Fluid shear stress‐induced transcriptional activation of the vascular endothelial growth factor receptor‐2 gene requires Sp1‐dependent DNA binding

Abstract

Hemodynamic forces play a fundamental role in the regulation of endothelial cell survival. As signaling via the vascular endothelial growth factor (VEGF) receptor-2 pathway has been previously demonstrated to impact endothelial cell survival, we hypothesized that laminar shear stress may facilitate survival in part by inducing VEGF receptor-2 expression. This study shows a time-and dose-dependent upregulation of endothelial VEGF receptor-2 expression by £uid shear stress in microvascular and large-vessel derived endothelial cells. A functional analysis of the 5P P-regulatory region of the VEGF receptor-2 promoter localized the shear stress-response element to a sequence between bp 3 360 and 3 337 that encompasses two adjacent consensus Sp1 transcription factor binding sites. Constitutive and shear stress-inducible Sp1-dependent complexes are bound to this element, indicating that £uid shear stress-induced transcriptional activation of the VEGF receptor-2 gene requires Sp1-dependent DNA binding. Together, these results suggest that biomechanical stimulation may lead to endothelial cell survival by upregulating VEGF receptor-2 expression. ß

Role of shear-stress-induced VEGF expression in endothelial cell survival

“…This is consistent with previous reports of flow upregulating VEGFR2 expression by ECs in culture (Abumiya et al, 2002;Shay-Salit et al, 2002;Urbich et al, 2003), but more importantly corroborates our observation of constitutive VEGFR2 phosphorylation in aortic endothelium in vivo (Maharaj et al, 2006). Considering the role of VEGF in pathological neovascularization, continuous VEGFR2 activation might be expected to lead to EC hyperproliferation, such as seen in hemangiomas.…”

Mechanosensitive mechanisms in transcriptional regulation

“…Activated NF-kB upregulates the expression of genes that encode proteins such as eNOS (Balligand et al, 2009;Davis et al, 2004;Ozawa et al, 2004), several cytokines (Allport et al, 2000;Martin et al, 1997) and adhesion molecules that are crucial for maintaining vascular homeostasis (Ahmad et al, 1998). In addition, fluid shear stress activates the transcription of the gene encoding VEGFR2 through binding of the transcription factor SP1 to its promoter, and this transcriptional regulation is crucial for endothelial cell survival (Urbich et al, 2003). During angiogenesis, VEGFR2 protein synthesis and the formation of new microvessels are regulated by changes in ECM mechanics (e.g.…”

PPARα Activators Inhibit Vascular Endothelial Growth Factor Receptor-2 Expression by Repressing Sp1-Dependent DNA Binding and Transactivation

“…Fenofibrate, Wy14643, ciglitazone, rosiglitazone, troglitazone, and 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) were obtained from Biomol; ciprofibrate and phorbol myristate acetate (PMA) were from Sigma-Aldrich.…”

“…12 The membranes were incubated with the indicated primary antibodies (VEGFR1, clone H-225, VEGFR2, clone A-3, Sp1, clone PEP2, all from Santa Cruz, Heidelberg, Germany; PPAR␣, clone 3B6, Alexis; Tubulin Ab from LabVision), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (anti-mouse or antirabbit IgG, Amersham). The blots were developed using the enhanced chemiluminescence detection system (ECL) according to the instructions of the manufacturer (Amersham).…”

“…Nuclear proteins were extracted as described previously. 12 The DNA binding reactions were performed with or without excess of unlabeled competitor, Sp1 consensus-oligonucleotide (Promega), PPAR␣, Sp1, Sp3, or RelA/NF-B antibody (Santa Cruz).…”