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

macrophages phagocytosing apoptotic cells, LO activity seemed not to be obligatory for the
response to OxLDL (or mmLDL). Most importantly, macrophages pre-incubated with mmLDL
exhibited a decreased ability to phagocytose apoptotic thymocytes. We speculate that OxLDL
and mmLDL carry oxidized lipids similar to the products of 12/15-LO catalysis. In turn, these
oxidized lipids promote exhausting actin polymerization in macrophages, interfering with
subsequent phagocytic activity. These findings could provide part of the explanation as to why
apoptotic cells are not efficiently cleared from atherosclerotic tissue.
Induction of Monocyte Chemotactic Activity by Leptin
Farhad Parhami, Jason Hwang, Susan Hama, Mohamad Navab, Yin Tintut, Linda L Demer.
UCLA Division of Cardiology, Los Angeles, CA
Leptin, the product of the ob gene, regulates food intake, energy expenditure, and other
physiological functions of the peripheral tissues. We have previously demonstrated leptin
receptor in mouse artery wall, leptin protein in atherosclerotic lesions of ApoE null and LDL
receptor null mice, and osteoinductive effects of leptin on calcifying vascular cells. Since
human monocytes express leptin receptor(s), and leptin causes the activation and proliferation
of these cells, we investigated other effects of leptin on human peripheral blood monocytes that
may impact atherogenesis. Treatment of these cells with 1 g/ml human recombinant leptin
resulted in formation of structures resembling lamellipodia of migratory cells. Furthermore,
leptin (0.5–4 g/ml) caused a dose-dependent increase in monocyte chemotactic activity in a
Boyden chamber chemotaxis assay (control2.50.5; leptin: 0.5 g/ml5.21; 1 g/
ml7.50.5; 2 g/ml11.52; 4 g/ml19.03 monocytes/field SD; p0.05 for
control vs. all leptin concentrations). Western blot anaylsis of STAT3 activation in monocytes
showed no induction of STAT3 phosphorylation by 1–4 g/ml leptin after 5–30 minutes of
treatment, whereas the positive control IL-6 (50 ng/ml) induced STAT3 activation in those cells.
In addition, cultured human peripheral blood monocytes expressed leptin mRNA and protein as
assessed by Northern blot analysis and ELISA, respectively. These results show that leptin may
be a chemotactic factor for monocytes, and its presence in the atherosclerotic lesion may
further promote monocyte transmigration into the artery wall. We hypothesize that this
observation may also at least in part explain the previously reported resistance of leptin and
leptin receptor deficient mice to atherosclerotic lesion formation.
Evidence for Involvement of the Plasminogen Activator System in the
Aetiology of Plaque Destabilization and Rupture
Kevin G Carson, Aernout Luttun, Helen Williams, Peter Carmeliet, Christopher L Jackson.
Bristol Heart Institute, University of Bristol, Bristol, UK; Center for Transgene Technology and
Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium
The brachiocephalic artery of the apolipoprotein E knockout mouse (apoE-/-) has been
established as a site of predilection for atherosclerotic plaque rupture. The aim of this study
was to test the hypothesis that the plasminogen activator system may be involved in the
process of plaque destabilization and rupture. Double knockout mice, deficient in the genes for
apoE and Plasminogen Activator Inhibitor-1 (apoE-/-:PAI-1-/-) on a mixed C57Bl6/129SvJ strain
background were created by intercrossing PAI-1-/- and apoE-/- mice. Eleven of these
apoE-/-:PAI-1-/- mice and twelve apoE-/- controls were fed a high fat diet, and after 25 weeks
they were terminated and perfused. Serial sections were taken throughout the length of the
brachiocephalic artery. These were examined for the presence of plaque ruptures, and
additionally, the thickness of the fibrous cap, the proportion of the plaque occupied by the lipid
core, and the number of fibrous layers within the plaque (previous healed ruptures) were also
determined. There were significantly more plaque ruptures in the apoE-/-:PAI-1-/- mice than
in the apoE-/- controls (6 out of 11 compared to 1 out of 12, p 0.027, Fisher’s exact test).
