ALPHA V SUPPRESSION BY SIRNA AUGMENTS OR INHIBITS VEGF AND VEGFR-2 EXPRESSION DEPENDING ON THE ORIGIN OF ENDOTHELIAL CELLS
Abstract number: P-S-416
Dardik1 R., Livnat1 T., Zivelin1 A., Rosenberg1 N., Seligsohn1 U.
1Amalia Biron Thrombosis and Hemostasis Research Institute, Sheba Medical Center, Tel Hashomer, Israel
How-to-cite Dardik R, Livnat T, Zivelin A, Rosenberg N, Seligsohn U. ALPHA V SUPPRESSION BY SIRNA AUGMENTS OR INHIBITS VEGF AND VEGFR-2 EXPRESSION DEPENDING ON THE ORIGIN OF ENDOTHELIAL CELLS. J Thromb Haemost 2007; 5 Supplement 2: P-S-416
Introduction: Mice lacking b3 and b5 exhibit pathological angiogenesis due to enhanced expression of VEGFR-2 (1), and av-null mice show elevated VEGF and VEGFR-2 levels (2). To further explore the role of avb3/ avb5 in regulation of VEGF/VEGFR-2 expression, we utilized the approach of av suppression using siRNA.
Methods: Using siRNA, av expression was suppressed by 70-80% in 2 endothelial cell (EC) lines (EAhy.926- a HUVEC cell line, and HMVEC- microvascular EC line), and one epithelial cell line (retinal pigmental epithelial (RPE) cells). av, VEGF and VEGFR-2 mRNA levels were studied by quantitative RT-PCR, and VEGF protein was analysed by ELISA. Flow cytometry and WB were used for analysis of av and VEGFR-2 protein levels.
Results: Following av suppression, VEGF expression was upregulated at the mRNA level in both EAhy.926 and RPE cells (by 1.7 and 1.9 fold, respectively). VEGF protein levels were also increased about 1.5 fold in both cell lines. Similarly, VEGFR-2 expression was upregulated in EAhy.926 cells by 2.6 fold. In contrast, in microvascular EC (HMVEC), both VEGF and VEGFR-2 were remarkably downregulated (2.5 and 3.3 fold reduction for VEGF and VEGFR-2 expression, respectively). The downregulation of VEGFR-2 was associated with a marked decrease in nuclear protein binding to Sp1, Sp3, -170 EBox, +184 EBox and Inr sites in the VEGFR-2 promoter, as shown by EMSA. The mechanism of transcriptional regulation of VEGF expression by av suppression in all 3 cell lines, and of VEGFR-2 expression in EC lines is under investigation.
Conclusions: Suppression of av integrins augments or inhibits VEGF and VEGFR-2 levels in EC of venous and microvascular origin, respectively. Conceivably, therapeutic avb3 inhibitors used for suppressing angiogenesis may act variably at different vascular sites.
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2. McCarty JH, Monahan-Earley RA, Brown LF et al., 2002, Mol Cell Biol., 22: 7667-77