2011 CVC Annual Report (PDF) - Medical College of Wisconsin

Cardiovascular diseases remain the leading cause ofdeath in the US for both men and women. Research inthis field is of critical importance to the health of ourcommunity and our nation. The overall goal of theCardiovascular Center (CVC) is to enable laboratorydiscoveries to move forward in the form of new treatments,products, preventions and cures to those whosuffer from diseases of the heart, blood vessels, kidneys,and lungs.The CVC plays a vital role as a campus incubator tostimulate the exchange of new ideas and to nurture thedevelopment of novel research programs that fosterthe understanding of the underlying basic mechanismsof cardiovascular diseases and the translation of thisbasic knowledge into improved diagnostics, prevention,and therapeutics for the citizens of Wisconsin and beyond.The laboratories of 20 investigators reside withinthe confines of the CVC, and many more Faculty areaffiliated with our Center throughout the MCW campus.More than 100 clinicians and more than 50 basicscientists from various departments conduct researchwith cardiovascular implications.Over the past year, progress has been made in coalescingscientific Affinity Groups among the membersof the CVC thereby fostering interdisciplinary collaborationsin areas that have been defined as focus areasfor growth at MCW such as heart failure and heart tissueregeneration. Work is now in progress to betteralign the focus of CVC research with those areas ofstrength within the MCW clinical service lines of cardiologyand surgery, both adult and pediatric. Towardthat end, over the past year, CVC has co-recruited anumber of new Faculty, including Jennifer Strande,M.D., Ph.D. (a cardiologist in the division of CardiovascularMedicine), Paul Goldspink, Ph.D. (cardiacphysiology), Aron Geurts, Ph.D. (cardiovascular genomics)and Scott Levick, Ph.D., (cardiovascular pharmacology).In order to support the expanding researchefforts of the CVC, an additional 5000 square feet ofnew laboratory space has been developed and a numberof the existing laboratories have undergone renovationsto provide better utilization of existing space.Greater space has been developed to accommodatestudents and research fellows, and a newly createdConference Center that enables seating up to 125 andis divisible for small group meetings, has been constructed.These activities represent important advancements to-Letter from the DirectorAllen Cowley, PhDDr. Allen Cowleyward the overall mission of the CVC “to further developstrong and nationally recognized interdisciplinarycardiovascular research programs at the MedicalCollege of Wisconsin and to promote comparable excellencein clinical care and education while developingcommunity outreach”.The front cover: Dr. Williams’ lab (left to right) Carmen Bergom, MD, PhD, AdamGastonguay, Nathan Schuld, Andrew Hauser, Elizabeth Ntantie, Ellen L. Lorimer

The Cardiovascular Center in 2010-2011Created in 1992, the Cardiovascular Center (CVC) is focusing onthe prevention, detection, treatment and cure ofthe large family of cardiovascular diseases.Under the direction of Dr. Cowley, the CVC is administered by the staff: (from left to right):Allen W. Cowley Jr., PhD, Jane Brennan Nelson, Jean-Francois Liard, MD, PhD, JoannaMcCormick, Cescilly Burk, April MaysThe Cardiovascular Center offers a number of services to its Faculty and to the MCW campus, including thefollowing cores: Information Technology, Engineering, and Microscopy.IT core: ( left to right) Andrew J. Milbrath, Greg McQuestion, Pedro MendezEngineering core: David Eick, Mike KloehnMicroscopy core: Glenn SlocumCardiovascular Center Annual Report 20111

Cardiovascular Center FY 2011Annual Revenue SourcesMCW Central Funds $ 465,062Dean Program Development $ 100,000Philanthropic $ 420,893Total Revenues $ 985,955The Cardiovascular Center in 2010-2011FinancialsThis past year has been marked by the recruitment of several new Faculty:♦ Paul Goldspink, PhD, Associate Professor, Physiology♦ Jennifer Strande, MD, PhD, Assistant Professor, Cardiovascular Medicine♦ Aron Geurts, PhD, Assistant Professor, Physiology♦ Scott Levick, PhD, Assistant Professor, PharmacologyThe CVC primary Faculty whose research is described in this annual report together with Dr. Cowley combinefor a total of $12,915,367 in NIH funding as listed in NIH RePORTER (fiscal year total cost).Extensive renovations were conducted in the CVC, with the creation of several new facilities, including over5000 square feet of laboratory space for basic scientists, a surgical facility suite, a stem cell culture facility, a 125-seat conference room, a dedicated room for a new confocal microscope purchased by the CVC, a freezer farm,and the consolidation of core equipment in rooms protected by electronic card readers.The CVC Scientific Advisory Committee was again of great help to us in achieving our objectives and willcontinue to provide guidance. This Committee is made of the following MCW Faculty:♦ Ellis Avner, MD, Associate Dean of Research, Children's Research Institute, Professor, Pediatrics♦ Joseph Besharse, PhD, Chair of Cell Biology, Neurosciences and Anatomy♦ William Campbell, PhD, Chair of Pharmacology♦ Michael Cinquegrani, MD, Chief of Cardiovascular Medicine♦ Stephen Duncan, PhD, Professor of Cell Biology♦ Andrew Greene, PhD, Director, Bioengineering and Biotechnology Center♦ David Gutterman, MD, Professor in Cardiology, Senior Associate Dean for Research♦ David Harder, PhD, Professor of Physiology, Associate Dean for Research Mentoring♦ Howard Jacob, PhD, Director of the Human and Molecular Genetics Center♦ Elizabeth Jacobs, MD, Chief of Pulmonary and Critical Care Medicine♦ Michael E. Mitchell, MD, Associate Professor Surgery, Division of Cardiothoracic Surgery♦ Ramani Ramchandran, PhD, Investigator Children's Research Institute, Associate Professor, Pediatrics♦ David Warltier, MD, Chair of Anesthesiology♦ Gilbert White, MD, Director of Research of Blood Research Center♦ Michael Widlansky, MD, Assistant Professor, Cardiovascular MedicineFurther details about the CVC are provided on the CVC website at http://www.mcw.edu/cvc.htm and on theKidney Disease Center website at http://www.mcw.edu/kdc.2 Cardiovascular Center Annual Report 2011

