Nanoscale extracellular matrix alters endothelial function under disturbed flow

Stanford University, Stanford CA

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Abstract

PROJECT SUMMARY With 25 million American suffering from at least one clinical manifestation of atherosclerosis, and approximately 500,000 deaths annually attributed to coronary artery disease, there is a pressing need to better understand the biological basis for the development of atherosclerosis. Within blood vessels, the endothelial cells (ECs) that line the intimal layer play an important atheroprotective role by inhibiting adhesion of lipogenic proteins and monocytes in the circulating blood that leads to atherogenesis. This proposal arises from my recent findings suggesting that spatial patterning plays an important role in modulating EC function. Retention of a healthy endothelium in the presence of pathologic flow, has important implications for developing cell- patterned vascular grafts that bridge the area of atherosclerotic obstruction and resist occlusion. Therefore, the objective is to examine how modulation of EC alignment using nanopatterning cues, resists the formation of atherosclerosis. The global hypothesis is that vascular grafts containing spatially patterned ECs with longitudinally oriented cell morphology will provide atheroprotective function and improved graft patency under conditions of disturbed flow, when compared to vascular grafts without cell patterning. This hypothesis will be assessed using two Specific Aims: 1) Evaluate the effect of varying shear stress on spatially patterned EC morphology, atheroprotective function, and identification of the signaling pathways that mediate patterning-induced atheroprotective properties in aligned ECs; 2) To evaluate the role of spatially patterned ECs in resisting atherosclerotic lesion formation in vivo. These aims will be evaluated using the following methods: ECs will be cultured on parallel-aligned or randomly oriented fibrillary scaffolds. Effects of complexing cellular patterning and disturbed flow on EC response to shear stress will be evaluated by cell alignment, shape, and migrational trajectories. Functional effects of the patterned ECs to resist inflammation, will be examined by monocyte and platelet adhesion assays and validated by analysis of inflammatory markers. The cytoskeletal mechanisms underlying cell patterning modulation of atherogenesis will be interrogated by gain- and loss-of-function studies using cytoskeletal inhibitors. To evaluate the role of spatially patterned ECs in resisting atherogenesis in vivo, a narrowing cuff model will be used to artificially create a disturbed flow model. Luciferase expressing ECs, longitudinally patterned on vascular grafts will be implanted into the rat carotid artery. Cell turnover will be assessed via bioluminescent imaging and ultrasound will be used to measure graft patency up to 2 months post-transplantation. Histological analysis will be performed to assess graft occlusion and atheroprotective function of the transplanted graft. The long term objective of these studies are to provide novel insights into the mechanisms of promoting and resisting atherogenesis that are mediated by spatial cell patterning, and have important translational potential in the generation of atheroprotective vascular grafts that enhance graft patency.