Endothelial Healing is Inhibited by Activation of TRPC6 Channels

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Cardiovascular disease is a devastating disorder that has a major impact on length and quality of life. According to the American Heart Association, approximately 121.5 million Americans carry the diagnosis of heart disease. Veterans have significantly higher rates of cardiovascular disease starting at younger ages and have 42% higher odds of having more cardiovascular diseases compared to non-veterans. Higher rates of cardiovascular disease in veterans with a higher likelihood of cardiovascular morbidity at a younger age leads to early onset cardiovascular mortality later in life. The number of heart and vascular procedures (balloon angioplasties and vascular grafts) that will be performed in 2040 is expected to be more than twice the number performed in 2008 and restenosis requiring reintervention occurs in 30-75% of procedures depending on the treatment area. Similar increases in number of vascular procedures performed and similar restenosis rates 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 specific TRPC6 inhibitor without success. Non- selective TRPC inhibitors have developed, but they impact TRPC3, TRPC6, and TRPC7 channels. We have discovered that lipid oxidation products induce TRPC6 externalization by activating phosphatidylinositol 3- kinase (PI3K), which generates phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 is anchored in the cell membrane and promotes TRPC6 translocation to the cell membrane and leads to increased [Ca2+]i. Based on our ongoing studies, the interaction between PIP3 and TRPC6 is mediated by an adaptor protein, and preliminary data suggest that this is Grb2-associated binding protein 1 (Gab1). Importantly, identification of this adaptor suggests a way to block TRPC6 activation and to restore EC migration, while minimizing off-target effects of PI3K inhibitors or non-selective TRPC channel inhibition. We hypothesize that TRPC6 activation by lipid oxidation products requires the interaction of an adaptor protein to link TRPC6 and PIP3 in the membrane. As a corollary, inhibition of the TRPC6-PIP3 interaction can block TRPC6 activation and restore EC migration in the presence of lipid oxidation products. To test this, we will 1) identify the mechanism that mediates PI3K- generated PIP3 anchorage of TRPC6 in the plasma membrane, specifically the role of an adaptor protein, and 2) determine the mechanism of lysoPC-induced, adaptor protein-mediated TRPC6-PIP3 interaction. The long-term goal is to improve the outcome of therapeutic vascular interventions promoting endothelial surfacing of angioplasty sites, stents, and vascular grafts. 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 and improve veterans’ quality of life.