1Ongoing research from CVC FacultyThis section summarizes research conducted by the primary Faculty of the CardiovascularCenter, including the Kidney Disease Center. The research of many other Faculty affiliatedwith the CVC is not summarized here.John Auchampach, PhDCharacterization of adenosine receptors: Dr.Auchampach and his team are focused on the study ofadenosine receptors, or proteins on the surface of cellsthat recognize adenosine and related compounds.Adenosine is produced by cells when the breakdownof adenosine triphosphate (ATP) is increased duringenergy utilization. Hence, adenosine levels are raisedwhen a cell is stressed, activated, or when there is notenough oxygen in the cells, as in ischemic heart diseaseand myocardial infarction.There are four different types of cell-surface receptorsfor adenosine. Dr. Auchampach’s research studiesthe function of two of the receptor subtypes that haveonly recently been discovered, the A 2B and A 3 adenosinereceptors. His group is testing the hypothesis thatthe A 3 adenosine receptor helps minimize muscledamage and reduce inflammatory processes that occurduring cardiac ischemia, whereas the A 2B adenosinereceptor that has low binding affinity for adenosinepromotes fibrotic responses in the cardiovascular systemcontributing to the development of heart failureand hypertensive disease. The research is aimed at developingnew drug therapies that target receptors foradenosine as potential treatments for patients with coronaryartery disease. In particular, it tests the hypothesis(see Figure below) that activating the A 3 adenosinereceptor in cardiomyocytes protects against ischemicinjury by improving mitochondrial function and redu-cing apoptosis, whereas activating the A 3 adenosinereceptor in immune cells during reperfusion is protectiveby suppressing inflammatory responses.Cardiac regeneration: in collaboration with colleaguesat MCW and the Medical College of Georgia, Dr.Auchampach’s research group is also investigating thetherapeutic potential of stem cells for regeneratingheart tissue that has been damaged following myocardialinfarction. Using embryonic and induced pluripotentstem cells, the hypothesis is being tested that transplantationof stem cells that have been predifferentiatedto specific cardiac lineages (i.e., cardiomyocytesor vascular precursor cells) will reduce thepotential for tumor formation and improve functionalrecovery of the damaged heart due to the incorporationof functional, electrically coupled myocytes intothe myocardium.Aron Geurts, PhDThrough genetic engineering in rats, the Geurts’ lab,along with colleagues in the Human and MolecularGenetics Center and the Department of Physiology,has made recent progress unraveling some of the complexgenetics of hypertension and kidney failure.Recent large-scale genetic studies in humans haveidentified many genes which play likely, yet poorly understoodroles in risk and progression of diseases likehypertension and chronic kidney disease. This hasshifted the focus of the Geurts’ lab and colleagues tounderstanding these specific genes as they may relateto these diseases. The laboratory rat is the preferredmodel to study diseases like hypertension and renalfailure because it is highly physiologically similar tohumans. In the past few years, the Geurts’ lab has developedseveral technological approaches to modifythe genome of rats to disrupt or ‘knock out’ specificgenes, something that was not previously possible. Thistechnological breakthrough has been named as one ofthe Top 10 scientific breakthroughs in both 2009 and2010 by “The Scientist” and “Science” magazines, respectively.Cardiovascular Center Annual Report 20113

Ongoing research from CVC FacultyAn effort is underway with other MCW laboratoriesto use these new approaches to knock out in the laboratoryrat 100 genes which have been identified in humanstudies. These models can then be studied to seeif disrupting these genes plays a potential role in hypertensionor kidney failure which lends support to thehuman findings and provide an animal model to understandgene function. The initial results are promisingas knocking out one gene, Sh2b3, which has beenstudied for several years in mice but never appreciatedfor its role in disease, demonstrates significant protectionfrom both hypertension and kidney failure, andpossibly other disease traits. This suggests that this is avery important gene in human disease which we cannow study further. It is the first time that a human diseasegene candidate has been experimentally validatedin a rat model of disease.The figure below illustrates the power of the zincfingernuclease (ZFN) technology in rats as used by Dr.Geurts’ lab for one of many genes it can be applied to.A gene called Rab38 controls trafficking of vesicles insidecells. When this gene is mutated in the rat usingZFN technology, it causes a pigmentation defect leadingto the fawn color that is evident in a subset of theanimals. Importantly, we have worked with HowardJacob’s lab to validate that this gene is also controllingtrafficking in the tubules of the kidney and mutationsin this gene can contribute to the end stage renal failure– a disease that affects millions of people worldwide.Paul Goldspink, PhDDr. Goldspink’s lab is presently investigating the influenceof insulin like growth factor-1 (IGF-1) isoformpeptides on the resident stem/progenitor cells populationswithin the heart. They are exploiting their findingsin a number of different ways. They (and collaboratorsfrom two other institutions) have developed atechnology that provides a microscopic physical scaffoldto deliver both the physical cues for tissue growthcombined with the capacity to deliver peptide therapeutics.They believe this “biomimetic” approach canbe used to optimize stem cell therapy either alone orin conjunction with autologous stem cells. This novelapproach of delivering implantable cell-sized“biomimetic devices” to instruct resident stem cells toenhance natural tissue repair and regeneration, is alsoadaptable to other tissues as well as the heart. Basedon this technology, Dr. Goldspink has formed an earlystage biomedical device company (Cell Habitats, Inc.)aimed at developing an easy-to-administer, micro devicethat allows the natural repair and regeneration ofdamaged tissue. Its first application is to restore normalcardiac function after a heart attack.The image below shows immunofluorescence stainingof small Troponin I positive cells (shown with arrows)in the heart of MGF E-domain peptide treatedmice following myocardial infarction. Split image collectedat 40X magnification on Nikon A1 confocal microscope.Green is an antibody against Troponin I, redis cell membrane stained with wheat germ agglutinin,blue is nuclei stained with DAPI.4 Cardiovascular Center Annual Report 2011

