Vascular endothelial growth factor (VEGF) is the critical factor responsible for blood vessel formation in normal conditions, such as in wound healing, and in pathological conditions, such as in tumor growth. Disparate endothelial cell (EC) responses to VEGF stimulation: cell migration, proliferation and increased vascular permeability are mediated in part via cell surface receptor VEGF Receptor 2 (VEGFR2). Small GTPase Rac1 is involved in each of the VEGF-mediated readouts in ECs. Our preliminary data indicate that small GTPase Rap1, acting via Rac1, is a key regulator of VEGF signaling in ECs. Our central hypothesis is that VEGF signaling via VEGFR2 that disrupts EC junctions leading to elevated vascular permeability is in part relayed via interactions between activated Rap1, Rac1 and Rap1 GEF (Guanine nucleotide Exchange Factor) C3G. Studies in Aim 1 will delineate isoform-specific functions of Rap1 in VEGF-induced VE-cadherin junction disassembly in vitro and in vivo. Basal and VEGF-induced vascular permeability in two EC-specific Rap1a and Rap1b-KO mice models will be examined. To gain mechanistic insight into how cellular processes leading to cell-cell junction dissolution are altered in the absence of Rap1 isoforms, VEGF-induced signaling converging at VE-cadherin and differential regulation of RhoA by Rap1 in response to VEGF and Epac activation will be examined in ECs isolated from Rap1-deficient mice. To identify how Rap1 activity is regulated in response to VEGF stimulation in Aim 2, involvement of two GEFs, C3G and Epac, in VEGF-induced Rap1 activation will be examined. Biomolecular Fluorescence Complementation (BiFC) will be used to visualize VEGF-induced interaction between Rap1 and C3G and between Rap1 and Epac. The effect of silencing expression of either GEF on VEGF-induced permeability and, using Rap1 biosensor, on Rap1 activity, will be examined in WT and in Rap1a- or Rap1b-deficient ECs. For in vivo determination of C3G and Epac involvement in angiogenesis, the effect of morpholino-based knockdown of each GEF on intersomitic vessel formation will be examined. Studies in Aim 3 will identify the signaling mechanism downstream from VEGFR2 that is mediated by Rap1 in vitro and in vivo. Our working hypothesis that Rap1 regulates dynamics of EC responses to VEGF by regulating localization of active Rac1 will be tested using Rac1 biosensor in WT and Rap1-deficient ECs, BiFC in VEGF-stimulated WT ECs to detect Rap1 and Rac1 colocalization and by analysis of Rac1-dependent signaling and actin cytoskeleton dynamics. Furthermore, the involvement of two Rac GEFs and a Rac1 effector IQGAP1 in Rap1-dependent Rac1 localization and function in response to VEGF stimulation will be examined. We expect that these aims will identify key control mechanisms through which Rap1 isoforms regulate VEGF- induced permeability in ECs. Knowledge gained through this research is expected to enable successful modulation of VEGF responses by manipulating specific Rap1 isoform activity.

Public Health Relevance

Formation of new blood vessels is important in physiological and pathological conditions. This project will help understand mechanisms underlying blood vessel formation to help create new therapies to either promote new blood vessels required for wound healing and tissue repair, such as after an ischemic heart injury, or prevent blood vessel formation, as in tumor progression.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL111582-01A1
Application #
8304895
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Gao, Yunling
Project Start
2012-04-16
Project End
2016-03-31
Budget Start
2012-04-16
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$429,212
Indirect Cost
$172,199
Name
Bloodcenter of Wisconsin, Inc.
Department
Type
DUNS #
057163172
City
Milwaukee
State
WI
Country
United States
Zip Code
53233
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
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
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
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