Furthermore, in those that had plaque ruptures, the lipid core occupied a significantly greater
proportion of the plaque than in those without (44 /- 3 % compared to 33 /-3%,p 0.01,
unpaired t-test with Welch correction), and there were significantly more fibrous layers within
the plaque (4.9 /- 0.3 compared to 3.4 /- 0.5, p 0.02, unpaired t-test with Welch
correction). These results suggest that the plasminogen activator system plays an important
role in the process of plaque destabilization and rupture. They also demonstrate that this model
has important correlates with human lesions, in that the proportion of the plaque occupied by
lipid and the presence of previous healed ruptures both act as markers of instability.
Plasmin-Induced Gene Expression: Possible Mechanisms and Potential
Implications
Usha R Pendurthi, Samir Mandal, Elizabeth Crump, Mylinh Ngyuen. UT Health Center at
Tyler, Tyler, TX
Proteases involved in coagulation and fibrinolysis play an important role in pathophysiology,
such as wound healing, tissue remodeling and tumor metastasis. Our recent studies show that
a number of proteases involved in clotting and fibrinolysis, such as VIIa, thrombin and plasmin,
induced the expression of Cyr61, a gene that encodes extracellular matrix signaling protein that
was shown to regulate cell migration and proliferation, in fibroblasts. In the present study, we
have investigated possible mechanisms by which plasmin upregulates Cyr61 gene expression
and the ability of plasmin to induce cell proliferation. To evaluate a posssible involvement of
specific protease activated receptors (PARs) in plasmin-mediated gene induction, we investigated
the ability of plasmin to induce Cyr61 expression in mouse fibroblasts that lack (or
express) specific PARs. Plasmin effectively induced Cyr61 gene expression in both wild-type
and PAR-2 deficient cells but not in PAR-1 deficient cells. Transfection of PAR-1 deficient cells
with PAR-1 plasmid conferred plasmin responsiveness to these cells. However, in addition to
PAR-1, an additional cell associated factor may be necessary for plasmin-induced gene
expression since COS-7 cells, which has functional PAR-1 and PAR-2, failed to respond to
plasmin. PD 98059, a specific inhibitor of p44/42 Downloaded MAPK, inhibited plasmin-induced from
Cyr61 gene
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expression. Consistent with this, plasmin is shown to induce p44/42 MAPK phosphorylation in
fibroblasts. Plasmin (up to 100 nM) failed to induce cytosolic calcium mobilization in WI-38
cells. In additional studies, we investigated the effect of plasmin on fibroblast proliferation using
3H-thymidine incorporation assays. The data show that plasmin (50 nM) increased 3Hthymidine
incorporation by 3-fold. In conclusion, our data suggest that plasmin-induced gene
expression involves PAR-1 and is mediated through p44/42 MAPK pathway, independent of
Ca2-signaling. The ability of plasmin to upregulate the expression of Cyr61, and probably
other growth factor-like molecules, which promote cell proliferation and migration, adds a new
dimension to how plasmin plays a role in tissue remodeling
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VEGF and bFGF Alter the Urokinase Receptor (uPAR) Associated Proteolytic
Activity on the Surface of Human Umbilical Vein Endothelial Cells
Gerald W Prager, Judit Mihaly, Florian Gruber, Bernd Binder. University of Vienna, Austria,
Vienna, Austria
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) play an
important role in the angiogenic process. Urokinase type plasminogen activator receptor (uPAR)
is tightly regulated by these cytokines in ECs and itself plays an important role for the
angiogenic process that is dependent on local proteolytic activity. Using flow cytometry we
could confirm previous data that VEGF or bFGF upregulate expression of uPAR in a time and
dose dependent manner via the MEK/ERK pathway, starting 8 hrs. after addition of growth
factors. We could inhibit this upregulation by using PD98059, a specific inhibitor of MEK-1. For
the first time we can show here an additional immediate short term effect of these growth
factors (GF) on the distribution of uPAR within the cell. When VEGF (50ng/ml) or bFGF (10ng/ml)
was added to the cells, a redistribution of uPAR was seen. The GFs induce internalization of
surface uPAR after 30 min with a maximum after 4 hrs. To proof a possible LRP dependent
internalization process, we used the Receptor Associated Protein (RAP), a specific inhibitor of
LDL-receptor family mediated internalization and found that the redistribution was RAP
dependent. To further delineate the initial events by GFs, we analyzed cell surface bound
urokinase by fluorometric cell assay and ELISA, using antibodies recognizing the inactive
pro-uPA as well as the active form uPA, respectively. We found a loss of pro-uPA on the cell
surface upon VEGF stimulation, while total uPA antigen followed the changes in uPAR.