David Gutterman, MDDr. Gutterman’s group is involved in a number ofprojects. The first one is a study of ballerinas, who experiencecardiovascular and bone health problems.Twenty-two professional ballet dancers volunteered todetermine the prevalence of the 3 components of thefemale athlete triad (disordered eating, menstrual dysfunction,low bone mineral density) and their relationshipswith brachial artery flow-mediated dilation. Seventeendancers (77%) had evidence of low/negativeenergy availability. Thirty-two percent had disorderedeating. Thirty-six percent had menstrual dysfunction.Twenty-three percent had evidence of low bone density.Sixty-four percent had abnormal brachial artery flow-mediated dilation. All of the 3 components of the triadplus endothelial dysfunction were present in 14% ofthe subjects. In conclusion, endothelial dysfunction wascorrelated with reduced bone mineral density, menstrualdysfunction, and low serum estrogen. Thesefindings may have profound implications for cardiovascularand bone health in professional women dancers.They were recently published by A.Z. Hoch et al. inClin. J. Sport Med. 2011; 21:119–125.Another project examined the hypothesis that hydrogenperoxide (H 2 O 2 ) serves as the endotheliumdependenttransferrable hyperpolarization factor(EDHF) in human coronary arterioles (HCAs) to shearstress. Two HCAs were cannulated in series, with adonor intact vessel upstream and an endotheliumdenudeddetector vessel downstream. Results showedOngoing research from CVC Facultythat flow induced endothelial production of H 2 O 2 ,which acts as the transferrable EDHF activating largeconductance Ca 2+ -activated K + channels on thesmooth muscle cells. This was published by Y. Liu etal. in Circ. Res. 2011; 108:566-573.Finally, Dr. Gutterman’s group conducts researchinvolving the age-dependence of flow-induced dilationof the coronary arterioles in man. Human coronaryarterioles (~150 μm internal diameter) were obtainedat surgery from patients with or without coronary arterydisease (CAD) and prepared for videomicroscopy. Afterconstriction with endothelin-1, vasomotor responsesto flow were evaluated in the presence, absence orcombination of the cyclooxygenase (COX) inhibitorindomethacin and the nitric oxide synthase inhibitor L-nitro-arginine methyl ester. The following age groupswere examined: children 0–18 years; adults 19–55years; older adults >55 years without CAD, and adultswith CAD (mean age: 64±8.2 years). It was found thatflow-induced dilation in children is mediated mostly byCOX; whereas, during the aging process or in the presenceof CAD its contribution is diminished. Thesenovel findings may have clinical implications for theuse of COX inhibitors in children and young adults.This was published by N.S. Zinkevich et al. in Circulation2010, 122, 21, A15764.Gutterman/Widlansky/Zhang’s labs: Back row: (left to right) Rong Ying, MD , Suelhem A. Mendoza, Kodlipet Dharmashankar, MD, David Zhang, PhD,David Gutterman, MD, Reina Watanabe, PhD, Matt Durand, PhD, Andreas Beyer, PhD, Xiadong Zheng, PhDFront row: (left to right ) Joe Hockenberry, Yoshinori Nishijima, PhD, Jingli Wang, PhD, Dana VollmerCardiovascular Center Annual Report 20115

