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

P152 WITHDRAWN
Oxidized Phospholipids Inhibit Angiogenesis In Vitro but Stimulate
Angiogenesis In Vivo: Potential Influence of Monocyte/Macrophages
Karol E Watson, Leslie A Caromile. UCLA School of Medicine, Los Angeles, CA
P153
Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) are a group of
oxidized phospholipids which have been shown to be the active compounds in minimally
oxidized LDL (MM-LDL). Emerging evidence suggests that lipoproteins may modulate
angiogenesis, we therefore tested the ability of Ox-PAPC to affect parameters of angiogenesis.
In our studies, Ox-PAPC inhibited in vitro proliferation, migration and tube formation of
endothelial cells. Adding exogenous recombinant VEGF at 100ng/ml restored EC proliferation
and migration to baseline. To confirm inhibition of angiogenesis by Ox-PAPC we performed an
in vivo angiogenesis assay. Surprisingly, Ox-PAPC stimulated angiogenesis in vivo (figure 1).
Further studies in vitro demonstrated that Ox-PAPC also stimulated VEGF production by
monocyte/macrophages. We conclude that Ox-PAPC directly inhibits angiogenesis in vitro, but
that in vivo this is overcome by the stimulation of VEGF produced by monocyte/macrophages.
These results may explain the co-localization of both atherosclerosis and angiogenesis in vivo
and form the basis for neovascularization of atherosclerotic plaques.
P154
Regulation of Class A Scavenger Receptor-Mediated Cell Adhesion by Gi/o Signaling Pathways
Steven R Post, Cecelia Gass, Stuart Rice, Dejan Nikolic, Reto Asmis. University of Kentucky,
Lexington, KY
Class A macrophage scavenger receptors (SRA) are multifunctional receptors with roles in
modified lipoprotein uptake and cell adhesion. Although not typically associated with a
signaling function, incubation of macrophages with acetylated LDL (AcLDL) was shown to
activate several intracellular signaling cascades. Activation of these signaling cascades by
AcLDL was inhibited by pertussis toxin (PTX) indicating that SRA activates a G i/o protein.
Recently, we reported that in mouse peritoneal macrophages (MPM), G i/o activation positively
regulated SRA-mediated lipoprotein uptake. The finding that SRA-mediated lipoprotein uptake
was PTX-sensitive led us to hypothesize that SRA-mediated cell adhesion might be similarly
regulated. Because macrophages express multiple classes of scavenger receptors, we
expressed SRA in HEK cells, cells that lack endogenous SRA expression. Similar to our results
using MPM, we found that SRA-mediated AcLDL uptake by transfected HEK cells was reduced
by 35% following PTX treatment. Using a standard adhesion assay in the presence of serum,
we found that cells expressing SRA displayed enhanced cell adhesion which was reduced by
42% following PTX treatment. The ability of the SRA antagonist polyinosine to reduce cell
adhesion to the level observed with HEK cells lacking SRA confirmed that the increased cell
adhesion was mediated by SRA. To rule out the potential involvement of integrins, adherant
cells were incubated in Ca 2 -free media. Under these conditions, 72% of cells that expressed
SRA remained attached, whereas only 4% of the cells that lacked SRA remained attached.
However, following PTX treatment only 36% of SRA expressing cells remained attached.
Because the actin cytoskeleton plays an active role in cell adhesion, we used phalloidin to stain
F-actin in HEK cells. We found that cells expressing SRA developed a phenotype characterized
by fine filopodial extensions; a phenotype similar to that observed in primary macrophages and
in cells transfected with activated Cdc42. Overall, our results demonstrate that the ability of
SRA to enhance cell adhesion is PTX-sensitive and correlates with changes in the actin
cytoskeleton.
P155
Pulse Pressure: Is it an Age-Independent Predictor of Mortality in Patients
with Angiographic Coronary Disease?
Keshav Chander, James S Zebrack, Tami L Blair, Robert R Pearson, Joseph B Muhlestein,
Benjamin D Horne, Jeffrey L Anderson. University of Utah, Salt Lake City, UT; LDS Hospital,
Salt Lake City, UT
Objective: Wide pulse pressure (PP) has been proposed as a stronger predictor of mortality than
systolic (SP) or diastolic blood pressure (DP). PP is known to widen with age; and its prognostic
value independent of age is uncertain. We evaluated its predictive value for mortality; alone,
and together with age and other standard riskDownloaded factors in a large, from
prospectively followed
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Poster Presentations a-27
population with documented coronary artery disease (CAD). Methods: We prospectively studied
1370 consecutive patients with angiographically defined CAD (1 stenosis 70%). Baseline
pulse pressure together with other standard risk factors was recorded. During 2.8Â1.2 years
of follow-up, 173 patients died (12.6%). Predictors of mortality were assessed using Cox
regression analyses. Findings: In univariate analysis, PP was a highly significant predictor of
mortality (relative hazard [RH]1.10, 95% CI1.03 - 1.18 / 10 mmHg,p0.005). In
multivariate analysis with SP and DP, the predictive value of PP improved (RH1.29 [1.11 -
1.50] / 10 mmHg, p0.0008). PP correlated moderately with age (Pearson†s correlation
coefficient 0.381, p.001). When forced into bivariate survival analysis, age (RH1.87 [1.56
- 2.24] / decade, p.0001), but not PP (RH0.99[0.92 - 1.07]) was significantly predictive of
mortality. Multivariate analysis (stepwise, backward conditional) including PP with the standard
risk factors of age, sex, diabetes, history of [h/o] hyperlipidemia, h/o hypertension, h/o
smoking, and family history was performed. The age(p.0001), h/o hyperlipidemia (p.0006),
and diabetes (p.002), but not PP came out as independent predictors. Conclusion: PP is a
strong univariate predictor of mortality in patients with CAD. However, it was not found to be
an independent predictor when adjusted for age in our population. Thus, the independent
predictive value of PP remains to be established for secondary risk assessment.
