Robin C. Looft-Wilson, Ph.D. - US grants

Vasomoter responses are conducted along arterioles and feed arteries through cell-to-cell coupling as enabled by gap junctions. The properties of the conduction differ between vascular beds that supply tissues that differ in structure, function, and sympathetic innervation. The Specific Aim of this study is to examine conducted vasomoter responses as related to connexin isoform expression in endothelial and smooth muscle cells of resistance microvessels (arterioles and feed arteries) that are innervated by sympathetic nerves (hamster retractor muscle) relative to microvessels that are not (hamster cheek pouch epithelium). Complementary experiments will determine how these parameters are effected by acute alterations in sympathetic neurotransmission. The long- term goal is to understand how the sympathetic nervous system influences the molecular determinants of conduction in microvessels that control oxygen transport to tissues. Understanding of such regulation of connexin expression and function will aid in the treatment of diseases involving gap junction dysfunction.

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this project is to understand the role of cell-to-cell communication in vascular remodeling. Remodeling is an important process that is both beneficial (e.g., enlargement of arteries during exercise training) and pathological,(e.g., narrowing of arteries during hypertension), and it is unknown what role cell- to-cell communication plays in this process. Cell-to-cell communication occurs through gap junctions that link the interiors of neighboring cells and allows coordinated responses to stimuli. This communication is important for coordinated contraction/relaxation of arteries and vascular development, and thus is likely to be involved in the coordination of vascular cells during remodeling. The specific goal of this project is to understand the effect of altered flow on cell-to-cell communication and expression of the proteins that constitute gap junctions. Altered flow is a primary stimulus for vascular remodeling, where increased flow promotes vessel lumen enlargement and decreased flow promotes narrowing. Using a cultured vessel model, early changes (24, 48 hrs) in cell-to-cell communication (assessed by evaluating conducted vasomotor responses, and vasodilation due to endothelium-derived hyperpolarizing factor) and gap junction protein expression (assessed by immunohistochemistry and quantitative PCR) will be measured in response to decreased or increased flow through the vessel lumen, while maintaining lumen pressure and tissue environment. The effects of chronically increased flow on these parameters will also be measured in vivo in gluteus maximus muscle 8 d after ligation surgery, which promotes increased flow and remodeling in collateral arteries. Vessel function will be assessed by intravital microscopy using transgenic mice with fluorescent vessels (i.e., endothelial cells express green fluorescent protein) to effectively measure vessel diameter responses. Because vascular remodeling is an important process for allowing blood vessels to meet the metabolic demands of tissues, understanding the ability of cells to communicate during this process will provide insight into the remodeling process and response to flow. Decreased cell-to-cell communication has been linked to vascular dysfunction, which is a major component of atherosclerosis. Little is known about how vascular cell-to-cell communication is regulated, and it is important to understand how a major physical force, such as flow, affects this important vascular function. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this project is to expand our understanding of how cells in the blood vessel wall transduce the physical force shear stress. Shear stress is the frictional force exerted on the vessel wall due to the flow of blood through the lumen, and is the primary stimulus that governs both acute changes in blood vessel diameter and chronic structural remodeling. These processes are critically important for ensuring appropriate blood flow to tissues, which is essential for human health. Remarkably, how the cells in the vessel wall transduce the shear stress into a dilation response or structural remodeling is not clearly understood. Therefore, the overall objective of this project is to uncover key molecular signaling events that control the vascular response to shear stress, with a focus on endothelial nitric oxide synthase (eNOS). eNOS is a key enzyme critical to blood vessel dilation and vascular remodeling. Uncovering its regulatory mechanisms during shear stress will be achieved through two specific aims. Aim 1 is to determine how eNOS is regulated acutely by shear stress in intact arteries, through examining key regulatory phosphorylation sites on eNOS (Tyr83, Ser116, Thr497, Ser617, Ser635, Tyr657, Ser1179), the role of upstream kinases, protein-protein interactions (caveolin-1, Hsp90), and concomitant diameter changes, using an isolated, cannulated, perfused mouse artery preparation and video microscopy. Arteries (mesenteric and carotid arteries) will be subjected to various magnitudes of shear stress (with or without specific kinase inhibitors), at sequential time-points, and analyzed for phosphorylation and protein-protein interaction using immunoblotting and immunoprecipitation. These experiments will illuminate how this enzyme is regulated during shear stress-induced dilation. Aim 2 is to determine how eNOS is regulated during chronically elevated shear stress in intact arteries, by examining eNOS phosphorylation and protein-protein interactions during the remodeling process. This will be measured: 1) in vitro using an innovative method in which isolated, cannulated mouse arteries are cultured for several days in a chronic perfusion system, and 2) in vivo using a surgical ligation model to chronically increase flow in the external carotid artery and the small mesenteric arteries of a mouse. These experiments will provide insight into when and how eNOS is activated during chronic shear stress-induced remodeling, which is currently unknown. PUBLIC HEALTH RELEVANCE: This project will examine, at the molecular level, how arteries respond to the physical force produced by blood flowing through the artery. This force influences both the structure and the health of the artery. Healthy arteries are critical to overall human health because when they are unhealthy, atherosclerosis and heart disease results.