced<br />
AAA formation in mice. In addition, calpain inhibition strongly suppressed adipose tissue<br />
inflammation in obese mice. The purpose of this study was to determine the functional contribution<br />
of calpain-2 in obesity-accelerated AAA.<br />
Methods and Results: Calpain-2 floxed mice that were hemizygous for β-actin Cre-ERT2 were<br />
produced by breeding male Cre-ERT2 to female calpain-2 floxed mice. At 8 weeks of age, male non-<br />
Cre littermates (Cre-) and Calp-2 x Cre-ERT2 (Cre+) mice were injected with tamoxifen (25 mg/kg,<br />
i.p.) daily for 5 consecutive days. After 2 weeks, Western blot analyses showed a complete depletion<br />
of calpain-2 protein in the aorta and periaortic adipose tissue from Cre+ mice compared to non-Cre<br />
littermates. Mice were fed a high fat diet (60% Kcal) for 20 weeks. After 16 weeks of diet feeding,<br />
mice were infused with AngII (1,000 ng/kg/min) by osmotic minipumps for 4 weeks. Depletion of<br />
calpain-2 had no effect on high fat diet-induced body weight gain, fat mass, glucose and insulin<br />
tolerance. Interestingly, calpain-2 depletion significantly attenuated AngII-induced expansion of exvivo<br />
maximal diameter of abdominal aortas in obese mice (Cre-: 1.4 ± 0.14; Cre+: 0.9 ± 0.04 mm;<br />
P
76<br />
Pancreatic Beta Cell Export of miR-375 to High-Density Lipoproteins is Regulated by Cellular<br />
Ion Fluxes.<br />
Leslie Sedgeman 1 • Carine Beysen, PhD 2 • Marisol Ramirez-Solano 3 • Quanhu Sheng, PhD 4 •<br />
Yan Guo, PhD 4 • Scott Turner, PhD 2 • Kasey Vickers, PhD 3<br />
1Molecular Physiology and Biophysics, Vanderbilt University • 2 Kinemed, Inc. • 3 Division of<br />
<strong>Cardiovascular</strong> Medicine, Department of Medicine, Vanderbilt University Medical Center •<br />
4Department of Cancer Biology, Vanderbilt University Medical Center<br />
Graduate Student<br />
microRNAs (miRNAs) are critical regulators of glucose metabolism and contribute to the<br />
pathogenesis of Type 2 Diabetes (T2D). Recently, we reported that high-density lipoproteins (HDL)<br />
transport and deliver functional miRNAs to recipient cells, including endothelial cells and<br />
hepatocytes. However, the mechanism of export is not understood. Since miR-375 expression in the<br />
islets is 10X greater than in other organs, we tested whether pancreatic beta cells have the ability to<br />
export miR-375 to HDL through in vitro export assays, incubating HDL with INS1 beta cells or<br />
primary human islets. Indeed, we found miR-375 to be readily exported to HDL from INS1 cells and<br />
primary islets in vitro. To determine if cholesterol transporters contribute to HDL-miR-375 export<br />
from beta cells, Abca1, Abcg1 and Scarb1 (SR-BI) were inhibited using siRNAs; however, we found<br />
that knockdown of each of these transporters failed to affect the beta cell’s ability to export miR-<br />
375 to HDL. On the other hand, inhibition of the KATP channel with tolbutamide resulted in the<br />
suppression of HDL-miR-375 export. Similarly, export of miR-375 to HDL was blunted from islets of<br />
two mouse models lacking functional KATP channels (Kir6.2 and SUR1 KO mice). Our work<br />
suggests that miR-375 export to HDL is regulated by cellular dynamics, including ion fluzes in the<br />
beta cell. We are currently investigating the relationship between HDL-miR-375 export, insulin<br />
secretion, and miRNA processing in pancreatic beta cells to further elucidate the mechanism(s)<br />
controlling HDL-miR-375 export. Collectively, results suggest that a large fraction of HDL-miRNAs<br />
originate from pancreatic beta cells and HDL-miRNAs are exported independent of cholesterol<br />
transporters.<br />
92
77<br />
The Role of ApoC-III in the immune system<br />
Alison Kohan, PhD 1 • Cayla Rodia 1<br />
1Nutritional Sciences, University of Connecticut<br />
Faculty<br />
While we know that CD4+CD25+Foxp3+ regulatory T cells (Tregs) are a powerful tool in the<br />
resolution of gut inflammation, and that their secretion of IL-10 is critical to inflammatory bowel<br />
disease (IBD) remission, there is a major gap in identifying mechanisms for increasing Tregs in the<br />
intestine. Recently, we have found that intestinal Tregs and plasma IL-10 are dramatically<br />
increased in mice overexpressing human apoC-III. These intestinal Tregs significantly protect apoC-<br />
III transgenic mice mice from experimental colitis, including weight loss, diarrhea, and TNF-α<br />
secretion. While apoC-III overexpression is protective, loss of apoC-III in apoC-III-/- mice severely<br />
impacts colitis sensitivity; more than 50% of apoC-III-/- mice die in response to dextran sodium<br />
sulfate (DSS)-colitis induction, compared to wild-type controls that all survive. Supporting our<br />
studies in these mouse models are human studies which show that both plasma and ileal apoC-III<br />
levels are reduced in patients during active IBD flare-ups. We have now determined that apoC-III<br />
acts specifically on Tregs and CD103+dendritic cells in the intestine and mesenteric lymph nodes,<br />
and that treatment of Tregs ex vivo with apoC-III-containing lipoproteins inhibits their ability to<br />
take up extracellular fatty acids. This inhibitory role of apoC-III on lipid uptake is well known, but<br />
this is the first time this role has been described in Tregs, thus defining a new and biologically<br />
important function of this apolipoprotein in the immune system. We hypothesize that a critical<br />
function of apoC-III is to regulate lipid uptake and metabolism in intestinal Tregs, which results in<br />
increased tolerogenicity in the gut.<br />
93
78<br />
Understanding inhibition of lipoprotein lipase by angiopoietin-like protein 4<br />
Aspen Gutgsell 1 • Saskia Neher, PhD 1<br />
1Biochemistry and Biophysics, UNC Chapel Hill<br />
Graduate Student<br />
More than three million cases of hypertriglyceridemia are diagnosed every year in the United<br />
States. Hypertriglyceridemia, or elevated levels of plasma triglycerides, is a major risk factor<br />
cardiovascular disease and the leading cause of death worldwide.<br />
Two major sources of plasma triglycerides are dietary fat and stored fat in adipose tissue.<br />
Triglycerides are packaged into lipoprotein particles and reahernmoved from circulation by<br />
lipoprotein lipase (LPL) in the capillaries of muscle, heart, and adipose tissue. In the absence of LPL,<br />
patients have severe hypertriglyceridemia, life-threatening pancreatitis, and fatty skin lesions. A<br />
family of proteins known as angiopoietin-like proteins (ANGPTL3, 4 and 8) temporally regulate LPL<br />
to control the delivery of fatty acids to certain tissues. More specifically, ANGPTL4 inhibits LPL in<br />
adipose tissue during periods of fasting. Recently, genome wide association and exome sequence<br />
studies identified individuals with loss of function mutations in ANGPTL4 as having a unique lipid<br />
profile that significantly decreases their risk for developing cardiovascular disease. Their profile<br />
consists of low plasma triglycerides and high levels of “good” cholesterol, commonly known as HDL.<br />
The direct relationship between LPL activity and plasma triglyceride levels lends LPL as an ideal<br />
target for triglyceride lowering therapeutics. More specifically, preserving LPL activity by blocking<br />
ANGPTL4 inhibition is hypothesized to generate a “cardio protective” lipid profile by lowering<br />
plasma triglycerides and increasing HDL cholesterol in patients with high triglycerides.<br />
However, the interaction between LPL and ANGPTL4 is poorly understood as no structural<br />
information is available. It was originally believed ANGPTL4 irreversibly inhibits LPL by<br />
dissociating it into inactive monomers. However, our group has shown that ANGPTL4<br />
noncompetitively inhibits LPL, while leaving the active dimeric structure intact. Since this<br />
discovery, little information has surfaced regarding exactly how or where ANGPTL4 and LPL<br />
interact. Therefore, our aim is to characterize the ANGPTL4/LPL interaction and generate a<br />
structural model for this complex.<br />
94
79<br />
Serum Levels of Dioxin-Like Pollutants Are Positively Associated With the Cardiometabolic<br />
Disease Risk Biomarker TMAO in Leaner Individuals<br />
Michael Petriello, PhD 1 • Richard Charnigo • Manjula Sunkara • Sony Soman • Marian Pavuk 2 •<br />
