Oral Presentations - Arteriosclerosis, Thrombosis, and Vascular ...

Species Differences in Endothelial Cell Injury Due to X-rays
Linda Hiebert, Pat Thomas, Tilly Ping, Mark Wickstrom, Bliss Tracy. University of
Saskatchewan, Saskatoon, Canada; Health Canada, Ottawa, Canada
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Our previous studies have demonstrated a marked species difference in response of cultured
endothelial cells to free radical injury. Bovine aortic endothelial cells were much more resistant
to injury from hydrogen peroxide (H 2O 2) and xanthine/xanthine oxidase than porcine aortic
endothelial cells (6 vs 0.25 mM H 2O 2 for fifty percent viable cells for bovine vs. porcine cells).
To determine if this same species difference could be demonstrated in response to radiation
injury, porcine and bovine aortic endothelial cells were exposed to increasing doses of x-rays.
Twenty-four hours later, death of irradiated cells was assessed by measuring percent viable
cells and total live cell number by trypan blue exclusion and the release of lactate
dehydrogenase (LDH) into cell medium. Clonogenic survival was determined by measuring the
survival fraction, i.e., the percentage of survivors forming colonies after 14 days. Surprisingly,
bovine cells were more sensitive than porcine cells to x-ray injury when slopes of the
dose-response curves were compared. The decrease in percent viable cells and live cell
number was 2.3–3.4 times greater for bovine vs. porcine cells and increase in LDH release was
8 times greater for bovine vs. porcine cells. Clonogenic survival (25%) was similar at a dose
of 4 Gy with porcine cells being more sensitive at lower doses and less sensitive at higher
doses than the bovine cells. These results suggest that these species differ in their ability to
resist and repair different types of free radical damage; bovine cells may have enhanced
amounts of cytoplasmic free radical scavengers, giving them resistance to damage from
diffusible H 2O 2compared to porcine cells. However damage from x-rays is concentrated along
specific tracks, to which porcine cells appear more resistant. While clonogenic survival curves
are somewhat similar, a species difference still exists, which may reflect differing abilities to
repair DNA damage. These results demonstrate that response to free radical injury depends on
the mode of free radical delivery and cannot be extrapolated between species.
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Inhibition of Neointima Formation by Co-Expression of Antisense Thrombin
Receptor Gene and p21waf1
Liguom Mi, Xianmin Meng, Xiuwen Zhao, Dongqing Liu, Jinfeng Ding, Runlin Gao. Molecular
Medicine Center for Cardiovascular Diseases, Cardiovascular Institute & Fu Wai Hospital,
Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China
Associated-adenovirus (AAV) mediated-gene transfer of antisense thrombin receptor gene
(ATR) or p21waf1 has shown to inhibit the proliferation of vascular smooth muscle cells
(VSMCs) and neointima formation in balloon-injured porcine coronary arteries. It is suggested
that co-expression of ATR and p21waf1 (AP) would enhance the effect by adjusting the
expression of thrombin receptor gene and p21waf1 in VSMCs. Here we constructed and
packaged the AAV vectors of expressing ATR (rAAV/ATR), p21waf1 (rAAV/p21), and coexpressing
ATR and p21waf1 (rAAV/AP). The cultured human aorta smooth muscle cells
(ASMCs) were infected with the rAAVs or control vector (rAAV/GFP) respectively, and the effects
of the genes were evaluated by MTT assay and flow cytometry. The rate of survival cells of
co-expression of ATR and p21waf1 was decreased (68.5% vs. 85.6% and 82.0%, AP/GFP vs.
ATR/GFP and p21/GFP), and the apoptosis cells were increased (8.24% vs. 4.84% and 5.17%,
AP vs. ATR and p21) at 48 hours after the gene transfer. In the balloon-injured rat carotid artery
model, both neointima formation and VSMCs of media were significantly inhibited by
co-expressing ATR and p21waf1 as compared with the expression of GFP (control) on days14
and 28 after the gene transfer (table). These data show that co-expression of ATR and p21waf1
can enhance the inhibitory effect of ATR or p21waf1 on proliferation of VSMCs and neointima
formation. This may have implications for developing strategies of gene therapy for restenosis.
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Reversal of Tissue Kallikrein Up-Regulation by Revascularization of Lower
Limb Ischemia
Costanza Emanueli, Paolo Porcu, Paolo Madeddu. Cardiovascular Medicine and Gene
Therapy Section National Laboratory INBB, Osilo, Italy; Vascular Surgery, University of
Sassari, Sassari, Italy
BACKGROUND: Tissue kallikrein (tK) and vascular endothelial growth factor (VEGF) are potent
angiogenic factors. Up-regulation of tK or VEGF was documented in animal models of acute
ischemia and impairment in endothelial growth factor surge has been report ed in animal
models of atherosclerosis. Yet, it remains unknown whether these endothelial cell mitogens are
modulated in patients with chronic peripheral vascular insufficiency and whether the levels of
expression correlate with clinical grading, collateral development, and revascularization. AIM:
To evaluate the expression of endothelial growth factors in the context of peripheral ischemia.
METHODS AND RESULTS: Circulating tK and VEGF were measured in 36 patients with
symptomatic peripheral vascular disease beforeDownloaded and after surgical from
revascularization. In 6
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patients without symptoms at rest, tK was assayed following exercise stress test. VEGF levels
fell within the normal range in all patients (9611 vs. 10913 pg/mL in healthy controls,
PN.S.) and remained unchanged after revascularization. In contrast, tK expression was
upregulated in 34 out 36 patients (1,107203 vs. 8510 pg/mL in controls, P0.05), with
no further increase after exercise. No correlation was found between growth factor expression
and clinical grading. However, TK levels in the venous effluent of ischemic limbs positively
correlated with the number of angiographically recognizable collateral vessels (r0.72,
P0.001). Follow-up studies documented reversal of tK upregulation following revascularization
(P0.01), whereas no change was observed in venous samples from untouched legs.