David Harder, PhDDr. Harder’s lab explores the dynamic control of cerebralblood flow and the interactive actions of cellswhich both require and impact delivery of oxygen tothe brain. Astrocytes play a crucial role in sensing andtransmitting neuronal signals to nearby cerebral microvesselsto regulate cerebral blood flow and thus deliveryof oxygen and nutrients to match the metabolicdemand of activated neurons. Studies are conducted todefine the signaling pathways impacting blood flow tometabolically active neurons. The hypothesis is thatsignaling pathways between astrocytes and the cerebralmicrovasculature are initiated via metabolites which actthrough second messengers, some of which requiretrigger Ca 2+ and/or phosphorylation, i.e. protein kinaseC, serine/threonine protein kinase (Akt), mitogenactivatedprotein kinases (P38), extracellular-signalregulatedkinases (ERKs), and inositol trisphosphate(IP 3 ). Among the cellular intermediates are epoxyeicosatrienoicacids (EETs), 20-hydroxyeicosatetraenoicacid (20-HETE), adenosine triphosphate (ATP), adenosine,nitric oxide (NO) and others. Finally, plasmamembrane ion channels comprise an important groupof the functional triggers mediating integration betweenneurons, astrocytes and the microvasculature. A systematicstudy of the signaling mechanisms mediatingthe functional role of astrocytes in regulating cerebralblood flow to match metabolic demand of neurons isof critical importance for understanding the mechanismregulating the cerebral circulation in differentconditions. A recent publication from Dr. Harder’s labOngoing research from CVC Facultydemonstrated that adenosine works through generationof free radicals (Gebremedhin et al., Journal ofCerebral Blood Flow and Metabolism, 2010; 30: 1777–1790). Dr. Harder also just published his 2009 WiggersAward lecture on the topic of the mechanisms ofautoregulation of cerebral blood flow (American Journalof Physiology 2011; 300(5):1557-1565).The Figure presented below was published in theWiggers Award lecture. It shows changes in morphologyof astrocytes and cerebral capillary endothelialcells when co-cultured: a) formation of capillary-likestructures (arrow) double labeled with DiI-Ac-LDL(red) and GFAP (green) in the co-culture of astrocytesand cerebral capillary endothelial cells; b) formation ofcapillary-like structures (arrow) triple labeled byPECAM-1 (green), GFAP (red), and DAPI (blue) inthe co-culture of astrocytes and cerebral capillary endothelialcells. Note the interaction between astrocytesand endothelial tubes (arrowheads in a and b); c) doubleimmunolabeling of blood vessels and astrocyteswith PECAM-1 (green) and GFAP (red) in a sectionfrom normal rat cortex showing astrocytes form footprocesses that impinge on vessels (arrows); d) cells (*)forming tube identified by DAPI staining (e) in cocultureexhibit punctate staining of connexin 43 ontheir cell-cell borders (arrow). Arrow points to areashown in the insert at higher magnification; f) cells (*)outside tubes in the same co-culture as in d show moderateto light cytoplasmic staining of connexin 43.6 Cardiovascular Center Annual Report 2011

John Imig, PhDDr. Imig works on therapeutically promising newcompounds for combating hypertension and cardiovasculardisease. With a team of Wisconsin and Texasscientists, he has recently discovered a promising newavenue that they strongly believe can be further developedto treat these diseases. The researchers are AbdulHye Khan, William B. Campbell and John D. Imig,from the Medical College of Wisconsin, and Vijaya L.Manthati, Jawahar L. Jat, and John R. Falck from theUniversity of Texas Southwestern Medical Center. Thefindings were discussed in April 2011 at the ExperimentalBiology meeting (“Novel EpoxyeicosatrienoicAcid Analogs Increase Sodium Excretion and LowerBlood Pressure in Hypertension”). Key to the researchis the role of endothelial cells, which line the inside ofthe blood vessels and the heart. Endothelial cells producearachidonic acid metabolites, a class of fatty acidsthat have biological actions beneficial for cardiovascularhealth. This production occurs through three primaryenzymatic pathways. Two of these pathways, the cyclooxygenaseand the lipoxygenase pathways, havebeen successfully targeted for the treatment of inflammation,pain, fever, and asthma. The third enzymaticpathway is the cytochrome P450 pathway that producesepoxyeicosatrienoic acids (EETs) as major biologicallyactive metabolites. EETs are endothelial-derived factorsthat significantly influence cardiovascular function.EETs can dilate blood vessels, lower blood pressureand have additional biologic actions including antiinflammatoryand anti-platelet aggregator activity. Thesebiological activities have made EETs a very attractivetherapeutic target for cardiovascular diseases.Ongoing research from CVC FacultyFor the last several years this research team has developedand synthesized an array of EET analogs, orchemical compounds that act as EETs. These EETanalogs have been tested for beneficial cardiovascularactions. For this study, 35 different EET analogs werescreened for their ability to dilate blood vessels. Thescreening produced five EET analogs that would befurther examined to determine their ability to lowerblood pressure in animal models of hypertension. Twoof the five EET analogs administered to hypertensiveanimals effectively lowered blood pressure and reducedkidney injury. This work is a major step forwardin developing novel EET analogs for the treatment ofcardiovascular disease.Elizabeth Jacobs, MD, MBADr. Jacobs and her colleagues are working on a projectwith important implications for the diagnosis andtreatment of ischemia-reperfusion (IR) lung injuries. IRlung injuries are commonly encountered clinically inconditions such as crush injury to the chest, lung transplantationand others. While the role of mitochondrialreactive oxygen species (mtROS) in IR injuries is generallyaccepted, less information is available regardinglung IR injury than cardiac or cerebral IR where thedensity of mitochondria is much higher. Dr. Jacobs willemploy an in vivo, rodent survival model of unilaterallung injury and cutting-edge imaging technology to mo-nitor and understand the role of mitochondrial complexI. Using this model, Dr. Jacobs and her colleagueshave excellent evidence that mitochondrial dysfunctiondrives lung IR injury. By varying the severity of IR andtracking changes in lung mitochondrial bioenergetics inthe first 2-72 hours, they will develop a profile whichpredicts recoverability by 7 days. Furthermore, theyintroduce two absolutely novel methods to quantifyapoptosis or an altered redox status in vivo at the bedside,which they will correlate with measures of mitochondrialfunction.The first method is in vivo SPECT/CT imaging,Cardiovascular Center Annual Report 20117

