The importance of the renin angiotensin system in cardiovascular disease has been proven by the positive clinical benefits associated with ACE inhibitors and angiotensin type 1 receptor (AT1R) blockers. Previously, we studied how the AT1R increases tyrosine phosphorylation since tyrosine kinase pathways are closely related to pathogenic events such as VSMC proliferation, expression of pro-inflammatory cytokines, and generation of oxidative stress. We found that a key mediator is the tyrosine kinase c-Src, which phosphorylates phospholipase-C3 (PLC3) and activates the ERK1/2 pathway. We next identified a 97 kD protein phosphorylated by c-Src, that is required for activation of PLC3and ERK1/2. This protein is GIT1 (G protein coupled receptor kinase-2 interacting protein 1). Then we showed that GIT1 is a scaffold for several key pathways including PLC3-CamKII , MEK1-ERK1/2, and PIX-PAK. In addition to the role for GIT1 in VSMC, we found that GIT1 mediated EC permeability and signaling by VEGF further defining the importance of GIT1 in vascular biology. Next, we created GIT1-knockout (KO) mice that exhibit a dramatic phenotype characterized by ~60% lethality within 7 days. Pathologic analysis showed that GIT1-KO mice appear normal except for a significant vascular abnormality with decreased capillary density. This defect is most apparent in the lung, characterized by hemorrhage in the parenchyma, increased alveolar spaces, decreased pulmonary microcirculation, and impaired basement membrane formation. These abnormalities resemble alveolar capillary dysplasia (ACD) and bronchopulmonary dysplasia (BPD). The GIT1-KO phenocopies the VEGF-120 mouse, suggesting that GIT1 deficiency abrogates VEGF receptor signaling. Because our preliminary data show essential roles for GIT1 in multiple VEGF signaling events, especially those mediated by the Delta4- Notch1 pathway, we propose that GIT1 is a novel mediator of VEGF-dependent signaling (via PLC3, ERK1/2 and Delta4-Notch) required for normal and pathologic angiogenesis. To prove this hypothesis we will show that 1) GIT1 is required for angiogenesis by regulating tip cell functions (polarity, migration, and adhesion) via assembly of structures (podosomes, filopodia) dependent on phosphoprotein complexes;2) GIT1 regulates Notch1 via specific effects on MEK1-ERK1/2 and PLC-CamKII in EC;3) GIT1 regulates VEGF-induced migration and tip cell function in the developing retina;and 4) GIT1 is required for angiogenesis in response to ischemia in the retina and hind-limb. Understanding how vascular remodeling is affected in the GIT1-KO may lead to discovery of novel tissue specific vascular pathways relevant to diseases such as BPD and proliferative retinopathy.

Public Health Relevance

The healing of tissues after surgery or trauma is critical for long-term survival. The proposed work will study novel mechanisms by which blood vessels grow in response to tissue injury.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL063462-13
Application #
8286924
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
OH, Youngsuk
Project Start
1998-12-25
Project End
2013-12-30
Budget Start
2012-07-01
Budget End
2013-12-30
Support Year
13
Fiscal Year
2012
Total Cost
$382,388
Indirect Cost
$134,888
Name
University of Rochester
Department
Internal Medicine/Medicine
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Yin, Guoyong; Sheu, Tzong-Jen; Menon, Prashanthi et al. (2014) Impaired angiogenesis during fracture healing in GPCR kinase 2 interacting protein-1 (GIT1) knock out mice. PLoS One 9:e89127
Majumder, Syamantak; Sowden, Mark P; Gerber, Scott A et al. (2014) G-protein-coupled receptor-2-interacting protein-1 is required for endothelial cell directional migration and tumor angiogenesis via cortactin-dependent lamellipodia formation. Arterioscler Thromb Vasc Biol 34:419-26
Pang, Jinjiang; Xu, Xiangbin; Wang, Xiaoqun et al. (2013) G-protein-coupled receptor kinase interacting protein-1 mediates intima formation by regulating vascular smooth muscle proliferation, apoptosis, and migration. Arterioscler Thromb Vasc Biol 33:999-1005
Shi, Xi; Yan, Chen; Nadtochiy, Sergiy M et al. (2011) p90 ribosomal S6 kinase regulates activity of the renin-angiotensin system: a pathogenic mechanism for ischemia-reperfusion injury. J Mol Cell Cardiol 51:272-5
Pang, Jinjiang; Xu, Xiangbin; Getman, Michael R et al. (2011) G protein coupled receptor kinase 2 interacting protein 1 (GIT1) is a novel regulator of mitochondrial biogenesis in heart. J Mol Cell Cardiol 51:769-76
Zhang, Ning; Cai, Weihua; Yin, Guoyong et al. (2010) GIT1 is a novel MEK1-ERK1/2 scaffold that localizes to focal adhesions. Cell Biol Int 34:41-7
Menon, Prashanthi; Deane, Rashid; Sagare, Abhay et al. (2010) Impaired spine formation and learning in GPCR kinase 2 interacting protein-1 (GIT1) knockout mice. Brain Res 1317:218-26
Menon, Prashanthi; Yin, Guoyong; Smolock, Elaine M et al. (2010) GPCR kinase 2 interacting protein 1 (GIT1) regulates osteoclast function and bone mass. J Cell Physiol 225:777-85
Wang, Jing; Yin, Guoyong; Menon, Prashanthi et al. (2010) Phosphorylation of G protein-coupled receptor kinase 2-interacting protein 1 tyrosine 392 is required for phospholipase C-gamma activation and podosome formation in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 30:1976-82
Pang, Jinjiang; Hoefen, Ryan; Pryhuber, Gloria S et al. (2009) G-protein-coupled receptor kinase interacting protein-1 is required for pulmonary vascular development. Circulation 119:1524-32

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