Linda Birnbaum 3 • Andrew Morris, PhD 4 • Bernhard Hennig 5<br />
1<strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky • 2 CDC Agency for Toxic Substances and<br />
Disease Registry • 3 NCI at NIEHS • 4 <strong>Cardiovascular</strong> Medicine, University of Kentucky • 5 Superfund<br />
<strong>Research</strong> Center, University of Kentucky<br />
Postdoc<br />
Trimethylamine N-oxide (TMAO) is a diet and gut microbiota-derived metabolite that has been<br />
linked to cardiovascular disease risk in human studies and animal models. TMAO levels show wide<br />
inter and intra individual variability in humans that can likely be accounted for by multiple factors<br />
including diet, the gut microbiota, levels of the TMAO generating liver enzyme Flavin-containing<br />
monooxygenase 3 (FMO3) and kidney function. We recently found that dioxin-like (DL)<br />
environmental pollutants increased FMO3 expression to elevate circulating diet-derived TMAO in<br />
mice, suggesting that exposure to this class of pollutants might also contribute to inter-individual<br />
variability in circulating TMAO levels in humans. To begin to explore this possibility we examined<br />
the relationship between body burden of DL pollutants (reported by serum lipid concentrations)<br />
and serum TMAO levels (n=340) in the Anniston, AL cohort, which was highly exposed to<br />
polychlorinated biphenyls (PCBs). TMAO concentrations in archived serum samples from the<br />
Anniston Community Health Survey (ACHS-II) were measured, and associations of TMAO with 28<br />
indices of pollutant body burden, including total dioxins toxic equivalent (TEQ), were quantified.<br />
Twenty-three (22 after adjustment for multiple comparisons) of the 28 indices were significantly<br />
positively associated with TMAO. Multivariate modeling revealed that total dioxins TEQ was<br />
significantly associated with TMAO among females (except at high BMIs) but not among males. Our<br />
results from this cross-sectional study indicate that exposure to DL pollutants may contribute to<br />
elevated serum TMAO levels. With the observation that dioxins were only associated with TMAO in<br />
leaner individuals, we have begun to mechanistically study the relationship between dioxin<br />
exposure, FMO3/TMAO, and cardiometabolic disease by characterizing a mouse model that<br />
develops atherosclerosis but not adipose tissue expansion. We examined the effects of PCB 126 on<br />
markers of systemic inflammation and atherosclerotic lesion size. Exposed mice exhibited<br />
significantly increased plasma cytokine levels and accelerated atherosclerotic lesion formation.<br />
More work needs to be completed to determine the role of TMAO and FMO3 in these processes<br />
(Supported in part by NIEHS/NIH grant P42ES007380).<br />
95
80<br />
Insulin signaling regulates apolipoprotein (Apo) AI secretion from hepatocytes<br />
Ailing Ji, PhD 1 • Xuebing Wang 1 • Lisa Bennett 2 • David Graf 2 • Gregory Graf, PhD 2 • Deneys van<br />
der Westhuyzen, PhD 1<br />
1<strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky • 2 Pharmaceutical Sciences, University of<br />
Kentucky<br />
Staff<br />
Insulin resistance is associated with increased risk for cholesterol gallstones as well as the<br />
development of diabetic dyslipidemia in which plasma levels of HDL cholesterol are reduced. HDL<br />
is the primary cholesterol carrier in the reverse cholesterol transport (RCT) pathway, the process<br />
by cholesterol is delivered from peripheral organs to the liver for elimination in the bile. Therefore,<br />
we hypothesized that insulin signaling is a regulator of hepatobiliary cholesterol clearance from the<br />
plasma and secretion into bile. To directly test the role of insulin signaling, we utilized mice<br />
harboring insulin receptor flanked by loxP sites (IRfl/fl) in combination with adenoassociated viral<br />
vectors containing no transgene (empty) or Cre recombinase to generate control and liver insulin<br />
receptor knock out mice (LIRKO), respectively. As with previous LIRKO strains (albumin-Cre,<br />
adenoviral-Cre), LIRKO mice showed markedly reduced insulin receptor mRNA and protein in liver,<br />
but not skeletal muscle or adipose tissue as well as impaired glucose tolerance when compared to<br />
controls. LIRKO mice had increased biliary cholesterol secretion as well as increased expression of<br />
the ABCG5 ABCG8 sterol transporter, the primary mediator of biliary cholesterol secretion. Levels<br />
of SR-BI, the primary HDL receptor were unchanged as were rates of HDL clearance from plasma<br />
and selective delivery of HDL cholesterol to the liver. None-the-less, plasma HDL cholesterol and<br />
ApoAI were reduced. Despite these reductions, ApoAI protein, but not mRNA, was increased in the<br />
liver of LIRKO mice. Immunofluorescence microscopy revealed that ApoAI accumulated in<br />
membrane-bound inclusions that stained positively for markers of early, late and recycling<br />
endosomes and of lysosomes. These structures persisted in primary hepatocyte cultures where<br />
rates of ApoAI secretion into the culture medium were reduced. To determine the intracellular<br />
itinerary of ApoAI, we developed a chimera of mCherry fluorescent protein and human ApoAI.<br />
mCherry ApoAI was capable of forming nascent HDL, associated with HDL in plasma, and partially<br />
restored HDL cholesterol in ApoAI-deficient mice. However, this mCherry-ApaAI failed to colocalize<br />
with a probe for acidic compartments in live cells, suggesting that the compartment is<br />
unique or that the accumulation of ApoAI neutralizes the endosomal/lysosomal vesicles in which it<br />
accumulates. Additional studies will be required to investigate the route by which ApoAI arrives<br />
and accumulates in cells with impaired insulin signaling.<br />
96
81<br />
Scavenging reactive aldehydes with 5'-O-pentyl-pyridoxamine (PPM) improves HDL function<br />
and reduces atherosclerosis in Ldlr deficient mice<br />
Jiansheng Huang, PhD 1 • Linda Zhang, PhD 2 • Patricia Yancey, PhD 1 • Huan Tao, PhD 1 • Lei Ding 3 •<br />
YouMin Zhang 1 • John Oates, MD 4 • Venkataraman Amarnath, PhD 5 • Jackson Roberts , PhD 2 •<br />
Sean Davies, PhD 2 • MacRae Linton, MD 4<br />
1Medicine, Vanderbilt University Medical Center • 2 Pharmacology, Vanderbilt University •<br />
3Vanderbilt University Medical Center • 4 Medicine and Pharmacology, Vanderbilt University<br />
Medical Center • 5 Pathobiology, Vanderbilt University Medical Center<br />
Postdoc<br />
Background: Lipid peroxidation products impair the cholesterol efflux capacity of high-density<br />
lipoprotein (HDL) and contribute to the development of atherosclerosis. The effect of inhibition of<br />
HDL dysfunction by scavengers on HDL function and whether scavenging reactive aldehydes with<br />
PPM protects against the development of atherosclerosis was examined.<br />
Methods and Results: HDL of familial hypercholesterolemia (FH) subjects has impaired ability to<br />
promote cholesterol efflux and FH-HDL contains 5-fold more malondialdehyde crosslinks (MDA-<br />
Lys) than control HDL. In vitro studies found that the reactive aldehyde malondialdehyde (MDA)<br />
crosslinks apolipoprotein AI (apoAI) and impairs the ability of HDL to promote cholesterol efflux<br />
from Apoe-/- macrophages in a MDA dose dependent manner. Western blot analysis of apoAI<br />
revealed that the reactive aldehyde scavenger 5'-O-pentyl-pyridoxamine (PPM) abolished MDAmediated<br />
crosslinking of apoA-I in HDL (at a molar ratio of MDA to HDL of 1:5) by 80% (P
82<br />
Protease-activated Receptor 2 Deficiency Attenuates the Formation of Atherosclerosis in<br />
Mice<br />
Shannon Jones 1 • Adrien Mann 1 • Kelsey Conrad, MS 1 • Keith Saum 1 • David Hall, MS 2 •<br />
Lisa McKinney 1 • Nathan Robbins, MS 1 • Joel Thompson, PhD 3 • Abigail Peairs, PhD 2 •<br />
Michael Tranter, PhD 1 • Nigel Mackman, PhD 4 • A. Phillip Owens III, PhD 1<br />
1Internal Medicine, University of Cincinnati • 2 Nutritional Sciences, University of Cincinnati •<br />
3Endocrinology and Molecular Medicine, University of Kentucky • 4 Medicine, UNC Chapel Hill<br />
Staff<br />
Objective – Protease-activated receptor 2 (PAR2)-dependent signaling results in augmented<br />
inflammation and has been implicated in the pathogenesis of several autoimmune conditions. While<br />
