Endothelial Healing is Inhibited by PI3 Kinase-Induced Activation of TRPC6

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Cardiovascular disease is a devastating disorder that has a major impact on length and quality of life. According to the CDC, approximately 27 million Americans carry the diagnosis of heart disease. The number of heart and vascular procedures (balloon angioplasties and vascular grafts) that will be performed in 2030 is ex- pected to be nearly twice the number performed in 2010. Similar increases will occur in the veteran population When a blood vessel is treated with angioplasty, the endothelial cells (EC) are removed. The cells must migrate from the edge of the injury into the area of injury to heal it. If healing is delayed, the chance of restenosis is increased. Lipid oxidation products accumulate in atherosclerotic arteries and at regions of injury, cause cellular dysfunction, and inhibit EC migration in vitro and in vivo. Limited re-endothelialization contributes to thrombogenicity, smooth muscle cell proliferation, and restenosis. Oxidized lipids cause an inappropriate increase in intracellular free calcium ion concentration ([Ca2+]i) through canonical transient receptor potential (TRPC) channels, specifically TRPC6. Activation of TRPC6 by causes an increase in [Ca2+]i that results in activation of TRPC5 and a prolonged increase in [Ca2+]i. The increased [Ca2+]i activates calpains that break down cytoskeletal proteins inhibiting EC migration. Studies in TRPC6-/- mice provide compelling evidence of the importance of this cascade in vivo. Re-endothelialization of injured carotid arteries is dramatically reduced in wild-type (WT) mice on a high fat diet compared with chow- fed mice, but in TRPC6-/- mice, hypercholesterolemia does not inhibit re-endothelialization of the injury. Considerable effort has been directed at identifying a TRPC6 inhibitor without success. Lipid oxidation products induce TRPC6 externalization by activating phosphatidylinositol 3-kinase (PI3K), which generates PIP3 (phosphatidylinositol (3,4,5)-trisphosphate). PIP3 is anchored in the cell membrane and binds to TRPC6, which promotes TRPC6 translocation to the cell membrane and leads to increased [Ca2+]i. We hypothesize that inhibition of PI3K can block the activation of TRPC6 channels by lipid oxidation products and restore EC migra- tion in vitro and promote EC healing of arterial injuries in vivo. To test this, we will 1) identify the PI3K iso- form(s) essential for TRPC6 activation, 2) investigate the effectiveness of isoform-specific PI3K inhibitors to re- store EC healing of arterial injuries in hypercholesterolemic animals, and 3) investigate the mechanism through which PIP3 interacts with TRPC6 to promote TRPC6 translocation to the membrane and activation to identify a more specific method of TRPC6 inhibition. The long-term scientific goal is to improve the outcome of ther- apeutic vascular interventions promoting endothelial surfacing of angioplasty sites, stents, and vascular grafts. In terms of a research career development, my short-term research goal is to develop a foundation in a focused research area that I can expand over the course of my career and which will serve as a complement to my clinical practice. My knowledge of vascular wall biology will increase through seminars and I will expand my experience in cellular molecular biology laboratory techniques over the course of the award, including protein extraction and analysis, small-inhibitory RNA transfection, and site directed mutagenesis. Over the course of the award, I will gain additional experience in biostatistics, scientific and grant writing, laboratory management and leadership, and mentoring. I will also continue to develop the critical thinking skills necessary for successful research plans. My primary long-term career goal is to transition to an independent investigator and compete for VA Merit Review and NIH funding. My long-term research goal is to more clearly define the mechanisms by which reactive oxygen species and the activation of TRPC channels inhibit endothelial cell healing. With progress in this area, mechanism-based treatment regimens can be developed, transitioned into clinical trials, and ultimately be carried into clinical practice to improve the long-term outcomes following vascular intervention.