CONCLUSIONS: Induction of tK could represent a compensatory response to chronic arterial
insufficiency, attempting to maintain an adequate tissue perfusion. Potentiation of this
mechanism may have important implications to therapeutic angiogenesis in limb ischemia.
Effect of Factor VIII on Tissue Factor-Initiated Spatial Clot Growth
Mikhail V Ovanesov, Euguene L Saenko, Fazoil I Ataullakhanov. National Research Center
for Hematology, Moscow, Russia; American Red Cross, Rockville, MD
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Intrinsic coagulation factor VIII (fVIII) is an essential component of blood coagulation cascade.
Inherited low levels of fVIII in plasma (Haemophilia A) are associated with bleeding tendency
whereas elevated levels of fVIII are likely to be an independent risk factor for venous
thrombosis. To gain an insight into the role of fVIII in clot formation, we used the novel in vitro
experimental system modelling the physiological spatial conditions in proximity of the damaged
blood vessel wall. Clotting was studied in recalcified plasma freshly drawn from normal donors
(N12) or patients with severe haemophilia A (N23) and supplemented with purified human
fVIII. The light scattering produced by TF-initiated clots formed on fibroblasts was recorded in
a thin layer of nonstirred plasma using time-lapse video microscopy, which allows to monitor
temporal evolution of the clot shape and to determine the clot growth rate. The initial kinetics
of clotting in a 0.2 mm-area near the activator surface was similar in both normal and
haemophiliac plasmas. However, the rate of spatial clot growth (from 0.2 to 1.7 mm width) was
approximately three times slower in haemophiliac plasmas than in normal ones. The clot
growth rate in haemophiliac plasmas increased from 16.11.7 up to 44.92.5 m/min
(normal value) with increasing fVIII concentration from 0.0001 to 0.1 UI. Remarkably, normal
clot formation was restored by addition of fVIII to 5 - 10 %, which is the threshold level defining
requirement for preventive treatment of Haemophilia A. At higher concentrations, fVIII had a
thrombogenic effect, since haemophilic plasmas tended to spontaneously coagulate. These
results support the hypothesis that the intrinsic, fVIII-dependent pathway is responsible for
expansion of the clotting area under conditions when TF remains cell membrane bound but not
present in plasma in its free form. Our experimental approach provides direct evidence of the
critical role of the intrinsic pathway in progression of spatial clot growth
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Functional Interactions between Hormone Nuclear Receptors Bound to a
Newly Identified HRE on the Hepatic Control Region-1 and an HRE on the
Proximal ApoC-II Promoter Account for the Transactivation of the ApoC-II
Promoter by HNF-4 and RXR/FXR Heterodimers in Response to Bile
Acids
Dimitris Kardassis, Anastasia Roussou, Paraskevi Papakosta, Costas Boulias, Iannis
Talianidis, Vassilis I Zannis. Department of Basic Sciences, University of Crete Medical
School, Heraklion, Crete, Greece; Institute of Molecular Biology and Biotechnology, FORTH,
Heraklion, Crete, Greece
We have shown previously that the hepatic control region-1 (HCR-1) enhanced the activity of
the human apoC-II promoter in HepG2 cells. This enhancement was mediated by two hormone
response elements B (-102/-81) and C (-156/-116) present in the apoC-II promoter that bind
specifically HNF-4 and RXR/T3R heterodimers respectively. We now report that HCR-1
mediates induction of the apoC-II promoter by chenodeoxycholic acid (CDCA), a natural ligand
of farnesoid X receptor (FXR). In addition, treatment of HepG2 cells with CDCA resulted in
significant induction (3.5-fold) in the steady state apoC-II mRNA levels. In contrast, HCR-1 could
not mediate induction of the apoE promoter by CDCA. Activation of the HCR-1/apoC-II promoter
by CDCA required the binding of RXR/FXR heterodimers to a novel hormone response
element present in HCR-1 which also binds the orphan nuclear receptors HNF-4 and ARP-1.
RXR/FXR heterodimers in the presence of CDCA as well as HNF-4 strongly transactivated the
HCR-1/apoC-II promoter whereas overexpression of ARP-1, which also binds to the element C,
abolished this transactivation. RXR/FXR heterodimers in the presence of CDCA also
transactivated an apoC-II promoter fused with the novel HRE of the HCR-1, whereas mutations
in this HRE which abolished binding of RXR/FXR heterodimers also abolished transactivation
by these nuclear receptors in the presence of CDCA. Finally, the transactivation of the
HCR-1/apoC-II promoter by RXR/FXR in the presence of CDCA was abolished specifically by
mutations in the hormone response element C of the apoC-II promoter. The findings a) establish
functional interactions between hormone nuclear receptors bound to element C of the apoC-II
promoter and the HRE of the HCR-1, and b) suggest that the HCR-1, in addition to its role as
a strong hepatic transcriptional enhancer of genes of the apoE/C-I/C-IV/C-II cluster, also serves
as a mediator of expression of the apoC-II gene in response to bile acids.
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The Region Comprising Kringle V and the Protease Domain is Critical for
the Binding of Human Apolipoprotein(a) to Fibronectin
Celina Edelstein, Olga Klezovitch, Angelo M Scanu. University of Chicago, Chicago, IL
In previous studies we have shown that the C-terminal domain (F2) of apolipoprotein(a),
apo(a),but not its N-terminus (F1), binds in vitro to fibrinogen and to the proteoglycans, decorin
http://atvb.ahajournals.org/ and biglycan, by both guest in their on April glycated4, and 2013 non-glycated forms. Fibronectin is also a component