PAR2 is present in coronary atherosclerotic lesions, the relevance of this finding has not been<br />
investigated in experimental models. The objective of this study was to determine the effect of<br />
PAR2 deficiency on the development of atherosclerosis.<br />
Approach and Results – PAR2 mRNA and protein expression is increased in human carotid artery<br />
and mouse aortic arch atheroma versus control carotid and aortic arch arteries, respectively. To<br />
determine the effect of PAR2 deficiency on atherosclerosis, male low density lipoprotein receptor<br />
deficient (Ldlr-/-) mice (8-12 weeks old) that were Par2+/+ or Par2-/- were fed a fat and<br />
cholesterol-enriched diet for 12 or 24 weeks. PAR2 deficiency attenuated atherosclerosis in the<br />
aortic sinus and aortic root after 12 and 24 weeks. PAR2 deficiency did not alter total plasma<br />
cholesterol concentrations or lipoprotein distributions. Bone marrow transplantation showed that<br />
PAR2 on non-hematopoietic cells contributed to atherosclerosis. PAR2 deficiency significantly<br />
attenuated levels of the chemokine monocyte chemoattractant protein 1 (MCP-1) in the circulation<br />
and macrophage content in atherosclerotic lesions. Mechanistic studies using ex vivo vascular<br />
smooth muscle cells showed that PAR2 deficiency is associated with reduced production of MCP-1<br />
mRNA and protein release into the supernatant resulting in less monocyte migration and<br />
infiltration.<br />
Conclusions – Our results indicate PAR2 deficiency is associated with attenuation of atherosclerotic<br />
inititation and reduces lesion progression by blunting MCP-1 induced monocyte infiltration.<br />
98
83<br />
Deoxysphingolipids - mediators in taxane-induced peripheral neuropathy<br />
Katrin Anne Becker, PhD 1 • Anne-Kathrin Uerschels, MD 2 • Jacek Bielawski, PhD 3 • Joan Colglazier 4<br />
• Erhard Bieberich, PhD 5 • Erich Gulbins, PhD 1 • Stefka Spassieva, PhD 5<br />
1Molecular Biology , University of Duisburg-Essen • 2 Neurosurgery, University of Duisburg-Essen •<br />
3Biochemistry and Molecular Biology , Medical University of South Carolina • 4 Medicine, Medical<br />
University of South Carolina • 5 Physiology, University of Kentucky<br />
Faculty<br />
Taxanes are chemotherapy drugs used in wide variety of cancers. However, there efficacy can be<br />
hindered by a major dose limiting side effect - peripheral neuropathy. Although, the clinical<br />
symptoms of taxane-induced peripheral neuropathy are well documented, the molecular<br />
mechanism is currently not well understood and there are no treatment options available.<br />
In our previous work, we have shown that in the plasma of breast cancer patients treated with<br />
taxane, the levels of minor class of lipids, the deoxysphingolipids, were associated with incidence<br />
and severity of neuropathy (Kramer et al, FASEB J, 2015). In our current study, we used a mouse<br />
model to test whether taxane treatment results in increased levels of deoxysphingolipids in the<br />
dorsal root ganglia and spinal cord. Mice were three times intraperitoneally injected with taxane.<br />
Lipids were extracted from the isolated ganglia and spinal cord and subjected to quantitative mass<br />
spectrometry analyses. We observed significant elevation of deoxysphingolipid levels only in the<br />
dorsal root ganglia. In addition, we compared in vitro in neuronal cultures, the toxic effect of<br />
deoxysphingosine, a deoxysphingolipid, and sphingosine, a structurally similar sphingolipid.<br />
Importantly, our in vitro data showed that only deoxysphingosine treatment resulted in<br />
morphological changes and toxicity in the neurons. Taken together our data suggest that in the<br />
dorsal root ganglia, the neurotoxic effect of the systemic taxane treatment is likely mediated by<br />
deoxysphingolipids.<br />
99
84<br />
Reduced Low-Density Lipoprotein Receptor-Related Protein-1 in Mature Mice Modestly<br />
Effects Hypercholesterolemia and Atherosclerosis<br />
Shayan Mohammadmoradi, MS 1 • Deborah A. Howatt 1 • Anju Balakrishnan 1 • Jessica<br />
J. Moorleghen 1 • Mark Graham 2 • Adam Mullick 2 • Hong Lu, PhD 1 • Alan Daugherty, PhD, DSc 1<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky • 2 Ionis Pharmaceuticals<br />
Graduate Student<br />
Background and Objectives:<br />
Low-density lipoprotein receptor-related protein-1 (LRP1) has been hypothesized to serve as a<br />
receptor for the removal of selected lipoprotein particles from plasma. However, mice with<br />
somatically engineered mutation of LRP1 have not provided a clear understanding of the specific<br />
lipoprotiens that are regulated by LRP1. Therefore, the aim of this study was to reduce LRP1 using<br />
antisense oligomers in mature mice to determine effects on lipoprotein metabolism and<br />
atherosclerosis.<br />
Methods and Results:<br />
LRP1 antisense oligonucleotide (ASO; 50 mg/kg, injected once per week) or its control ASO was<br />
administered subcutaneously to 8-week old male low-density lipoprotein (LDL) receptor deficient<br />
mice (n=10/each group). All groups were fed a saturated fat-enriched diet for 8 weeks started 1<br />
week after the first injection of ASOs. There was no difference in body weight between the two<br />
groups. Profound reduction of LRP1 was confirmed by qPCR and Western blotting. Whole body<br />
inhibition of LRP1 elicited a modestly increased total plasma cholesterol compared to its control<br />
group. Size exclusive chromatography analyses demonstrated the increased cholesterol was due to<br />
increased VLDL and LDL-cholesterol. Despite augmented hypercholesterolemia, whole body<br />
inhibition of LRP1 did not increase atherosclerotic lesion size. LRP1 was also reduced in a<br />
hepatocyte-specific manner by administration of an LRP1 ASO coupled with GalNac to target the<br />
asialofeutin receptor. LDL receptor deficient mice were injected with GalNAc LRP1 ASO or its<br />
control ASO (5 mg/kg/week). Hepatic-specific inhibition of LRP1 did not change plasma cholesterol<br />
concentration, lipoprotein distribution and atherosclerosis.<br />
Conclusion:<br />
Profound reductions of LRP1 in mature mice modestly increased hypercholesterolemia and had no<br />
effect on atherosclerosis in LDL receptor deficient mice.<br />
100
85<br />
Oxidized neutral lipid lipolysis as a novel regulator of insulin signaling during acute stress<br />
Katelyn Ahern 1 • Garrett Mullins, PhD 1 • Vidisha Raje, PhD 1 • Vlad Serbulea 1 •<br />
Norbert Leitinger, PhD 1 • Thurl Harris, PhD 1<br />
1Pharmacology, University of Virginia<br />
Graduate Student<br />
The acute stress response following an injury or surgery often results in systemic insulin resistance<br />
(IR) and hyperglycemia which can lead to increased morbidity and mortality. Post-operative IR is<br />
downstream of stress hormones such as catecholamines, but the underlying mechanisms driving IR<br />
are unknown, thus limiting therapeutic development. We have previously demonstrated that β-<br />
adrenergic stimulation of adipocytes causes inhibition of the mTOR complexes and leads to the<br />
development of IR in a lipolysis-dependent manner. Therefore, we hypothesize that a product of<br />
lipolysis is responsible. We find that lipid extracts isolated from forskolin-stimulated 3T3-L1<br />
adipocytes inhibit the activity of the mTOR complexes in vitro. However, when tested, “traditional”<br />
products of lipolysis had no effect on mTOR complexes. Interestingly, stimulating oxidation of fatty<br />
acids using tert-butyl hydroperoxide further exacerbates mTOR inhibition, while antioxidant<br />
treatment reverses this effect, suggesting that the active species is an oxidized fatty acid. In fact,<br />
incubation of mTOR complex with in vitro auto-oxidized fatty acids is sufficient to inhibit kinase<br />
activity. These findings reveal a previously unrecognized mechanism of oxidized fatty acid signaling<br />
and mTOR complex regulation in the development of peripheral IR during acute stress. Our future<br />
work will focus on identifying these species in vivo using our novel targeted liquid<br />
chromatography-mass spectrometry method and characterizing their physiological role in<br />
response to stress-inducing events known to stimulate reactive oxygen species production.<br />
101
86<br />
Pharmacological inhibition of HuR improves survival and reduces adverse cardiac<br />
