R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling.

Keywords:
VEGF signaling, R-Ras, Angiogenesis, Endothelial cells
1.
Holmes K, Roberts OL, Thomas AM, Cross MJ: Vascular endothelial growth factor receptor-2: Structure, function, intracellular signalling and therapeutic inhibition. Cell Signal 2007;19:2003-2012.
2.
Ferrara N: Vascular endothelial growth factor. Arterioscler Thromb Vasc Biol 2009;29:789-791.
3.
Gavard J, Gutkind JS: VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin. Nat Cell Biol 2006;8:1223-1234.
4.
Darland DC, D'Amore PA: Blood vessel maturation: vascular development comes of age. J Clin Invest 1999;103:157-158.
5.
Carmeliet P, Jain RK: Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Nat Rev Drug Discov 2011;10:417-427.
6.
Jain RK: Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005;307:58-62.
7.
Behl T, Kotwani A: Exploring the various aspects of the pathological role of vascular endothelial growth factor (VEGF) in diabetic retinopathy. Pharmacol Res 2015;99:137-148.
8.
Said SS, Pickering JG, Mequanint K: Advances in growth factor delivery for therapeutic angiogenesis. J Vasc Res 2013;50:35-51.
9.
Mughal NA, Russell DA, Ponnambalam S, Homer-Vanniasinkam S: Gene therapy in the treatment of peripheral arterial disease. Br J Surg 2012;99:6-15.
10.
Giacca M, Zacchigna S: VEGF gene therapy: therapeutic angiogenesis in the clinic and beyond. Gene Ther 2012;19:622-629.
11.
Sawada J, Urakami T, Li F, Urakami A, Zhu W, Fukuda M, Li DY, Ruoslahti E, Komatsu M: Small GTPase R-Ras regulates integrity and functionality of tumor blood vessels. Cancer Cell 2012;22:235-249.
12.
Komatsu M, Ruoslahti E: R-Ras is a global regulator of vascular regeneration that suppresses intimal hyperplasia and tumor angiogenesis. Nature Medicine 2005;11:1346-1350.
13.
Marte BM, Rodriguez-Viciana P, Wennstrom S, Warne PH, Downward J: R-ras can activate the phosphoinositide 3-kinase but not the map kinase arm of the ras effector pathways. Curr Biol 1997;7:63-70.
14.
Lowe DG, Goeddel DV: Heterologous expression and characterization of the human r-ras gene product. Molecular and Cellular Biology 1987;7:2845-2856.
15.
Zhang Z, Vuori K, Wang H, Reed JC, Ruoslahti E: Integrin activation by r-ras. Cell 1996;85:61-69.
16.
Mazzone M, Dettori D, Leite de Oliveira R, Loges S, Schmidt T, Jonckx B, Tian YM, Lanahan AA, Pollard P, Ruiz de Almodovar C, De Smet F, Vinckier S, Aragones J, Debackere K, Luttun A, Wyns S, Jordan B, Pisacane A, Gallez B, Lampugnani MG, Dejana E, Simons M, Ratcliffe P, Maxwell P, Carmeliet P: Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. Cell 2009;136:839-851.
17.
Meadows KN, Bryant P, Pumiglia K: Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation. J Biol Chem 2001;276:49289-49298.
18.
Meadows KN, Bryant P, Vincent PA, Pumiglia KM: Activated Ras induces a proangiogenic phenotype in primary endothelial cells. Oncogene 2004;23:192-200.
19.
Lakshmikanthan S, Sobczak M, Chun C, Henschel A, Dargatz J, Ramchandran R, Chrzanowska-Wodnicka M: Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin αvβ3. Blood 2011;118:2015-2026.
20.
Westenskow PD, Kurihara T, Aguilar E, Scheppke EL, Moreno SK, Wittgrove C, Marchetti V, Michael IP, Anand S, Nagy A, Cheresh D, Friedlander M: Ras pathway inhibition prevents neovascularization by repressing endothelial cell sprouting. J Clin Invest 2013;123:4900-4908.
21.
Sawada J, Li F, Komatsu M: R-Ras protein inhibits autophosphorylation of vascular endothelial growth factor receptor 2 in endothelial cells and suppresses receptor activation in tumor vasculature. J Biol Chem 2015;290:8133-8145.
22.
Katsogiannou M, Andrieu C, Rocchi P: Heat shock protein 27 phosphorylation state is associated with cancer progression. Front Genet 2014;5:346.
23.
Kobayashi M, Nishita M, Mishima T, Ohashi K, Mizuno K: MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration. EMBO J 2006;25:713-726.
24.
Rousseau S, Houle F, Huot J: Integrating the VEGF signals leading to actin-based motility in vascular endothelial cells. Trends Cardiovasc Med 2000;10:321-327.
25.
Rousseau S, Houle F, Kotanides H, Witte L, Waltenberger J, Landry J, Huot J: Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase 2 (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase. J Biol Chem 2000;275:10661-10672.
26.
Huot J, Houle F, Spitz DR, Landry J: Hsp27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res 1996;56:273-279.
27.
Guay J, Lambert H, Gingras-Breton G, Lavoie JN, Huot J, Landry J: Regulation of actin filament dynamics by p38 MAP kinase-mediated phosphorylation of heat shock protein 27. J Cell Sci 1997;110(Pt 3):357-368.
28.
Rousseau S, Houle F, Landry J, Huot J: p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene 1997;15:2169-2177.
29.
Piotrowicz RS, Hickey E, Levin EG: Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration. FASEB J 1998;12:1481-1490.
30.
Huot J, Houle F, Marceau F, Landry J: Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 1997;80:383-392.
31.
Lamalice L, Le Boeuf F, Huot J: Endothelial cell migration during angiogenesis. Circ Res 2007;100:782-794.
32.
Noria S, Xu F, McCue S, Jones M, Gotlieb AI, Langille BL: Assembly and reorientation of stress fibers drives morphological changes to endothelial cells exposed to shear stress. Am J Pathol 2004;164:1211-1223.
33.
Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C: Vegf guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 2003;161:1163-1177.
34.
McMullen ME, Bryant PW, Glembotski CC, Vincent PA, Pumiglia KM: Activation of p38 has opposing effects on the proliferation and migration of endothelial cells. J Biol Chem 2005;280:20995-21003.
35.
Zheng C, Lin Z, Zhao ZJ, Yang Y, Niu H, Shen X: MAPK-activated protein kinase-2 (MK2)-mediated formation and phosphorylation-regulated dissociation of the signal complex consisting of p38, MK2, Akt, and Hsp27. J Biol Chem 2006;281:37215-37226.
36.
Cote MC, Lavoie JR, Houle F, Poirier A, Rousseau S, Huot J: Regulation of vascular endothelial growth factor-induced endothelial cell migration by LIM kinase 1-mediated phosphorylation of annexin 1. J Biol Chem 2010;285:8013-8021.
37.
Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC: SB 203580 is a specific inhibitor of a map kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 1995;364:229-233.
38.
Parsons JT, Martin KH, Slack JK, Taylor JM, Weed SA: Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement. Oncogene 2000;19:5606-5613.
39.
Chiu JJ, Chien S: Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev 2011;91:327-387.
40.
Jain RK: Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Cancer Cell 2014;26:605-622.
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2016
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