Internalization of uPAR and activation of uPA in response of VEGF was absent in the presence
of PI3-kinase inhibitors, but could be restored adding exogenous active uPA to the PI3-kinase
inhibited cells. Because a specific inhibitor of gelatinases (MMP-2, MMP-9) could inhibit the
redistribution of uPAR and the loss of pro-uPA induced by growth factors in ECs, this indicates
that the MMP-protease dependent pro-uPA activation by VEGF is the part being PI3-kinase
dependent. Such mechanism might play a role in redistribution of uPAR to the leading edge and
fine tuning of surface bound proteolytic activity.
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Identification of Cell Adhesion Sites in Angiopoietin-1 and Angiopoietin-2
Timothy R Carlson, Kwesi O Mercurius, Yuezhong Feng, Milan Mrksich, Godfrey S Getz, Alex
O Morla. University of Chicago, Chicago, IL
Angiopoietin-1 (Ang-1) knockout animals are embryonic lethal and display profound defects in
angiogenesis. Angiopoietin-2 (Ang-2), an Ang-1 homologue, is upregulated in the vasculature
of many human tumors and has been shown to potentiate angiogenesis induced by VEGF and
bFGF in various angiogenesis models. However, the precise mechanism of angiopoietin action
remains unknown. Previously, we showed that cells adhere to both Ang-1 and Ang-2 and that
this adhesion is mediated primarily by 1 and v5 integrins. Herein, we report the
identification of sequences of Ang-1 and Ang-2 that support cell adhesion. The amino acid
sequences of Ang-1 and Ang-2 are about 60% identical and both contain an N-terminal
coiled-coil domain and a C-terminal fibrinogen-like domain. Cells plated onto surfaces coated
with recombinant fibrinogen-like domain do not adhere, even though this domain binds Tie2,
an endothelial-specific receptor tyrosine kinase. On the other hand, recombinant coiled-coil
domains of Ang-1 and Ang-2 produced in E. Coli support adhesion of fibroblasts and endothelial
cells. To further localize the cell binding sites, 100 amino acid polypeptides were produced
and tested for adhesivity. We find that Ang-1 (103–202) supports strong cell adhesion and
spreading. Ang-1 (21–120) and Ang-2 (21–121) demonstrate weak adhesive activity, while
Ang-1 (182–284) and Ang-2 (103–202 & 183–282) appear non-adhesive. To further localize
the cell binding sites, Ang-1 (103–202) was spanned with eleven 15mer peptides, and each
peptide was immobilized to an inert surface and tested in adhesion experiments. Two of these
peptides support cell adhesion and spreading comparable to that observed on surfaces
presenting a control Arg-Gly-Asp sequence. Thus, we have identified cell adhesion sites within
Ang-1 and Ang-2. Interestingly, the sites are physically separable from the sequences required
to bind Tie2.
Involvement of RhoA/Rho Kinase-Signaling in VEGF-Induced Endothelial
Cell Migration and Angiogenesis in Vitro
Geerten P Van Nieuw Amerongen, Amanda Versteilen, Erna A Peters, Rick Van Leeuwen,
Pieter Koolwijk, Victor W Van Hinsbergh. VU University Medical Center, Amsterdam,
Netherlands; TNO-Prevention& Health, Leiden, Netherlands
Vascular Endothelial Growth Factor (VEGF) potently stimulates endothelial cell (EC) migration
and angiogenesis. Reorganization of the F-actin cytoskeleton and cell-matrix adhesion is
essential in these processes. In the present study we investigated whether RhoA and its
downstream target Rho kinase are involved in the VEGF-induced EC changes. VEGF induced a
rapid and prolonged reorganization of the F-actin cytoskeleton in ECs, manifested by the
formation of stress fibers and focal adhesions. This was accompanied by a targeting of RhoA
to the cell membrane. 5 g/mL C3 transferase and 10 mol/L Y-27632, specific inhibitors of
RhoA andby Rho guest kinaseon respectively, April 4, 2013 completely abolished the VEGF-induced cytoskeletal
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