remodeling following left-ventricular pressure overload<br />
Sarah Anthony 1 • Xiaoqing Wu, PhD 2 • Lisa Green 1 • Michelle Nieman 3 • John Lorenz, PhD 3 •<br />
Jack Rubinstein, MD 1 • Liang Xu, PhD 2 • Michael Tranter, PhD 1 • Burns Blaxall, PhD 4<br />
1Division of <strong>Cardiovascular</strong> Health and Disease, University of Cincinnati • 2 Molecular Biosciences,<br />
University of Kansas • 3 Pharmacology and Systems Physiology, University of Cincinnati •<br />
4Department of Pediatrics, Division of Molecular <strong>Cardiovascular</strong> Biology, Heart Institute, Cincinnati<br />
Children's Hospital<br />
Staff<br />
Human antigen R (HuR) is a widely expressed RNA binding protein that has been implicated in<br />
numerous human diseases including cancer, neurological disorders, and cardiovascular<br />
disease. We have previously shown that HuR is both necessary and sufficient for induction of<br />
hypertrophic signaling pathways in isolated primary myocytes. In addition, data from our lab is the<br />
first to suggest that HuR expression and activation is increased in failing human hearts. To<br />
determine the role of HuR in hypertrophic signaling in vivo, we created an inducible<br />
cardiomyocyte-specific HuR deletion mouse, and showed that genetic deletion of HuR reduces<br />
pathology using a transverse aortic constriction (TAC) model of pressure-overload-induced<br />
hypertrophy, adverse cardiac remodeling, and heart failure. In this work, we sought to recapitulate<br />
this reduction in pathology using KH-3, a novel pharmacological inhibitor of HuR, to determine the<br />
translational potential of HuR inhibition as a viable therapeutic target.<br />
Twenty wild-type mice were randomized to either vehicle or KH-3 at four weeks post-TAC, a timepoint<br />
consistent with substantial development of cardiac hypertrophy, and monitored via serial<br />
echocardiography for a further seven weeks. Our results show that treatment with KH-3 increased<br />
survival relative to vehicle controls. In addition, as compared with vehicle, KH-3 treatment<br />
significantly abated the continued progression of left ventricular (LV) hypertrophy. This was<br />
accompanied by a significant preservation of LV ejection fraction and reduction in LV chamber<br />
dilation in KH-3 treated mice. Importantly, chronic KH-3 treatment had no effects on systemic<br />
blood pressure and no observable adverse reactions. In conclusion, these results suggest that<br />
inhibition of HuR is a promising therapeutic approach to treat pathological LV hypertrophy.<br />
102
87<br />
Perioperative high density lipoprotein particle changes and the risk of acute kidney injury<br />
after cardiac surgery<br />
Loren Smith, MD, PhD 1 • Derek Smith, PhD 2 • Alan Remaley, MD, PhD 3 • MacRae Linton, MD 4 •<br />
Frederic Billings IV, MD 5<br />
1Anesthesiology, Vanderbilt University Medical Center • 2 Biostatistics, Vanderbilt University<br />
Medical Center • 3 National Heart, Lung, and Blood Institute, National Institutes of Health •<br />
4Medicine and Pharmacology, Vanderbilt University Medical Center • 5 Anesthesiology and<br />
Medicine, Vanderbilt University Medical Center<br />
Faculty<br />
Acute kidney injury (AKI) after cardiac surgery occurs in up to 30% of patients and increases the<br />
risk of postoperative myocardial infarction and death. No effective treatments exist. Increased<br />
intraoperative oxidative stress independently predicts AKI after cardiac surgery. High density<br />
lipoproteins (HDL) have anti-oxidant capacity. We hypothesize that perioperative HDL cholesterol<br />
concentration (HDL-C), HDL particle concentration (HDL-P) and HDL composition are associated<br />
with the risk of postoperative AKI.<br />
We analyzed data from a prospective, 393-subject trial of perioperative atorvastatin to prevent AKI<br />
after cardiac surgery. Statin-using patients were randomized to placebo or 80mg atorvastatin the<br />
morning of surgery and 40mg on postoperative day 1. Stain-naïve patients were randomized to<br />
placebo or 80mg the day prior to surgery and 40mg daily thereafter during hospitalization. The<br />
associations between 1) HDL-C, 2) small HDL particle concentration, and 3) the change in HDL-P<br />
from anesthetic induction to postoperative day two with maximum serum creatinine change from<br />
baseline in the first 48 postoperative hours were assessed using two-component latent variable<br />
mixture models adjusted for known AKI risk factors. Regression analyses assessed interactions of<br />
chronic statin use, perioperative atorvastatin treatment, and HDL-C on AKI risk. We quantified HDL<br />
particle size by NMR Lipoprofile test in 90 subjects. HDL-C and HDL-P change over time were<br />
assessed with mixed effects models adjusted for AKI risk factors. We quantified myeloperoxidase<br />
(MPO) activity of apolipoprotein B-depleted serum in 30 subjects using a bioluminescence assay.<br />
A higher preoperative HDL-C was independently associated with a decreased postoperative serum<br />
creatinine change (p=0.02). The association between a high HDL-C and an attenuated increase in<br />
serum creatinine was strongest in chronic statin-using patients (p=0.008) and was enhanced with<br />
perioperative atorvastatin treatment (p=0.004) and increasing chronic statin dose (p=0.003). HDL-<br />
C did not change during the perioperative period (p=0.60), however, HDL-P decreased (p
88<br />
The Effects of Hypercholesterolemia on Wound Healing and Endothelial Angiogenic<br />
Properties<br />
Yedida Bogachkov 1 • Lin Chen, MD, PhD 2 • Myung-Jin Oh, PhD 3 • Luisa A. DiPietro, PhD 2 •<br />
Irena Levitan, PhD 4<br />
1Pharmacology, University of Illinois at Chicago • 2 Periodontics, University of Illinois at Chicago •<br />
3Medicine, University of Chicago • 4 Pulmonary, Critical Care, Sleep and Allergy, University of Illinois<br />
at Chicago<br />
Graduate Student<br />
Nearly 31 million adults in the US have elevated total cholesterol levels over 240 mg/dL, and almost<br />
32% of US adults have LDL cholesterol levels of 200mg/dL or above. Our study focuses on the<br />
impact of dyslipidemia on wound healing and wound angiogenesis. Using ApoE-/- mice, a known<br />
dyslipidemic model and a skin punch biopsy wound healing assay, we show that ApoE -/- mice<br />
show significantly delayed wound closure on days 2-7 post-wounding compared to control mice.<br />
These same ApoE-/- also display significantly decreased angiogenesis on days 10 and 14 post<br />
wounding, based on PECAM staining for endothelial cells, indicative of endothelial cell infiltration of<br />
the wound bed and new blood vessel growth. Furthermore, macrophage infiltration is also reduced.<br />
This significant suppression of wound angiogenesis is also seen when LDL is directly applied to the<br />
wounds via subcutaneous injections on C57BL6 mice. Additionally, using a matrigel plug assay, we<br />
show significantly suppressed angiogenesis in C57BL6 mice fed a Western Diet for 40 weeks. These<br />
results utilize an Isolectin B4 probe, which is indicative of endothelial cell infiltration into the<br />
matrigel plug and new blood vessel growth. Finally, in vitro studies on human aortic endothelial<br />
cells were pursued showing LDL significantly decreases endothelial cell proliferation, whereas its<br />
oxidized form, oxLDL, shows an increase in proliferation. In sum, we have shown in vivo that<br />
hypercholesterolemic mice have a decreased angiogenic potential, and in vitro, excess LDL leads to<br />
a decrease in angiogenic potential of aortic endothelial cells.<br />
104
89<br />
Coarse-Grained Molecular Dynamics Simulations of Kir2.2 Interactions with an Ensemble of<br />
Cholesterol Molecules<br />
Nicolas Barbera, MS 1 • Manuela Ayee, PhD 1 • Belinda Akpa, PhD 2 • Irena Levitan, PhD 3<br />
1Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at<br />
Chicago • 2 Department of Molecular Biomedical Sciences, North Carolina State University •<br />
3Department of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of<br />
Illinois at Chicago<br />
Graduate Student<br />
Hypercholesterolemia, elevated plasma levels of cholesterol, is a major risk factor in the<br />
development of atherosclerosis. Our previous studies have shown that inwardly rectifying K+ (Kir)<br />
channels, which play an important role in endothelial function, are suppressed by enriching cells<br />
