In response to shear stress of flowing blood, endothelial cells secrete a number of factors, including nitric oxide (NO), which play a key role in regulating vascular homeostasis. Defects in NO release lead to endothelial dysfunction and contribute to cardiovascular disease, including hypertension, restenosis and atherosclerosis. Our pioneering studies during the previous cycle identified Rap1 as a novel, critical regulator of endothelial cell shear sensing and nitric oxide release. The physiological significance of our finding is underscored by the phenotype of endothelial cell (EC)-specific Rap1 knockout mice, which include endothelial dysfunction and hypertension. Our pilot studies also suggest that Rap1 is required for transmission of shear stress signals from Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) to Vascular Endothelial Growth Factor 2 (VEGFR2) transactivation and downstream signaling to endothelial NO Synthase (eNOS). The goal of this proposal is to examine the role of Rap1 in transducing shear stress signals required for maintaining EC homoeostasis. The overall hypothesis is that Rap1 promotes shear stress-induced signals from the mechanosensing receptor PECAM-1, via its effector, Afadin, to VEGFR2 and downstream to eNOS. Further, disruption of Rap1 function promotes pro-inflammatory endothelial phenotype and exacerbates atherosclerosis. The hypothesis will be tested in three aims.
Aim 1 will examine molecular mechanisms of Rap1 activation in response to shear stress. Studies utilizing immortalized human endothelial cells expressing PECAM-1 mutants will determine the involvement of Rap1 activator (Rap1 GEF) C3G and Rap1 effector, Afadin, in transmission of signals from PECAM-1 to VEGFR2 activation and downstream signaling.
Aim 2 will investigate the role of Rap1 in transducing laminar and disturbed flow signals. Utilizing Rap1-deficient ECs in vitro and vessels from endothelial-specific Rap1 knockout mice ex vivo, the effect of shear on acute signaling and long-term pro-inflammatory gene expression will be examined.
Aim 3 will examine the effect of disrupted Rap1 signaling as a factor exacerbating endothelial function leading to a pro-inflammatory state in vivo. The studies will investigate the effect of endothelial Rap1 deletion on progression of atherosclerosis in a mouse model in vivo. Proposed studies will uncover novel, previously unexpected mechanisms governing EC responses to shear and may lead to a new direction in restoring EC function by controlling Rap1 signaling.

Public Health Relevance

The complications of atherosclerosis remain major killers of the American population. This project will help understand mechanisms through which endothelial cells respond to the flow of blood and how defects in these responses contribute to atherosclerosis. These are the first necessary steps in developing new strategies to restore endothelial function to prevent the progression of atherosclerosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL111582-05
Application #
9196891
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Gao, Yunling
Project Start
2012-04-16
Project End
2020-06-30
Budget Start
2016-07-05
Budget End
2017-06-30
Support Year
5
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Bloodcenter of Wisconsin, Inc.
Department
Type
DUNS #
057163172
City
Milwaukee
State
WI
Country
United States
Zip Code
53233
Chrzanowska-Wodnicka, Magdalena (2017) Rap1 in endothelial biology. Curr Opin Hematol 24:248-255
Rana, Ujala; Liu, Zhong; Kumar, Suresh N et al. (2016) Nogo-B receptor deficiency causes cerebral vasculature defects during embryonic development in mice. Dev Biol 410:190-201
Wang, Haibo; Han, Xiaokun; Bretz, Colin A et al. (2016) Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization. Mol Ther Methods Clin Dev 3:16056
Lakshmikanthan, Sribalaji; Zheng, Xiaodong; Nishijima, Yoshinori et al. (2015) Rap1 promotes endothelial mechanosensing complex formation, NO release and normal endothelial function. EMBO Rep 16:628-37
Kumar, Sachin; Xu, Juying; Kumar, Rupali Sani et al. (2014) The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation. J Exp Med 211:1741-58
Lakshmikanthan, Sribalaji; Zieba, Bartosz J; Ge, Zhi-Dong et al. (2014) Rap1b in smooth muscle and endothelium is required for maintenance of vascular tone and normal blood pressure. Arterioscler Thromb Vasc Biol 34:1486-94
Wang, Haibo; Jiang, Yanchao; Shi, Dallas et al. (2014) Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization. FASEB J 28:265-74
Wittchen, Erika S; Nishimura, Eiichi; McCloskey, Manabu et al. (2013) Rap1 GTPase activation and barrier enhancement in rpe inhibits choroidal neovascularization in vivo. PLoS One 8:e73070
Chrzanowska-Wodnicka, Magdalena (2013) Distinct functions for Rap1 signaling in vascular morphogenesis and dysfunction. Exp Cell Res 319:2350-9