Ongoing research from CVC Facultyusing 99m Tc-duramycin to detect apoptosis. Its use inrats is demonstrated in Figure 3, which illustrates thatduramycin (DU) uptake is increased in IR lungs bysingle photon emission computed tomography(SPECT). 99m Tc-duramycin was administered throughan IV line and images acquired at steady-state. Withoutrelocation of the rat, an injection of 99m Tc-MAAwas made and rats were reimaged. 99m Tc-MAA wasused as a pulmonary perfusion marker as the particlesrange in size from 10-40 microns and lodge in the pulmonarycapillaries in proportion to flow. The Figurebelow shows representative parametric SPECT images,depicting DU uptake normalized to flow (based onMAA), of a control rat (3i) and one rat each subjectedto IR of the left lung 24 (3ii) and 48 (3iii) hours earlier.The left-right ratio of flow normalized 99m Tc-duramycinuptake was increased by ~35% 24 hours after IR andgreater than 60% 48 hours after IR (n=1 each IR, n=3control).increased mtROS which lead to apoptosis and decreasedcell survival, b) SPECT/CT and optical imagingcan detect levels of apoptosis and altered mitochondrialbioenergetics in an IR survival model in amanner which predicts subsequent viability and c) targetingmitochondria with an agent that protects mitochondrialbioenergetics in lung IR will mitigate injuryand preserve lung function as detected by SPECT/CTor optical imaging. Dr. Jacobs and colleagues will attemptto define early alterations in mitochondrial bioenergeticsand mtROS generation in a rodent model oflung IR which predict the potential of lung to recover.They will also examine the role of mtROS in cell apoptosis/survivaland lung function. The second aim is toassess apoptosis and redox ratios in left and right lungsof IR rats using SPECT/CT and optical imaging respectively,and compare these data to measurements,obtained before, to define a profile of imaging datawhich predict lung tissue survival. Finally, they will determinehow and if agents including 20-HETE or theCSA-like immunosuppressive sanglifehrin A offer protectionfrom IR and if this protection can be predictedby SPECT/CT or optical imaging. With their novelmeans to detect apoptosis and redox injury in vivo andin a survival model of IR injury, Dr. Jacobs and colleaguesare poised to use SPECT/CT and optical imagingendpoints to define the first bedside tests that distinguishin vivo apoptosis or oxidoreductive state, andthe potential of treatments to rescue “irreversible” lunginjury. These studies will provide data to predict whichlung injuries are destined to recover, and which willprogress to non-viability days later.The second novel method introduced by Dr..Jacobs and her colleagues is optical imaging to detectthe NADH/FAD (redox) ratios in intact lungs. Earlychanges in these non-destructive measurements willprovide the first data to predict later recovery or loss oflung viability. Preliminary data demonstrate prosurvivaleffects of cyclosporine A (CSA) and 20-hydroxyeicosatetraenoic acid (20-HETE); protection byeither agent depends upon an action on mitochondria.Dr. Jacobs and colleagues hypothesize that: a) ischemia-reperfusionresults in complex I dysfunction and8 Cardiovascular Center Annual Report 2011

Girija Ganesh Konduri, MDImproving our understanding and treatment of newbornbabies with respiratory failure is the overall goalof the research program in the neonatology laboratoryof the CVC. Every year, newborn babies that fail toachieve normal respiratory and cardiac function atbirth are referred to the Neonatal Intensive care unit atChildren’s Hospital. These babies are cared for by thephysicians in the Neonatology Division of the MedicalCollege of WI. The Chief of the Neonatology Division,Dr. Girija Konduri, has established a researchprogram to investigate the mechanisms of respiratoryfailure that can occur at birth. Dr. Konduri’s researchis focused on the mechanism of a condition that affectsnewborn babies called Persistent Pulmonary Hypertensionof the Newborn (PPHN). When babies are insidetheir mother’s womb, their lungs are filled with liquidand do not participate in getting oxygen into the blood.Since the fetal lungs are not respiring, they receive onlya small fraction of the blood pumped by the heart.When the baby comes out and the umbilical cord isclamped, the lungs have to fill with air and take overthe function of providing oxygen supply to the body.This requires establishing circulation of blood throughthe lungs to transport oxygen from the lungs to the baby’sorgans. In babies with PPHN, the circulation ofblood to the lungs does not get established and the babybecomes blue. The affected infants suffer from consequencesof decreased oxygen supply to vital organsand have an increased risk of death or long term disabilities.Dr. Konduri’s research is focused on understandingthe normal mechanisms by which circulationgets established in the lungs at birth and why this processdoes not occur in babies with PPHN. He foundOngoing research from CVC Facultythat constriction of an important blood vessel that connectsheart and lungs in the fetus, called ductus arteriosus,leads to development of PPHN. He also foundthat blood vessels in the lung have increased amountsof oxygen free radicals in PPHN. This research is evaluatingthe reason for the narrowing of the ductus arteriosusin the fetus and a possible approach to clearingthe oxygen free radical accumulation from the bloodvessels in the lungs of babies with PPHN. These studieswill lead to improved survival and decreased disabilityrates in affected infants.Over the last 4 years, novel observations made in Dr.Konduri’s laboratory found that the cells lining theblood vessels have dysfunction of the mitochondriathat normally consume oxygen to provide ATP, thecellular source of energy. New studies proposed by theinvestigators in the laboratory lead to the award of 4new grants from NIH over the last 18 months to studyalteration in lung biology in pulmonary hypertension.In addition to Dr. Konduri’s, two other neonatologyfaculty members are actively investigating lung biology.Dr. Ru-Jeng Teng studies oxidative stress and its effectson angiogenesis in the lung as well as tetrahydrobiopterinand the function of nitric oxide synthase inthe lung. Dr. Venkatesh Sampath’s work is describedin another section. In addition to these 3 faculty members,there are currently 4 neonatology trainees that areconducting research in the CVC neonatology laboratory.The support of the CVC with its collection of scientistsand core facilities and resources is critical to thesuccess of the overall mission of the Neonatology section.Konduri/ Sampath Labs: (back row: left to right) Ping Ye, PhD, Ru-Jeng Teng, MD, Everett Tate, Ivane Bakhutashvili, MD, PhD;(front row: left to right): Venkatesh Sampath, MD, MRCPCh, Girija Ganesh Konduri, MD, Annie EisCardiovascular Center Annual Report 20119