with cholesterol. Additionally, earlier work done by our group and others has shown that<br />
cholesterol regulates Kir2 channels, a sub-family of Kir channels expressed in endothelial cells,<br />
through direct interactions at non-annular interaction sites. However, while a putative binding site<br />
has been identified, the dynamics of the binding process and cholesterol’s structural effect on the<br />
protein remain poorly understood. To address these questions, we used coarse grained molecular<br />
dynamics simulations of Kir2.2, a subfamily of Kir2, in model membranes containing cholesterol<br />
and POPC to interrogate their molecular interactions at the microsecond timescale. Our simulations<br />
show that rather than single ligand-channel binding, interactions between cholesterol and the<br />
channel are both complex and numerous, with an average of 15-20 cholesterol molecules<br />
interacting with the protein at any given time. These interactions occur at a range of timescales and<br />
at distinct annular and non-annular sites on the surface of the protein. Additionally, we observed<br />
spontaneous diffusion between these sites and between the protein surface and the surrounding<br />
membrane. At present, we are exploring the functional significance of these various interactions<br />
and the impact of this collective action on the structure-function relationships governing Kir2<br />
channel activity.<br />
105
90<br />
Reduced HDL in mice overexpressing SR-BI is associated with alterations in the acute<br />
inflammatory response to endotoxemia<br />
Yanzhang Li, PhD 1 • Ailing Ji, PhD 2 • Xuebing Wang 2 • Andrea Trumbauer 2 • Maria C. de Beer, PhD 3<br />
• Frederick C. de Beer, MD 4 • Nancy R. Webb, PhD 5<br />
1IM-Endocrinology, University of Kentucky • 2 <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of<br />
Kentucky • 3 Physiology, University of Kentucky • 4 Internal Medicine, University of Kentucky •<br />
5Pharmacology and Nutritional Sciences, University of Kentucky<br />
Faculty<br />
Objectives: Serum amyloid A (SAA) is an acute phase protein (APP) produced mainly by the liver<br />
during an acute phase response (APR). During the APR, virtually all of SAA circulating in the blood<br />
is associated with HDL. SR-BI is an HDL receptor and also can bind SAA. Previous reports showed<br />
that in SR-BI transgenic mice, hepatic uptake of SAA is increased, and this uptake is involved in the<br />
pro-inflammatory effects of SAA. Whether SR-BI regulates SAA expression in response to the<br />
inflammatory stimulation is not yet investigated.<br />
Methods/Results: Ten week-old male C57BL/6 mice were administered 1x1011 particles of a<br />
replication-defective adenoviral vector expressing mouse SR-BI (AdSR-BI) or an adenoviral vector<br />
containing no transgene (Adnull) as control. At 72h after adenovirus infusion, the mice were<br />
injected i.p. with 1mg /kg body weight LPS. Plasma and tissues were collected at selected time<br />
points after LPS injection. As expected, overexpression of SR-BI produced a dramatic reduction in<br />
HDL-C (56.52 ± 2.338 mg/dL vs 2.08 ± 1.555 mg/dL for Adnull versus AdSR-BI). Plasma levels of<br />
inflammatory cytokine IL-1b, TNFa and IL-6 were robustly higher in AdSR-BI-injected mice<br />
compared to control mice. Lympohcyte numbers were also signicantly higher in AdSR-BI-injected<br />
mice compared to control mice 12h after LPS injection. In contrast, SAA in plasma was much lower<br />
in AdSR-BI-injected mice compared to control mice. Before LPS injection, SAA could only be<br />
detected at very low levels in the plasma by ELISA (20.7±8.3 ug/ml). LPS induced a robust increase<br />
in plasma SAA levels as early as 2h post-LPS treatment in Adnull-treated mice (1100.0±108.7<br />
ug/ml). Notably, increases in plasma SAA were significantly lower in AdSR-BI-treated mice<br />
(122.7±9.5 ug/ml) 2h after LPS injection. At 12h after LPS injection, plasma levels of SAA were 10-<br />
fold lower in AdSR-BI-treated mice compared to control mice (12.9±0.24 mg/ml vs 1.2±0.21<br />
mg/ml). The results from western blot and real-time RT-PCR showed that hepatic SAA expression<br />
was significantly decreased in the liver of SR-BI overexpressed mice but significantly increased in<br />
hepatocytes of SR-BI knock out mice compared to that of control mice.<br />
Conclusions: Our results demonstrate that decreased levels of HDL in SR-BI-overexpressing mice<br />
are associated with alterations in acute inflammatory responses. Future studies will investigate the<br />
relationship between reduced SAA and increased inflammatory cytokines in mice with increased<br />
hepatic SR-BI overexpression.<br />
106
91<br />
A patient with very high plasma level of high-density lipoprotein cholesterol (HDLc) and<br />
sudden cardiac arrest<br />
Wenliang Song, MD 1 • Ginger Milne, PhD 2 • John Fahrenholz, MD 3 • John McPherson, MD 1 •<br />
John Oates, MD 4 • Dan Roden, MD 1 • MacRae Linton, MD 1<br />
1Cardiology, Vanderbilt University Medical Center • 2 Clinical Pharmacology, Vanderbilt University •<br />
3Medicine, Vanderbilt University Medical Center • 4 Medicine, Vanderbilt University<br />
Postdoc<br />
Synopsis:<br />
A 58-year-old female with plasma HDLc of 110-150 mg/dL suffers from a very debilitating facial<br />
flushing for the past several years. She also had two episodes of sudden cardiac arrests.<br />
Purpose:<br />
To find a pathological condition that can explain the constellation of symptoms this patient has,<br />
which may provide important insights of HDL metabolism<br />
Methods:<br />
Extensive clinical work-ups to evaluate her constellation of symptoms and basic laboratory<br />
experiments including HPLC/MS/MS to evaluate specific biomarkers for rare orphan diseases<br />
Results:<br />
This patient's coronary angiogram was normal. However, acetylcholine challenge produced<br />
significant coronary spasm. Bone marrow biopsy excluded systemic mastocytosis. Urinary<br />
prostaglandin D2 metabolites increased after flushing attacks, which suggests the patient has a<br />
condition of mast cell activation syndrome. Her clinical constellation of symptoms mimic the<br />
niacin's pharmacological effect. Genetic investigation may shed lights on the mechanism of HDL<br />
metabolism and niacin's therapeutic effect.<br />
Conclusions:<br />
Isolated mast cell activation syndrome may cause coronary spasm. Rare gene mutation may be<br />
responsible for her condition. Future genetics investigation may shed lights on the mechanism of<br />
HDL metabolism.<br />
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92<br />
Human Antigen R (HuR) Regulates Structure and Function of Brown Adipose Tissue<br />
Lindsey Lanzillotta 1 • Zach Taylor 1 • Kaila Yamamoto 1 • Sarah Anthony 1 • Shannon Jones 1 •<br />
George Yoshida 1 • A. Phillip Owens III, PhD 1 • Michael Tranter, PhD 1<br />
1Internal Medicine, University of Cincinnati<br />
Graduate Student<br />
Human antigen R (HuR) is an RNA binding protein widely expressed throughout the body, including<br />
in both white (WAT) and brown (BAT) adipose tissue. Recent work from our lab has shown that<br />
adipocyte-specific HuR knockout (adipo-HuR-/-) mice gain less weight following a high fat diet<br />
(HFD) compared to wild type (WT) controls. In this study, we focused on the role of HuR deletion<br />
on the function of BAT, which mediates non-shivering thermogenesis.<br />
Histological analysis revealed that, while the BAT of the chow fed adipo-HuR-/- mice appeared<br />
normal, the BAT of the HFD fed adipo-HuR-/- mice appears less dense with a cell size that more<br />
closely resembles WAT. Upon further examination of BAT function, our results show that, when<br />
subjected to cold stress (4C), adipo-HuR-/- mice are less cold tolerant compared to their WT<br />
counterparts . We also show that, compared to the WT mice, adipo-HuR-/- mice have decreased<br />
mitochondrial density and expression of uncoupling protein-1 (UCP-1) in their BAT. This may be<br />
caused by a HuR-dependent decrease in expression of peroxisome proliferator-activated receptor<br />
gamma coactivator 1 alpha (PGC-1a), which plays a key role in mitochondrial biogenesis and is<br />
upregulated during exposure to cold (4C).<br />
In conclusion, our results indicate that HuR regulates both the structure and function of BAT, at<br />
