Ushio-Fukai Lab Research
Angiogenesis, the process of formation of new blood vessels from pre-existing ones, plays an important role in physiological process such as development and wound repair as well as for treatment of ischemic heart and limb diseases. Uncontrolled angiogenesis contributes to pathophysiology such as cancer. Signaling by binding of VEGF to VEGF receptor type2 (VEGFR2) plays an important role in angiogenesis in endothelial cells (ECs). Over the past several years, the focus of my research is to understand the role of reactive oxygen species (ROS) especially hydrogen peroxide (H2O2) in mediating receptor-coupled signal transduction, so called “redox signaling”. Major sources of ROS are NADPH oxidase (NOX) and mitochondria. Phagocyte NOX consists of 2 membrane-bound subunits, gp91phox (NOX2) and p22phox which form the flavocytochrome b558 complex, together with the cytosolic subunits p47phox, and p67phox p40phox and the small GTPase Rac. NOXs have several homologs of NOX2 including Nox1, Nox3, Nox4, and Nox5, as well as the Dual oxidases (Duox), Duox1 and Duox2. Our laboratory is one of the first to demonstrate that ROS derived from NADPH oxidase (NOX) play an essential role in VEGFR2-mediated signaling linked to EC proliferation and migration as well as postnatal angiogenesis in vivo.
Role of Reactive Oxygen Species (ROS) in VEGF Signaling and Angiogenesis:
Compartmentalization of VEGF Redox Signaling through NADPH oxidase-derived ROS:
Since ROS are highly diffusible, we also study the importance of compartmentalization of NOX-derived ROS in activation of specific redox signaling events involved in angiogenesis. Using newly-developed Cys-OH trapping reagents, we recently demonstrated Cys-OH formation of IQGAP1, a VEGFR2 binding scaffold protein involved in ROS-dependent directional EC migration and post-ischemic angiogenesis. We showed that localized Cys oxidation of IQGAP1 and its binding proteins at the lamellipodial leading edge where it colocalizes with NOX is required for ROS-dependent directional EC migration as well as revascularization in response to ischemic injury. Oxidation responses to VEGF signaling within caveolae/lipid rafts are also observed in ECs, where lipid rafts-associated extracellular superoxide dismutase (ecSOD)-derived H2O2 induce oxidative inactivation of tyrosine phosphatases (PTP1B and DEP-1), thereby promoting VEGFR2 activation and angiogenesis. We currently plan to identify additional key molecular targets of ROS involved in angiogenesis in vitro and in vivo using Cys oxidation-reactive probes and redox proteomics approach.
Redox Regulation of Stem/ProgenitorCells and Bone Marrow Microenvironment (Niche):
Our laboratory investigates redox regulation of stem and progenitor cell function as well as stem cell niche. ROS play important roles in regulating stem and progenitor cell function in various physiologic and pathologic responses. The low level of H2O2 in quiescent hematopoietic stem cells (HSCs) contributes to maintain their stemness, whereas a higher level of H2O2 within HSCs or their niche promotes differentiation, proliferation, migration, and survival of HSCs or stem/progenitor cells. Bone marrow (BM)-derived stem and progenitor cell functions including self-renewal, differentiation, survival, migration, proliferation and mobilization are regulated by unique cell-intrinsic signals and -extrinsic signals provided by their microenvironment, also termed the ‘niche’. We demonstrated that in response to ischemic injury, ROS derived from NADPH oxidase are increased in the BM microenvironment, which is required for hypoxia and HIF1a expression and expansion throughout the BM. This, in turn, promotes progenitor cell expansion and mobilization from BM, leading to reparative neovascularization and tissue repair. Understanding the molecular mechanisms of how ROS regulate the functions of stem and progenitor cells and their niche in physiological and pathological conditions will lead to the development of novel therapeutic strategies.
We use various knockout and transgenic mice models, and perform gene transfer or cell-based therapy in animal model of angiogenesis in normal and disease conditions such as diabetes, atherosclerosis and hypertension. We also perform analysis of protein-protein interaction, signal transduction, cell imaging, cell fractionation analysis, molecular biology, cell biology, and in vivo imaging. Our study should advance the understanding of molecular mechanism by which ROS are involved in angiogenesis and stem/progenitor cell function, which will lead to the development of new therapeutic strategies for treatment of various ischemic and cardiovascular diseases as well as cancer.