P156
Oxidized Phospholipids Inhibit Inflammatory Effects of Endotoxin Both In
Vitro and In Vivo
Valery N Bochkov, Alexandra Kadl, Joakim Huber, Florian Gruber, Bernd R Binder, Norbert
Leitinger. Inst. of Vascular Biology and Thrombosis Research, Univ. of Vienna, Vienna,
Austria
Minimally modified LDL and their active components - oxidized phospholipids - are known to
regulate expression of inflammatory genes in endothelial cells (EC). Oxidized 1-palmitoyl-2arachidonoyl-
sn-glycero-3-phosphorylcholine (OxPAPC) is known to up-regulate tissue factor
and interleukin-8 in human EC, but on the other hand to suppress induction of E-selectin after
stimulation of EC by LPS or TNF. In this work, we have studied mechanisms and in vivo
relevance of this inhibitory action. We have found that OxPAPC only partially inhibited elevation
of E-selectin levels in TNF or IL-1-stimulated human umbilical vein endothelial cells. In
contrast, OxPAPC completely blocked LPS-induced up-regulation of E-selectin, ICAM-1 and
VCAM-1. The inhibitory effect of OxPAPC could not be overcome by raising LPS concentration
100-fold above saturation, suggesting that the mechanism of inhibition is more complex than
competition between OxPAPC and LPS for the LPS receptor(s). The anti-endotoxin effect of
OxPAPC was not mimicked by unoxidized PAPC, lysoPC or arachidonic acid. OxPAPC inhibited
activation of the NFB pathway, namely LPS-induced phosphorylation and degradation of IB,
activation of p65/DNA binding and stimulation of 5xNFB-luciferase reporter construct. In
C57BL/6 mice injected intraperitoneally with LPS, simultaneous injection of OxPAPC inhibited
accumulation of blood-borne cells in the peritoneal cavity, expression of E-selectin mRNA in
peritoneal tissue, heart and aorta, and blocked oedema formation in peritoneal tissue. Finally,
we have found that OxPAPC significantly reduced lethality in mice injected with high doses of
LPS. We hypothesize that oxidized phospholipids accumulating at the sites of acute bacterial
inflammation due to high concentrations of reactive oxygen species generated by neutrophils,
can function as a negative feedback to down-regulate the process of acute inflammation. Thus,
oxidized phospholipids represent a novel class of compounds with anti-endotoxin activity which
potentially can be used for treatment of Gram-negative sepsis.
P157
Heterodimerization of the and Isoforms of the Human Thromboxane
Receptor
Paul Sullivan, Emer M Smyth. University of Pennsylvania, Philadelphia, PA
Two splice variants of the human thromboxane receptor (TP) have been identified. TP and
TP are 89% homologous, have similar ligand binding and signaling properties but
demonstrate divergent patterns of sequestration. Heterodimerization of highly homologous G
protein-coupled receptors has been reported, but has not been tested for TP/TP. HEK 293
cells were stably transfected with hemagglutinin (HA) tagged TP (HEK-TP), myc tagged TP
(HEK-TP) or both (HEK-TP/), and heterodimerization examined. Protein complexes were
cross-linked (2mM DSP, 20 min), cell membranes prepared and solubilized in 2% digitonin and
TP immunoprecipitated with an anti-Myc antibody. Immunoprecipitates were resolved by
SDS-PAGE and HA-TP revealed with an HRP-anti-HA antibody. HATP was present in
anti-Myc immunoprecipitates from HEK-TP/ , but not HEK-TP, demonstrating that
co-expression of TP with TP led to the formation of TP/ heterodimers. TP signaling was
examined by measurement of inositol phosphate (InsP) generation. IBOP, a TP agonist,
stimulated InsP generation in all three cell lines (Table 1). The isoprostane iPF2-III, a free
radical catalyzed by guest product on April of arachidonic 4, 2013 acid present in atherosclerotic plaque, mediates