least in part through modulation of PGC-1a mRNA expression.<br />
Support or Funding Information:<br />
This work was supported by a University of Cincinnati Heart, Lung, and Vascular Institute Near<br />
Horizons Grant (MT, APO).<br />
108
93<br />
Impact of miR-33 antagonism on atherosclerotic plaque size and cholesterol content in<br />
nonhuman primates<br />
Tong Li, MD 1 • Lei Cai, PhD 1 • Sierra Paxton 1 • Courtney Burkett 1 • Peter Hecker, MS 1 •<br />
Ryan Temel, PhD 1<br />
1CVRC, University of Kentucky<br />
Graduate Student<br />
Introduction: Elevated LDL cholesterol (LDL-C) is a major risk factor for coronary heart disease<br />
(CHD). Statins are used to lower LDL-C and CHD risk but do not completely eliminate CHD events<br />
caused by atherosclerotic lesion rupture. By stimulating macrophage cholesterol efflux and<br />
promoting anti-inflammatory macrophage polarization, antagonism of microRNA-33a (miR-33a) in<br />
mice reduces the cholesterol content and size of atherosclerotic lesions. Mouse studies have limited<br />
translational value since mice do not develop coronary artery atherosclerosis and express only one<br />
of the two miR-33 family members found in humans. Since nonhuman primates (NHPs) have miR-<br />
33a and miR-33b and develop coronary artery atherosclerosis, NHPs are the best preclinical model<br />
for determining the therapeutic potential of miR-33 antagonism. We hypothesize that NHPs treated<br />
with miR-33 antagonist will have decreased plaque size and cholesterol content.<br />
Methods: Male cynomolgus monkeys (n=36) were fed for 20 months a high fat/high cholesterol<br />
diet, which caused severe hypercholesterolemia. At the end of the 20-month progression phase, a<br />
subset of monkeys (n=12) were euthanized to collect tissues. The remaining monkeys were<br />
switched to a cholesterol-lowering “chow” diet and treated with either vehicle (n=12) or anti-miR-<br />
33 (n=12) for 6 months. Formalin-fixed right coronary arteries (RCA) were split into five ~3 mm<br />
sections, paraffin embedded, and cut into 5 µm slices. Lesion area, defined as the region between<br />
the lumen and internal elastic lamina, was measured for each RCA section and then averaged for<br />
each animal. Cholesterol was extracted from a section of the fixed thoracic aorta and the free and<br />
total cholesterol levels were determined by GC-FID. Esterified cholesterol (EC) = total cholesterol –<br />
free cholesterol (FC).<br />
Results: No significant difference among the progression, vehicle, and anti-miR-33 groups were<br />
observed for RCA lesion area (P=0.60) and thoracic aorta FC (P=0.24). Thoracic aorta EC was<br />
significantly increased in the progression versus the regression groups (P
94<br />
Impact of miR-33 antagonism on coronary artery atherosclerotic plaque composition in<br />
nonhuman primates<br />
Lei Cai, PhD 1 • Tong Li, MD 1 • Sierra Paxton 1 • Courtney Burkett 1 • Peter Hecker, MS 1 • Ryan Temel<br />
, PhD 1<br />
1CVRC, University of Kentucky<br />
Staff<br />
Introduction: Elevated LDL cholesterol (LDL-C) is a major risk factor for coronary heart disease<br />
(CHD). Statins are used to lower LDL-C and CHD risk but do not completely eliminate CHD events<br />
caused by atherosclerotic lesion rupture. By stimulating macrophage cholesterol efflux and<br />
promoting anti-inflammatory macrophage polarization, antagonism of microRNA-33a (miR-33a) in<br />
mice causes atherosclerotic lesions to acquire a more stable composition. Mouse studies have<br />
limited translational value since mice do not develop coronary artery atherosclerosis and express<br />
only one of the two miR-33 family members found in humans. Since nonhuman primates (NHPs)<br />
have miR-33a and miR-33b and develop coronary artery atherosclerosis, NHPs are the best<br />
preclinical model for determining the therapeutic potential of miR-33 antagonism. We hypothesize<br />
that miR-33a/b antagonism will stabilize coronary artery atherosclerotic plaques of NHPs.<br />
Methods and Results: Male cynomolgus monkeys (n=36) were fed for 20 months a high fat/high<br />
cholesterol diet, which caused severe hypercholesterolemia. At the end of the 20-month<br />
progression phase, the hearts from 12 animals were perfused with formalin under physiological to<br />
preserve the structure of the coronary arteries. The remaining monkeys were switched to a<br />
cholesterol-lowering “chow” diet and treated with either vehicle (n=12) or anti-miR-33 (n=12) for<br />
6 months. LDL-C for both treatment groups was rapidly reduced by the chow diet and returned to<br />
baseline levels by the 3rd month of the regression phase. The anti-miR-33 versus the vehicle group<br />
had a sustained and significant elevation in HDL-C that was linked to a significant increase in<br />
hepatic ABCA1, a miR-33 target. Following dissection and paraffin embedding of the right coronary<br />
arteries (RCA), 5 µm slices were analyzed by histology and immunohistochemistry (IHC). The RCA<br />
atherosclerotic lesions from both the progression and regression groups were characterized by<br />
intimal smooth muscle cell proliferation, collagen deposition, elastin degradation, and medial layer<br />
destruction. While necrotic cores were a common feature, there was a minimal amount of plaque<br />
calcification. Macrophages were found at high levels in the progression group lesions but were<br />
almost absent from the plaques of both the anti-miR-33 and vehicle groups.<br />
Conclusions and Future Directions: The qualitative histology and IHC results show that the<br />
monkeys developed RCA atherosclerotic plaques similar to early fibroatheromas observed in<br />
humans. By quantifying the area occupied by the various plaque components, we will determine<br />
whether antagonism of miR-33a/b allowed the lesions to attain a more stable composition. In<br />
addition, to determine whether anti-miR-33 changes the amount or type of macrophages in lesions,<br />
we will analyze coronary arteries from monkeys subjected to 6 weeks, as opposed to 6 months, of<br />
regression.<br />
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95<br />
Targeting of HB-EGF against hypertension and renal damage<br />
Lihua Yang, MS 1 • Seonwook Kim 1 • Debra Rateri 1 • Richard Lee, PhD 2 • Mark Graham, PhD 2 •<br />
Sangderk Lee, PhD 1<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky • 2 <strong>Cardiovascular</strong> Antisense Drug<br />
Discovery Group, Ionis Pharmaceuticals<br />
Staff<br />
Study goal: Previous reports suggested that the heparin binding EGF-like growth factor (HB-EGF),<br />
which is an EGFR ligand, is positively involved in the development of renal disease. In this study, we<br />
tested the effects of the HB-EGF targeting using antisense oligonucleotide (ASO) administration on<br />
the blood pressure and renal damage induced by AngII infusion in mouse model systems.<br />
Experimental design and results: To test the effects of the HB-EGF ASO administration on the AngIIinduced<br />
hypertension, C57BL/6 mice (male, 10 weeks of age) were pretreated with control and HB-<br />
EGF ASO for 2 weeks and cotreated with AngII infusion (1,000ng/min/kg) for additional 4 weeks.<br />
We monitored blood pressure changes during 6 weeks using standard tail-cuff procedure. We<br />
observed that the HB-EGF ASO administration significantly downregulated basal and AngII-induced<br />
blood pressure in the system. The HB-EGF targeting also reduced the kidney size. To test the effects<br />
of the HB-EGF targeting on the renal damage, the LDLR KO mice (male, 16 weeks of age) were<br />
pretreated with control and HB-EGF ASO and cotreated with high fat diet (HFD; 42% calorie from<br />
fat, 0.2% cholesterol) and AngII-infusion (1,000ng/min/kg) for additional 4 weeks. At the<br />
termination step, we compared plasma creatinine and BUN levels as markers of the status of the<br />
renal damage. The result showed that the HB-EGF ASO administration effectively protected against<br />
HFD and AngII-induced renal damage in the mouse model system.<br />
Conclusion: These results suggested that the HB-EGF signaling positively involved in regulating<br />
blood pressure and renal damage. The targeting of the HB-EGF signaling might be an approach to<br />
delay the development of the hypertension associated with renal damage.<br />
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96<br />
