Reactive oxygen species (ROS) function as signaling molecules through reversible oxidation of reactive cysteine (Cys) residues in specific target proteins. Because ROS are highly diffusible, developing new probes to detect and quantify ROS or the initial key Cys oxidation product, cysteine sulfenic acid (Cys-OH), with high degree of spatial and temporal resolution, is essential to understand the molecular mechanisms of redox signaling. Angiogenesis is dependent on ROS and important for treating ischemic heart/limb diseases, common causes of morbidity and mortality. Thus, enhancing efficacy of the angiogenesis response is likely to be an effective therapy for ischemic vascular disease. VEGF induces autophosphorylation of VEGFR2 (VEGFR2-pY), which initially occurs in part at caveolae/lipid rafts (C/LR), followed by its further activation at intracellular compartments, leading to endothelial cell (EC) proliferation. We demonstrated that ROS derived from NADPH oxidase are required for VEGF-induced VEGFR2-pY, EC proliferation and postnatal angiogenesis in vivo. Further, we discovered "IQGAP1" as a novel VEGFR2 binding scaffold protein involved in ROS-dependent VEGFR2 signaling linked to EC proliferation as well as post-ischemic neovascularization. However, how ROS and Cys-OH formation regulate VEGF signaling and can promote postnatal angiogenesis remain unknown. Preliminary studies using newly-developed Cys-OH detecting probes found that VEGF increased Cys-OH formation of IQGAP1, VEGFR2, and PTP1B and their disulfide bond formation in human ECs, which was inhibited by a thiol antioxidant, N-acetylcysteine, and confirmed in mice hindlimb ischemia model. We showed that PTP1B negatively regulates VEGF-induced VEGFR2-pY and EC proliferation. Although oxidative inactivation of PTP1B by H2O2 is shown, the mechanism and role of Cys oxidation of PTP1B and other proteins involved in VEGF redox signaling is largely unknown. Our preliminary data are consistent with the hypothesis that IQGAP1 senses VEGF-induced ROS signal via Cys oxidation, which facilitates redox-sensitive disulfide bond formation with VEGFR2 and PTP1B to compartmentalize ROS-dependent VEGFR2 signaling, thereby promoting effective postnatal angiogenesis.
Aim 1 will determine whether NADPH oxidase-derived ROS mediate Cys oxidation of IQGAP1, VEGFR2 and PTP1B in VEGF-stimulated ECs and hindlimb ischemia model in vivo, and identify their Cys oxidation sites.
Aim 2 will determine whether disulfide bond formation of Cys oxidized IQGAP1 with VEGFR2 or PTP1B in ECs is essential to compartmentalize ROS-dependent VEGFR2 activation at C/LR and ER/endosomes, thereby inducing EC proliferation. The long-term goal is to understand the molecular mechanisms through which ROS regulate angiogenesis. Proof of this entirely novel theory will suggest that targeting Cys oxidized proteins and/or their molecular interaction at specific signaling microdomains offers a new therapeutic strategy for promoting the efficacy of therapeutic angiogenesis needed for treatment of ischemic cardiovascular diseases.

Public Health Relevance

Reactive oxygen species (ROS) play an important role in the process of new blood vessel growth in ischemic heart and limb diseases. This proposal will not only advance the field to understand molecular mechanism through which ROS are involved in postnatal angiogenesis, but also provide strong impact to discover novel therapeutic targets and strategies for treatment of various angiogenesis-dependent cardiovascular diseases PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HL112293-02
Application #
8620710
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Charette, Marc F
Project Start
2013-02-15
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
2
Fiscal Year
2014
Total Cost
$179,438
Indirect Cost
$66,938
Name
University of Illinois at Chicago
Department
Pharmacology
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
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