Research Project
10117552
Vascular homeostasis is critically dependent upon vasodilator factors released from the endothelium. The most prominent of these factors is nitric oxide (NO), which is the main barometer of endothelial function and becomes impaired in a broad range of diseases including coronary artery disease (CAD). In the human coronary and adipose microcirculation, we have demonstrated a novel process where loss of NO-dependent flow-mediated dilation (FMD) in subjects with CAD is compensated by the production of hydrogen peroxide (H2O2) from endothelial mitochondria and subsequent H2O2-dependent dilation. Although both are vasodilators, H2O2, in opposition to NO, generally promotes cell activation, inflammation, and atherosclerosis, and thus understanding mechanisms responsible for this transition from NO to H2O2 may be key to developing novel strategies to improve endothelial function in patients with CAD. The overall goal of this project is to elucidate the signaling mechanisms that regulate the vasodilator switch from NO to H2O2 during CAD. Building on findings from the last cycle, this proposal is designed to determine intracellular pathways responsible for a previously unappreciated gain of function of endothelial transient receptor potential vanilloid 4 (TRPV4) channels and its contribution to vasodilator switch in CAD. We will test the central hypothesis that a synergy of shear-sensitive phospholipase A2-derived arachidonic acid and NADPH oxidase signaling promotes TRPV4 activation and subsequent H2O2-dependent dilation while cross-inhibiting NO-dependent dilation in CAD arterioles. Further, NADPH oxidases as novel aging- and CAD-associated upstream regulators play a critical role in initiating the switch. This application brings together expertise in vasomotion regulation, human microcirculation, and ion channel structural biology to identify novel molecular mechanisms and interactions that regulate vasodilator switch during CAD. Specific Aims: (1) we will determine the molecular mechanism of TRPV4 activation and arteriolar dilation by flow; and (2) we will determine how NADPH oxidases regulate TRPV4 activation and conversion from NO to H2O2 as mediator of FMD in CAD arterioles. Studies will be conducted on freshly isolated human arterioles and endothelial cells as well as in vivo animal models, using a multifaceted approach incorporating isolated vessel reactivity, Ca2+ imaging, patch-clamping electrophysiology, mass spectrometry, RNA-Seq, mutagenesis, and ion channel molecular modeling. Significance: our proposed studies will provide insight into fundamental mechanisms regulating human microvascular function in health and disease and potentially impact our approach to coronary microvascular dysfunction associated with CAD and a variety of other vascular pathologies.