HB-EGF is a Key Positive Regulator of Hepatic VLDL Secretion<br />
Seonwook Kim 1 • Lihua Yang, MS 1 • Debra Rateri 1 • Ryan Temel, PhD 1 • Richard Lee, PhD 2 •<br />
Mark Graham, PhD 2 • Sangderk Lee, PhD 1<br />
1Saha <strong>Cardiovascular</strong> <strong>Research</strong> Center, University of Kentucky • 2 <strong>Cardiovascular</strong> Antisense Drug<br />
Discovery Group, Ionis Pharmaceuticals<br />
Staff<br />
Study goal: Unbalanced hepatic VLDL production (assembly/secretion) is a risk factor for the<br />
development of hypertriglyceridemia or non-alcoholic fatty liver disease (NAFLD) under metabolic<br />
stress conditions like metabolic syndrome. Previous reports showed that the circulatory heparinbinding<br />
EGF-like growth factor (HB-EGF), which is a ligand of EGFR, is correlated with cholesterol<br />
level and risk of coronary artery disease in human. From these previous data, we tested the<br />
hypothesis that the HB-EGF signaling is a positive regulator of VLDL production in the liver.<br />
Experimental procedure and results: In Hep-G2 cells, the recombinant HB-EGF induced increase of<br />
the apoB secretion, which is an essential component of VLDL particle. In C57BL/6 mice, the<br />
administration of recombinant HB-EGF increased hepatic VLDL secretion; in contrast, the HB-EGF<br />
antisense oligonucleotide (ASO) administration or the tail-vein injection of EGFR blocker BIBX1382<br />
induced significant suppressions of the hepatic VLDL secretion. The HB-EGF ASO administration<br />
effectively downregulated circulatory lipid levels (TG and cholesterol) associated with VLDL<br />
particles in multiple mouse strains (C57BL/6, LDLR KO, and apoE KO). At the same time, the<br />
targeting induced accumulation of neutral lipids in the liver, specifically, TG and cholesterol ester.<br />
Conclusion: These results indicate that the HB-EGF signaling positively regulates VLDL secretion in<br />
the liver under normal and metabolic stress condition. Future study of the interaction with insulin<br />
signaling for the regulation would be interesting in understand the pathology of metabolic<br />
symptom.<br />
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97<br />
Anti-ApoA-I antibodies induced using an epitope-specific immunostimulatory liposomal<br />
formulation exacerbate atherosclerosis in dyslipidemic mice<br />
David Henson 1 • Robert Kline IV, MS 1 • Vincent Venditto, PhD 1<br />
1Pharmaceutical Sciences, University of Kentucky<br />
Graduate Student<br />
Autoantibodies targeting apolipoprotein A-I (ApoA-I) have been identified in patients with chronic<br />
inflammatory diseases including rheumatoid arthritis, lupus and obesity and correlate with<br />
cardiovascular disease progression. Although correlated, the exact role of these antibodies has not<br />
been fully elucidated. Induction of antibodies in mice through immunization, rather than passive<br />
administration of antibodies derived from other species, offers a unique opportunity to explore the<br />
impact of anti-ApoA-I antibodies in the context of atherosclerosis. Furthermore, the ability to<br />
modulate epitope-specific immune responses offers a strategy to study the impact of antibody<br />
responses toward specific domains within the full protein. To achieve these objectives, we prepared<br />
an immunostimulatory liposomal formulation containing peptides derived from the LCAT domain<br />
of ApoA-I. We first determined that mice immunized with the complete formulation induce robust<br />
antibody responses toward the epitope, as well as the full-length protein. These data suggest that<br />
antibody responses toward ApoA-I are either not controlled by immunological tolerance<br />
mechanisms or our formulation was capable of breaking tolerance. Although successful at inducing<br />
a robust immune response, the antibodies alone failed to induce atherosclerosis in wild-type mice.<br />
We then studied the role of antibodies in the context of dyslipidemia utilizing the AAV-PCSK9 gainof-function<br />
mutant and western diet. After establishment of dyslipidemia, mice were immunized<br />
with the immunostimulatory liposomal formulations. Once again, mice immunized with the<br />
complete formulation achieved robust antibody responses, which persisted for 150 days.<br />
Furthermore, mice immunized with the complete formulation had a statistically significant<br />
elevation in atherosclerosis over control mice. These data highlight our ability to modulate the anti-<br />
ApoA-I antibody response using epitope-specific liposomal formulations and provides a platform to<br />
study the role of these antibodies in an immunologically intact system.<br />
This work was supported by a pilot grant through the Center for <strong>Research</strong> in Obesity &<br />
<strong>Cardiovascular</strong> Disease COBRE, NIH (P20GM103527) and a Scientist Development Grant from the<br />
American Heart Association (17SDG32670001). DH is supported by a UK Center for Clinical and<br />
Translational Science training grant from the National Center for Advancing Translational Sciences,<br />
NIH (TL1TR001996).<br />
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98<br />
HDL-miR-223 Communication Pathway in vivo.<br />
Carrie Wiese 1 • Leslie Roteta 1 • Ryan Allen, PhD 2 • Wanying Zhu 2 • Kasey Vickers, PhD 2<br />
1Molecular Physiology & Biophysics, Vanderbilt University • 2 <strong>Cardiovascular</strong> Medicine, Vanderbilt<br />
University Medical Center<br />
Graduate Student<br />
High-density lipoproteins (HDL) stably transport microRNAs (miRNAs) through the blood and<br />
facilitate a HDL-miRNA communication pathway. Previously, we demonstrated that macrophages<br />
and other myeloid cells export miR-223 to HDL in vitro. Furthermore, HDL-miRNAs, including miR-<br />
223, are delivered to recipient human coronary artery endothelial cells (HCAECs) where miR-223 is<br />
not transcribed. Upon transfer of miR-223 to endothelial cells, the validated miR-223 target<br />
intracellular adhesion molecule 1 (Icam-1) expression is significantly suppressed. While HDLmiRNA<br />
transfer and cellular gene regulation have been demonstrated in vitro, intercellular<br />
communication has not been established in vivo. We utilized bone marrow transplantation between<br />
C57/B6J (WT) and Mir223-/- mice to define the physiological role of HDL-miRNA pathway in<br />
regulating cellular gene expression. To restore the HDL-miR-223 communication pathway within<br />
Mir223-/- mice, we transplanted WT bone marrow into lethally irradiated Mir223-/- mice resulting<br />
in a significant increase in HDL-miR-223 levels. Subsequently, a significant increase in miR-223 was<br />
identified in recipient tissues including whole liver, hepatocyte isolates, and aortic endothelium.<br />
Conversely, HDL-miR-223 pathway was depleted by transplanting Mir223-/- bone marrow into<br />
lethally irradiated WT mice, which resulted in a significant reduction of miR-223 on HDL. The<br />
reduction in HDL-miR-223 levels correlated with reduced miR-223 levels in recipient cells<br />
including endothelial and hepatocyte. Furthermore, altering miR-223 levels in endothelial cells and<br />
hepatocytes resulted in altered mRNA levels of putative targets, which we have validated as miR-<br />
223 targets. In conclusion, myeloid cells export miR-223 to extracellular carriers such as HDL,<br />
which results in delivery of miR-223 to recipient cells where it regulates gene expression.<br />
114
99<br />
Dynamic hepatic oxidized phospholipid composition promotes non-alcoholic fatty liver<br />
disease progression<br />
Clint Upchurch 1 • Vlad Serbulea 1 • Norbert Leitinger, PhD 1<br />
1Pharmacology, University of Virginia<br />
Graduate Student<br />
Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, affects<br />
25% of individuals within the United States. NAFLD is a progressive pathology initiating with<br />
accumulation of fat within the liver (hepatic steatosis) followed by inflammation (steatohepatitis)<br />
and liver fibrosis. Currently, drivers of NAFLD progression are poorly understood. We propose that<br />
oxidized phospholipids (OxPLs) within the liver promote NAFLD pathophysiology. We developed a<br />
liquid chromatography-mass spectrometry method to quantify oxidation products of 1-palmitoyl-2-<br />
arachidonoyl-sn-glycero-3-phosphatidylcholine (PAPC) in liver tissue and observed an increase in<br />
