Vascular Endothelial Growth Factor Induction of the Angiogenic Phenotype Requires Ras Activation

Abstract

We investigated the role of Ras in vascular endothelial growth factor (VEGF)-mediated signal transduction and the promotion of angiogenic changes primary endothelial cells. We find that VEGF potently induces Ras activation and that this step is essential for the stimulation by VEGF of several cellular changes associated with angiogenesis, including proliferation, migration, and branching morphogenesis in three-dimensional culture. Inhibition of Ras signaling induced subtle changes in the actin architecture but had no effect on the phosphatidylinositol 3-kinase (PI3K) or p38 signaling pathways. In contrast, activation of ERK was largely dependent on Ras. Although inhibiting ERK activity completely suppressed cell proliferation and partially blocked in vitro differentiation, neither ERK nor PI3K activity was required for VEGF-induced migration. These data provide the first direct demonstration that inhibition of Ras signal transduction is anti-angiogenic. Interestingly, VEGF signal transduction bifurcates both upstream and downstream of Ras, with different Ras-dependent signals controlling endothelial cell proliferation and migration, essential components of the angiogenic response.

VEGF121 and VEGF165 Regulate Blood Vessel Diameter Through Vascular Endothelial Growth Factor Receptor 2 in an in vitro Angiogenesis Model

“…These data are consistent with previous reports showing a role for the MEK-ERK1/2 pathway downstream of VEGF in regulating cell proliferation but not necessarily survival (Meadows et al, 2001;Takahashi et al, 1999). Our data suggest, therefore, that whereas cell survival seems to be dependent on PI3K and p38, vessel diameter regulation by VEGF is more directly dependent on cell proliferation, likely through a pathway involving MEK-ERK1/2 activation.…”

“…Numerous downstream targets of VEGFR-2 are known to be phosphorylated upon activation of the receptor, including phosphatidylinositol 3-kinase (PI3K), MAP and ERK Kinase-1 (MEK-1), p38 mitogen-activated protein kinase (MAPK), phospholipase C-␥ (PLC-␥), Ras GTPase-activating protein, and several others (Bernatchez et al, 2001;Gerber et al, 1998;Meadows et al, 2001;Rousseau et al, 2000;Takahashi et al, 1999). It is possible that the increased diameter of vessels in response to higher concentrations of VEGF represents a graded response of second messenger pathways to increased VEGFR-2 signaling.…”

“…Several reports have demonstrated a critical role for ERK downstream of MEK and VEGFR-2 in mediating DNA synthesis and cell proliferation (Kanno et al, 2000;Meadows et al, 2001;Takahashi et al, 1999); however, PD98059, a specific inhibitor of MEK-1, has consistently failed to block EC migration in response to VEGF. In our system, PD98059 treatment led to a decrease in the shape index of the capillary sprouts (Fig.…”

Activation of p38 Has Opposing Effects on the Proliferation and Migration of Endothelial Cells

“…The cDNA for dominant negative p38␣ (DNp38␣) was from Roger Davis (University of Massachusetts Medical School). Adenoviruses were all created using the Adeasy system, essentially as described previously (10,15,23). The mutant form of Hsp27 (mutHsp27) adenovirus was a generous gift of William Gerthoffer (University of Nevada School of Medicine).…”

“…Functionally, these kinases are also thought of as distinct. ERK is involved in cell proliferation and survival responses in a variety of cell types, including endothelial cells (8,10). Activation of p38 is implicated in inflammation, cell growth control, cell differentiation, cell migration, and apoptosis (6).…”