Redox Proteomics and Visualization of ROS targets in VEGF Signaling Linked to Angiogenesis and Vasculogenesis

Reactive oxygen species (ROS) are crucial component of signaling events linked to neovascularization in response to injury. Detecting ROS with high degree of spatial and temporal resolution by the current available ROS reacting probes remains a challenge. Thus, development of new technology to detect molecular targets of ROS as chemical footprint of endogenous ROS is essential. Reactive cysteine (Cys) residues in proteins such as protein tyrosine phosphatases (PTPs) become the primary targets of oxidative modifications. Cysteine sulfenic acid (Cys-SOH), the initial product of ROS-mediated reactive Cys oxidation, is a key intermediate in the functional modulation of enzymes and proteins involved in ROS-mediated signaling. Thus, trapping of Cys-SOH upon its formation in cellular proteins using a detectable reagent is critically important for identifying and visualizing redox-responsive proteins in cell signaling. We previously demonstrated that ROS derived from Nox2-based NADPH oxidase play an important role in VEGF signaling in endothelial cells (ECs) and ischemia-induced angiogenesis and endothelial progenitor cells (EPCs)-mediated vasculogenesis in vivo. However, underlying mechanisms remain unclear. Recently, cell permeable, biotin- or fluorescein-conjugated Cys-SOH reactive reagents have been developed by a collaborator of this grant. We will apply this new tool to facilitate and accelerate the identification and characterization of molecular targets of ROS that play a crucial role in VEGF signaling linked to postnatal angiogenesis and vasculogenesis. In this grant proposal, we will employ the state-of-art redox proteomics approaches using newly-developed Cys-SOH trapping reagents to identify novel molecular targets of ROS in VEGF-stimulated ECs and EPCs in vitro as well as ischemic tissues and EPCs obtained from mice hindlimb ischemia model in vivo. We will also visualize the Cys-SOH formation to localize ROS targets signaling molecules in cells (ECs and EPCs) with live cell imaging and tissue (ischemic muscle and BM), and will validate the functional significance of these oxidized proteins in ROS-dependent VEGF signaling in ECs and EPCs. These studies should discover the novel molecular targets of ROS involved in postnatal angiogenesis and vasculogenesis, which may lead to developing new direction in angiogenesis research as well as new therapeutic strategies to promote revascularization in ischemic heart and limb diseases. (AHA Program: Innovative Research Grant)