Meetha Medhora, PhDEffects of ionizing radiation on the lungs. Dr.Medhora’s group is identifying agents to mitigate injuryto the lungs that may occur after radiotherapy, a nuclearaccident or radioterrorism. Using a rat model,they have observed radiation to the thorax resulting inlung injury after 6-8 weeks. This recovers and is followedby fibrotic remodeling of lung tissue starting 7months after exposure. They measure acute andchronic injuries to the pulmonary vasculature. Currentlythey are testing a number of pharmaceutical reagentsin an effort to identify mitigators and treatment for theradiation lung injury. In the past year they have beenfunded to study the effect of combined injuries by radiationto the lung and skin. This is a collaborative projectbetween the Departments of Radiation Oncology,Dermatology and Medicine at MCW and the RadiationCenter at the University of Rochester.The Figure below demonstrates the effect of radiationon the lung and the mitigating effect of drugtreatment with captopril on the injury. Sections of ratOngoing research from CVC Facultylungs stained with hematoxylin and eosin show radiationinduced increase in cellularity and loss of alveolarstructure (middle panel). This injury is mitigated bytreatment with captopril started soon after irradiation.Vasoreactive and antiapoptotic actions of EETs and 20-HETE (products of the essential fatty acid arachidonicacid). The endogenous fatty acids EETs and 20-HETEhave potent vasoreactive and other properties. In collaborationwith the laboratory of Dr. Elizabeth Jacobsthey have demonstrated that EETs protect pulmonaryarteries ex vivo using vessels from three species, human,mouse and rat. 20-HETE also protects rat pulmonaryand cerebral arteries from hypoxia and reoxgygenationinjury ex vivo. Dr. Medhora and her groupare currently studying the intracellular mechanisms forprotection by these fatty acids.Effects of ionizing radiation on the lungs.Daisy Sahoo, PhDDr. Sahoo and her group are measuring HDL functionand developing a clinical assay that will revolutionizeassessment of cardiovascular risk.Despite the years of epidemiological evidence thatsupports an inverse relationship between HDLcholesterollevels and cardiovascular disease (CVD),the anti-atherogenic benefits of raising HDL levels arecurrently controversial. For example, geneticallymodifiedmice that have high HDL levels are moreprone to atherosclerosis. Further, recent large-scaleclinical studies revealed that patients who had normallevels of HDL-cholesterol still suffered cardiovascularevents. Moreover, use of a drug that nearly doubledHDL levels in a phase III human clinical trial came toan abrupt end due to high patient mortality. For thesereasons, there is now a greater emphasis on measuring“HDL function” as a better predictor of CVD than lowlevels of HDL-cholesterol.Basic scientists of the Atherosclerosis AffinityGroup of the Cardiovascular Center, Drs. KirkwoodPritchard, Hao Zhang and Daisy Sahoo, are currentlydeveloping novel in vitro assays of HDL function thathave the potential to revolutionize the way physiciansdiagnose and treat patients with cardiovascular risk.Recent studies suggest that the oxidation status ofHDL plays an important role in determining how thislipoprotein prevents or promotes atherosclerosis. Assuch, Drs. Pritchard, Zhang and Sahoo hypothesize10 Cardiovascular Center Annual Report 2011