full-length PAPC oxidation products (OxPAPC) in mice fed a high fat diet (HFD). HFD-fed mice<br />
exhibit extensive hepatic steatosis, the first stage of NAFLD. Separately, using<br />
immunohistochemistry we observed an increase in liver OxPLs in mice treated with carbon<br />
tetrachloride, a model of liver damage. Furthermore, hepatic stellate cells, the major contributor to<br />
fibrosis, show an increase in fibrotic gene expression in response to treatment with OxPAPC. Taken<br />
together, OxPLs play a role in stellate cell over-activation in response to liver damage and<br />
subsequent fibrosis.<br />
115
100<br />
Short-Term Exercise Training Differently Modifies Aspects of HDL Function in Lean, Obese<br />
and Diabetic Subjects<br />
Lin Zhu, PhD 1 • Nicolas Musi, MD 2 • John Stafford, MD, PhD 1<br />
1Diabetes, Vanderbilt University Medical Center • 2 Diabetes, University of Texas Health Science<br />
Center<br />
Faculty<br />
The triglyceride (TG)-lowering effects of exercise have been well documented. In contrast,<br />
mechanisms for the cardiovascular protective effects of exercise are not well established. We<br />
previously showed that short-term exercise training improves insulin sensitivity but does not<br />
inhibit inflammatory pathways in immune cells from insulin-resistant subject. The purpose of this<br />
study is to evaluate metabolic changes of lipoproteins after short-term exercise training in lean,<br />
obese and type 2 diabetic (T2D) subjects. Lean, obese nondiabetic, and obese T2D individuals (n=6<br />
per group) underwent a high intensity aerobic exercise program for 15 days. Blood lipid<br />
parameters and HDL functions were analyzed before and after exercise. Blood cholesterol levels<br />
were higher in obese and T2D than in lean subjects. Exercise training primarily reduced LDL- and<br />
HDL-cholesterol (17%-21%, P
101<br />
Identification of a novel lipoprotein microRNA carrier in plasma<br />
Wanying Zhu 1 • Danielle L. Michell 1 • Ryan M. Allen 1 • Kasey C. Vickers 1<br />
Vanderbilt University Medical<br />
Staff<br />
Recently, multiple groups have detected of microRNAs (miRNA) in plasma by high-throughput<br />
sequencing. Circulating sRNAs are likely protected from RNases in plasma through their association<br />
with lipid carriers. Our lab previously reported that lipoproteins (HDL and LDL) transport miRNAs<br />
in plasma within potential intercellular communication networks. Based on these previous studies,<br />
we sought to define the distribution of miRNAs across lipid carriers in plasma.<br />
Methods: To specifically precipitate carriers, whole plasma and pre-purified HDL (density-gradient<br />
ultracentrifugation) treated with lipid removal agents (LRA and Cleanascite) for the separation of<br />
lipid-associated miRNAs. Plasma samples from humans and mice were fractionated using sizeexclusion<br />
chromatography (FPLC, Superdex 200 Increase in triplicate). Each fraction was assessed<br />
for total cholesterol, triglycerides, protein, phospholipids, and markers for lipoproteins and<br />
extracellular vesicles by western blotting. Carrier sizes in each fraction were quantified using<br />
dynamic light scattering. Total RNA was isolated from whole fractions and separated contents, lipid<br />
pellets and supernatants, after centrifugation. Real-time PCR and high-throughput sequencing were<br />
used to quantify miRNAs.<br />
Results: Results demonstrated that Cleanascite efficiently removed lipids from plasma without<br />
damaging lipoproteins, as determined by the lack of phospholipids and apolipoproteins in the<br />
supernatants. Candidate miRNAs (miR-24-3p, miR-26a-5p, miR-146a-5p, miR-92a-3p, and miR-<br />
223-3p) were detected in fractions corresponding to HDL and LDL based on the distribution of<br />
cholesterol and triglycerides. Strikingly, candidate miRNAs also entirely separated with lipids in<br />
fractions that contained low cholesterol and high phospholipid content with a mass corresponding<br />
to very small HDL. These fractions were found to contain apoA-I and spherical particles
102<br />
The Ral-Exocyst Pathway Regulates Platelet Secretion<br />
Jinchao Zhang 1 and Sidney W. Whiteheart 1<br />
1<br />
Department of Molecular and Cellular Biochemistry, College of Medicine, University of<br />
Kentucky<br />
Postdoc<br />
In Platelets, Exocyst complex has been identified as an octamer consisting of Sec3, Sec5, Sec6,<br />
Sec8, Sec10, Sec 15, Exo70 and Exo84 in platelets. The complex plays an essential role in<br />
tethering vesicles to the plasma membrane and regulating dense core granule secretion in<br />
platelets. RalA/B (family members of GTPases) are regarded as the upstream regulator of the<br />
Exocyst complex, and RalA/B binds to Sec5 and Exo84 in a GTP-dependent manner.<br />
RalA/B, sharing over 88% identity in sequences, directly interact with Exo84 and Sec5 in the<br />
Exocyst complex. The role of RalA/B are interchangeable, but RalA has the higher ability to bind<br />
to the Exocyst components in GTP binding manner in cells. Several groups suggest that<br />
interrupting the association Sec3 and Exo70 with plasma membrane could disrupt exocytosis in<br />
other cells. However, it is little known how the role of the Exocyst complex in platelet secretion.<br />
Here we ask the question whether inhibitors of RalA/B could affect granule secretion or not in<br />
platelets. And what is the exact role of the Exocyst complex in granule secretion? How is the<br />
Exocyst complex regulating vesicles tethering in secretion process? Firstly, specific RalA/B<br />
inhibitors, RBC8 and BQU57, were used to investigate the role of RalA/B in granule secretions.<br />
From the biochemical perspective, these inhibitors affect RalA/B bound to their downstream<br />
effector (RLP76) due to the Ral-GDP bound forms. We also found the inhibitors of RalA/B<br />
affects platelet spreading (data are not shown here). At the same time, we also answer the<br />
questions of the localization of Rals and Exo70 (one of the Exocyst component) as well as their<br />
association with F-actin.<br />
118
103<br />
Alternating heights of the R wave in ECG: possible link with depolarization<br />
alternans<br />
Sahar Varnoosfaderani 1 , David Wasemiller 1 , Siqi Wang 1 , Paul Anaya 2 , and Abhijit<br />
Patwardhan 1 .<br />
1<br />
Department of Biomedical Engineering and 2 Division of <strong>Cardiovascular</strong> Medicine. University of<br />
Kentucky.<br />
Alternans of the T wave in the ECG (TWA) is widely investigated as a potential predictor of<br />
ventricular arrhythmia, however, clinical trials show that TWA has high negative predictive<br />
value but poor positive predictive value. A possible reason that TWA has a large number of false<br />
positives is that a pattern of alternans, concordant alternans, may not be as arrhythmogenic as<br />
discordant alternans. Currently, it is not possible to discern the pattern of alternans using<br />
clinical ECGs. However, our research using tissue and cellular level electrophysiological<br />
measurements suggest that depolarization alternans which affects the pattern of alternans may<br />
manifest itself in the amplitude of the R wave of the ECGs. In order to investigate the link<br />
between depolarization alternans and changes in ECGs, we used a mathematical model which<br />
simulated ECGs from the cellular level changes observed in our experimental studies. These<br />
results suggest that the changes in ECGs should appear as alternating pattern of the heights of<br />
the R wave. However, there are a variety of factors which may also cause the R wave heights to<br />
change. We use signal analysis and statistical modeling to determine the link between the<br />
observed changes in R wave heights and depolarization alternans. Results from ECGs recorded<br />
from patients show that heights of the R wave can change as predicted by our experimental<br />
results and mathematical models. These results support further exploration of the link between<br />
depolarization and repolarization alternans as it has the potential to improve the positive<br />
predictive value of TWA.<br />
Supported by grants from the Kentucky Science and Engineering Foundation (KSEF RDE18)<br />
and NSF EPSCoR RII Track-2.<br />
119
NOTES<br />
120
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