Support is requested to analyze the interaction between the Ras family small GTPase R- Ras and a novel effector molecule, RLIP76;the effect of this interaction on R-Ras signaling;the mechanisms by which R-Ras/RLIP76 interactions and signaling regulate vascular cell spreading, migration, proliferation and differentiation;and the roles of this signaling node in angiogenesis and injury-induced neointimal hyperplasia leading to arterial restenosis. Ras GTPases regulate cell proliferation, differentiation, apoptosis, adhesion and migration. The binding of specific effectors to activated Ras proteins propagates signaling cascades resulting in unique outcomes depending on the Ras family isotype. R-Ras in particular functions to regulate adhesiveness, motility, proliferation and differentiation of cells of the vessel wall, and to modulate these processes during angiogenesis and vascular regeneration in response to injury. Thus R-Ras signaling has unique, pleiotropic effects in vascular cells and in vascular functions, but the specific pathways involved are unknown. We identified RLIP76 in a proteomic screen for R-Ras effectors and found that RLIP76 regulates some of the unique cellular functions of R-Ras including cell signaling, spreading, migration, and angiogenesis in vitro. This is the first identification of a specific, unique effector for R-Ras with these distinct cellular functions. We hypothesize that R-Ras/RLIP76 binding controls distinct signaling pathways to mediate vascular cell functions and the unique effects of R-Ras in the vasculature. To test this hypothesis we will map the binding sites in R-Ras and RLIP76 and will identify mutations which will selectively disrupt this interaction. We will use these mutants to determine the effect of blockade of R- Ras/RLIP76 interaction on R-Ras signaling in cells of the vessel wall. We will then evaluate a putative scaffolding function for RLIP76 and will map the protein components of the RLIP76/R- Ras signaling complex in vascular cells. Next we will investigate the contributions of R- Ras/RLIP76 binding and the characterized signaling pathways to R-Ras-mediated vascular cell spreading, migration, proliferation and differentiation. Lastly, we will investigate a role for this signaling axis in R-Ras functions in vivo, namely blockade of angiogenesis, and injury-induced neointimal hyperplasia and arterial restenosis. These studies will characterize the regulation and mechanisms of novel signaling pathways that control cell signaling, migration, proliferation and vascular function. Therefore these studies will provide new insights into processes essential in physiological and pathophysiological function and may indicate new therapeutic targets in the treatment of cardiovascular disease.

Public Health Relevance

We propose to investigate the molecular and cellular mechanisms of a process regulating the growth and migration of cells in two major pathological responses in the vasculature: 1) new blood vessel formation (angiogenesis), which occurs at sites of injury and during tumor development, in which new blood vessels are required for tumor growth, and 2) arterial restenosis, a vessel-occluding response to injury resulting from arterial bypass, stent insertion and balloon dilatation surgeries, which affects as many as 400,000 people in the U.S. per year. We have identified candidate molecular signaling pathways which we will examine as unique and selective agents of tumor angiogenesis and post-surgical restenosis. Thus we will use a combination of biochemical, cell biological and physiological approaches in mouse models of angiogenesis and arterial restenosis to identify and characterize novel therapeutic targets for the treatment of human cardiovascular disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Vascular Cell and Molecular Biology Study Section (VCMB)
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Olive, Michelle
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Temple University
Internal Medicine/Medicine
Schools of Medicine
United States
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Wurtzel, Jeremy G T; Lee, Seunghyung; Singhal, Sharad S et al. (2015) RLIP76 regulates Arf6-dependent cell spreading and migration by linking ARNO with activated R-Ras at recycling endosomes. Biochem Biophys Res Commun 467:785-91
Lee, Seunghyung; Goldfinger, Lawrence E (2014) RLIP76 regulates HIF-1 activity, VEGF expression and secretion in tumor cells, and secretome transactivation of endothelial cells. FASEB J 28:4158-68
Silver, Lee; Michael, James V; Goldfinger, Lawrence E et al. (2014) Activation of PI3K and R-Ras signaling promotes the extension of sensory axons on inhibitory chondroitin sulfate proteoglycans. Dev Neurobiol 74:918-33
Lee, Seunghyung; Wurtzel, Jeremy G T; Goldfinger, Lawrence E (2014) The RLIP76 N-terminus binds ARNO to regulate PI 3-kinase, Arf6 and Rac signaling, cell spreading and migration. Biochem Biophys Res Commun 454:560-5
Lee, Seunghyung; Barbe, Mary F; Scalia, Rosario et al. (2014) Three-dimensional reconstruction of neovasculature in solid tumors and basement membrane matrix using ex vivo X-ray microcomputed tomography. Microcirculation 21:159-70
Kim, Soochong; Dangelmaier, Carol; Bhavanasi, Dheeraj et al. (2013) RhoG protein regulates glycoprotein VI-Fc receptor ?-chain complex-mediated platelet activation and thrombus formation. J Biol Chem 288:34230-8
Goldfinger, Lawrence E; Lee, Seunghyung (2013) Emerging treatments in lung cancer - targeting the RLIP76 molecular transporter. Lung Cancer (Auckl) 2013:61-69
Wurtzel, Jeremy G T; Kumar, Puneet; Goldfinger, Lawrence E (2012) Palmitoylation regulates vesicular trafficking of R-Ras to membrane ruffles and effects on ruffling and cell spreading. Small GTPases 3:139-53
Lee, Seunghyung; Wurtzel, Jeremy G T; Singhal, Sharad S et al. (2012) RALBP1/RLIP76 depletion in mice suppresses tumor growth by inhibiting tumor neovascularization. Cancer Res 72:5165-73
Bhavanasi, Dheeraj; Kim, Soochong; Goldfinger, Lawrence E et al. (2011) Protein kinase C? mediates the activation of protein kinase D2 in platelets. Biochem Pharmacol 82:720-7

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