that oxidation impairs HDL function by altering itsability to bind biomolecules (i.e. cargo) that are involvedin inflammation and HDL-dependent cholesterolmetabolism, thus making it atherogenic and unableto efficiently participate in reverse cholesteroltransport.Using biolayer interferometry (BLI), a new label-freetechnique for measuring biomolecular protein-proteininteractions, these investigators are now able to measurethe rates at which functional and non-functional(i.e. oxidized) HDL binds key proteins that are criticalfor cholesterol removal from the body. BLI analyzesthe interference pattern of light reflected from two surfaces.The first surface is the layer of immobilized proteinthat serves as an internal reference. As the numberof biomolecules binding to the biosensor increases, theaqueous protein layer increases, causing a shift in interferencethat can be measured in real-time. The OctetRed is a fully automated, 8 channel BLI instrument. Itrequires extremely small sample sizes and is highly reproducible.The design of the Octet Red is equivalentto having 8 BiaCore instruments, which use a similartechnology (surface plasmon resonance) to analyzeprotein-protein interactions. The Octet Red can measureprotein concentration (via determining rates ofantigen-antibody binding) in 96 samples at once, in lessthan 20 min and at a cost of ~30¢/sample. Furthermore,HDL binding affinity for different proteins atthe same time can be determined quickly and in realtime.The Figure below is a diagram of BLI biosensorloaded with anti-apoA-I antibody to capture HDL. ImmunocapturedHDL is then probed with specific antibodies,pro- and anti-oxidant enzymes, cholesterol metabolizingenzymes or the extracellular domains of differentreceptors and transporters.Ongoing research from CVC FacultyAlready, preliminary data demonstrate that oxidizedHDL (generated in vitro) directly impacts its ability tobind proteins associated with inflammation and cholesteroltransfer as compared to HDL that was not oxidized.More importantly, HDL isolated from hypercholesterolemicmice displayed increased binding affinitiesfor pro-oxidant enzymes compared to HDLisolated from healthy mice. Therefore, these data providestrong support for the concept that BLI assays areable to distinguish between control HDL and HDLfrom established murine models of atherosclerosis.Drs. Pritchard, Zhang and Sahoo have recently submittedan R01 grant proposal to the National Institutesof Health to support the continuation of these highlyinnovative studies. The objectives of this proposal areto determine how oxidized lipid and protein compositionof reconstituted HDL influences HDL’s interactionswith key biomolecules as cholesterol is transportedfrom the arteries to the liver for disposal. Thesestudies will be performed in parallel using cell cultureassays and BLI methodologies. Critically, BLI assayswill be used to analyze samples from healthy patients,as well as those with clinically-documented atherosclerosis,to determine if and the extent to which atherosclerosisimpairs HDL interactions with biomoleculesthat mediate HDL metabolism. Together, these excitingstudies will, for the first time, test the hypothesisthat HDL binding affinity can be used to distinguishbetween functional and dysfunctional HDL.Successful completion of the above studies will laythe foundation for the development of new clinical assaysfor determining HDL “function”. The BLI assayswill allow us to perform large clinical studies to fullytest the idea that dysfunctional HDL is a better indicatorof atherosclerotic risk. Further, these standardized,validated, accurate and robust assays have high diagnosticpotential as they can be performed quickly, andin a cost-effective manner in a clinical laboratory setting.As large-scale application of our assays holdsgreater promise for predicting CVD than HDLcholesterollevels, physicians may be able to preventcardiovascular events in patients with normal HDLcholesterolbecause, for the first time, they will be ableto identify which patients have dysfunctional HDL.Cardiovascular Center Annual Report 201111

Venkatesh Sampath, MD, MRCPChOur laboratoryseeks to understandthe role of innateimmune signalingin the pathogenesisof diseases ofpremature infants.Our research focuseson how lipopolysaccharide(LPS)mediated endothelialinjury contributesto vascular remodelingin bronchopulmonarydys-Venkatesh Sampath, MD, MRCPChplasia (a chronicform of inflammatory lung disease in premature infants).We have identified NADPH oxidase (Nox) as acritical regulator of LPS mediated endothelial injuryusing in vitro techniques. We propose to expand onthis research by identifying the specific isoforms ofNox that mediate LPS-dependent endothelial toxicityand examining the biological mechanisms underlyingNox-dependent endothelial injury and microvascularOngoing research from CVC Facultyremodeling. This work is supported by a R03 grantfrom the National Institutes of Child Health and Development(NICHD) entitled “LPS-mediated oxidativestress and pulmonary vascular injury in bronchopulmonarydysplasia”.On the translational aspect, we are investigatingwhether functional genetic variaton in the Toll-like receptorspathway genes (innate immune genes) modulatesusceptibility to bronchopulmonary dysplasia andother diseases of the preterm infants. Our preliminarydata suggest that loss of function polymorphisms in thispathway does increase susceptibility to lung disease inpremature infants. This research is funded by aNIEHS Children’s Environmental Health Sciencespilot award entitled “TLRs-modulators of environmentallung injury in premature infants”, which will determinewhether TLR/Nrf2 genetic variation alters susceptibilityto oxidative stress and susceptibility to bronchopulmonarydysplasia in preterm infants.Jennifer Strande, MD, PhDDr. Strande researches the role of thrombin and itsreceptors in the inflammation and fibrosis that occursin the heart after ischemic events. While reducing risksusing preventive strategies is important in combattingheart disease, we still need new treatment strategies forprevention of the adverse effects of heart disease.The clotting pathway culminates in the formation ofthrombin, which is best known for acting on its cellularreceptors to cause platelet aggregation and thrombosis.One of the primary treatments strategies for patientswith coronary artery disease is to acutely stop this process,in the condition of acute myocardial infarction, orto chronically inhibit this process to prevent recurrentheart attacks. Thrombin also acts through its receptorson other cell types and organs such as liver, lung andkidney to cause effects such as inflammation and fibrosis.Inflammation and fibrosis contribute to left ventricularremodeling and eventually cardiomyopathy.Dr. Strande has shown that in rats treated with eitherdirect thrombin inhibitors or thrombin receptor antagonists,the heart is protected against ischemia by a reductionof the damaging effects of free radicals andinflammation. Furthermore, she has shown that in longterm studies, a thrombin receptor antagonist also limitscardiac fibrosis and protects the heart from adverseremodeling after myocardial infarction. As a physician,Dr. Strande sees first-hand how heart damage resultingfrom heart attacks affects the mental and physical wellbeingof patients and their families. By recognizing thegaps in treatments for people affected by heart attacksand heart failure, she can identify new targets for potentialtherapies. She also has the basic research trainingto perform the research studies needed to advancethese new potential therapies which may have amajor impact on keeping patients with cardiovasculardisease feeling better.The Figure below shows typical photographs of myocardialslices from three control (top row) and three12 Cardiovascular Center Annual Report 2011

Carol Williams, PhDAtherosclerosis and its complications cause almostone million deaths per year in the United States. Atherosclerosisresults from excessive proliferation and migrationof vascular smooth muscle cells, which promotesplaque formation and blockage of blood flow inmajor vessels. The Williams’ laboratory is investigatingnovel ways to inhibit the proliferation and migration ofvascular smooth muscle cells, which will help definenew ways to prevent and treat atherosclerosis.Dr. Williams and her team are focusing their researchon a group of proteins called “small GTPases”.Small GTPases are emerging as therapeutic targets inatherosclerosis, because these proteins promote themigration and proliferation of vascular smooth musclecells. There are many different types of smallGTPases, including the proteins known as Rho, Rac,Ras, and Rap. Since there are so many different typesof small GTPases, inhibiting the activity of only onetype of small GTPase might not be enough to stop theexcessive proliferation and migration of vascularsmooth muscle cells during atherosclerosis. Instead,simultaneously blocking the activity of multiple smallOngoing research from CVC FacultyGTPases at one time might be a better way to inhibitatherosclerosis. The Williams’ laboratory is investigatingthis new strategy of simultaneously inhibiting multiplesmall GTPases at one time, by conducting researchon the unique protein known as SmgGDS.SmgGDS can interact with multiple small GTPases,including Rho, Rac,Ras, and Rap. This unique featuremakes SmgGDS a master regulator of these smallGTPases. The Williams’ laboratory is testing the hypothesisthat blocking the activity of SmgGDS in vascularsmooth muscle cells will block the activity of allsmall GTPases in these cells, and diminish the migrationand proliferation of vascular smooth muscle cells.The Figure below illustrates how vascular smoothmuscle cells make many types of small GTPases, includingRho, Rac, Ras, and Rap. These small GTPasescan promote the proliferation and migration of vascularsmooth muscle cells, which can promote atherosclerosis.SmgGDS is a unique protein that regulatesall of these small GTPases. Stopping the interaction ofSmgGDS with small GTPases might offer a new therapeuticapproach to prevent and treat atherosclerosis.14 Cardiovascular Center Annual Report 2011

2CVC Advisory Board and PhilanthropyThe general mission of the CVC Advisory Board is to serve as advocates in the community,thereby supporting the primary objectives of the CVC. These objectives are to contributeto the eventual elimination of heart disease, stroke and other cardiovascular diseases ashuman health problems through coordinated research, clinical treatment, control and education,and to provide the citizens of Southeastern Wisconsin with state-of-the-art therapy,research, control and educational programs for all aspects of cardiovascular disease. Thespecific purpose of the CVC Advisory Board is to provide financial support for the researchand clinical programs carried on by the CVC and to educate the community in cooperationwith the Board of Trustees of The Medical College of Wisconsin on the causes, prevention,diagnosis and treatment of cardiovascular disease. Bruce E. Jacobs, a lifetime member ofthe CVC Advisory Board, was the Founding Chair. Other lifetime members include ByronFoster, Gordon Gunnlaugsson and William H. Levit, Jr.Current composition of the BoardChair:Nancy J. SennettVice-chair:Johan C. R. SegerdahlMembers:Ned W. BechtholdPriscilla BoelterCharles P. BrumderMarybeth BudischCellene ByrneKristine Cleary, Esq.Dominic ColonnaFrancis R. CroakMichael J. CudahyGael Garbarino CullenMichael S. ErwinByron T. FosterFrederic G. FriedmanEllen GlaisnerLinda Gorens-LeveyEckhart GrohmannBarry GrossmanGordon H. GunnlaugssonStanley F. HackCristina Hernandez-MalabyMikel HoltBruce E. JacobsRobert H. JenkinsSarah Wright KimballJohn KirchgeorgDr. Vincent KuttemperoorWilliam H. Levit, Jr.William M. MaslowskiDaniel F. McKeithan, Jr.Daniel M. MuchinJeffrey J. NohlJohn K. SchultzBruce M. SmithSonia Shields StoweNicholas C. WilsonKenneth F. YontzGary V. Zimmerman, FAIAEmeritus Members:James D. BellWilliam D. BrowneJohn J. Burke, Jr.Cardiovascular Center Annual Report 201119

Cullen runThis event supports heart research at the Cardiovascular Center. To date, Cullen Run/Walk proceeds havehelped several research programs, including studies to identify genetic risk factors of heart disease, to investigatethe Female Athlete Triad and its potentially life-threatening link to heart disease, and to better understandthe correlation between plaque buildup and coronary artery disease. The Cullen Run/Walk is an 8-kilometerUSA Track and Field competitive run and a 2-mile fun run/walk following a scenic path along UnderwoodParkway in Wauwatosa. Sponsored by the Cullen family and the Badgerland Striders, the event includes famousCullen Family & Friends Chili, refreshments, food, door prizes, an awards ceremony and live music forthe entire family. The event is held in memory of Steve Cullen, a former Milwaukee alderman, who died in1995 at age 40 of sudden cardiac arrhythmia. His father, at age 41, and two brothers, ages 53 and 51, also diedof heart disease. The Cullen Run/Walk has grown in attendance by over 500% since its inception, encouragingheart healthy lifestyles for all participants, their families and friends and contributing over $120,000 to cardiovascularresearch and awareness.

Geurts’ lab ( from back to front): Jason Klotz, Bradley Endres, Molly Corbett, Sheila Reagles, Michael Grzybowski, Sheng Yang, PhD8701 Watertown Plank Road Milwaukee, WI 3226 414-456-8296