Endothelial cell (EC) migration is essential to heal arterial injuries such as those that occur during balloon angioplasty or bypass grafting. Oxidized low-density lipoprotein (oxLDL) and lysophosphatidylcholine (lysoPC), a product of LDL oxidation and the major lysophospholipid in oxLDL, accumulate in atherosclerotic arteries and at sites of arterial injury, and inhibit EC migration. Limited EC healing contributes to thrombogenicity, smooth muscle cell proliferation, and eventual restenosis. Rapid EC healing has been shown to limit intimal hyperpla- sia after angioplasty or prosthetic graft implantation. Lipid oxidation products cause a prolonged rise in intracellular free calcium ion concentration ([Ca2+]i) that inhibits EC migration. We discovered that oxLDL and lysoPC activate canonical transient receptor potential 6 (TRPC6) channels, and this initiates a series of events leading to activation of TRPC5 that is responsible for the prolonged increase in [Ca2+]i that inhibits EC migration. The importance of the TRPC6 to TRPC5 activation cascade in vivo is shown by: 1) the activation of TRPC6 and TRPC5 by a high cholesterol diet, and 2) the inhi- bition of arterial healing in wild-type mice on a high cholesterol diet, but dramatically less inhibition in TRPC6-/- or TRPC5-/- mice. Unfortunately, no TRPC6 inhibitor has been developed for clinical use. We have discovered, however, that TRPC6 activation requires influx of calcium through arachidonic acid-regulated calcium (ARC) channels. Based on our preliminary data, we postulate that lipid oxidation products activate TRPC6 channels by activating phospholipase A2 (PLA2) to release cellular arachidonic acid that opens ARC channels. The Ca2+ that enters through the ARC channels activates a Src kinase that leads to the opening of TRPC6 chan- nels. To test this hypothesis we will determine the mechanism of the initial lysoPC-induced increase in [Ca2+]i, investigating the role of PLA2 activation and ARC channels and the specific type of PLA2 involved. We will also explore the mechanism by which the increase in [Ca2+]i activates TRPC6, investigating the specific Src kinase that is activated. The effect of PLA2 and Src kinase inhibitors on TRPC6 activation and EC migration in vitro will be studied, and the ability of these inhibitors to block TRPC activation and promote EC healing and inhibit intimal hyperplasia will be explored in animals on a chow or high fat diet using a mouse carotid injury model and a rabbit vascular graft model. The proposed studies will lead to a better understanding of the mechanisms by which lipid oxidation prod- ucts inhibit EC migration and promote intimal hyperplasia. This will allow development of targeted interventions to inhibit TRPC6 activation and promote EC migration into angioplasty sites or onto cardiovascular implants to improve the outcome after vascular interventions in patients with cardiovascular disease.

Public Health Relevance

For arteries to heal after balloon angioplasty and stenting or bypass grafting, endothelial cells (cells that normally line blood vessels) must move into the area of injury, but oxidized lipids block this movement, in part by opening specific calcium channels (TRPC6 channels). We have found that a localized increase in calcium is required to activate TRPC6 channels. We propose to identify the source of this calcium and block it to promote endothelial migration. The results of these studies will provide direction in the development of new treatments, such as an inhibitor of the calcium influx, to promote endothelial cell healing after vascular interventions for the benefit all patients who require cardiovascular interventions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL064357-16
Application #
10094069
Study Section
Bioengineering, Technology and Surgical Sciences Study Section (BTSS)
Program Officer
Lundberg, Martha
Project Start
2000-08-01
Project End
2021-11-30
Budget Start
2020-12-01
Budget End
2021-11-30
Support Year
16
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Chaudhuri, Pinaki; Rosenbaum, Michael A; Birnbaumer, Lutz et al. (2017) Integration of TRPC6 and NADPH oxidase activation in lysophosphatidylcholine-induced TRPC5 externalization. Am J Physiol Cell Physiol 313:C541-C555
Chaudhuri, Pinaki; Rosenbaum, Michael A; Sinharoy, Pritam et al. (2016) Membrane translocation of TRPC6 channels and endothelial migration are regulated by calmodulin and PI3 kinase activation. Proc Natl Acad Sci U S A 113:2110-5
Rosenbaum, Michael A; Chaudhuri, Pinaki; Graham, Linda M (2015) Hypercholesterolemia inhibits re-endothelialization of arterial injuries by TRPC channel activation. J Vasc Surg 62:1040-1047.e2
Rosenbaum, Michael A; Chaudhuri, Pinaki; Abelson, Benjamin et al. (2015) Apolipoprotein A-I mimetic peptide reverses impaired arterial healing after injury by reducing oxidative stress. Atherosclerosis 241:709-15
Rosenbaum, Michael A; Miyazaki, Keiko; Graham, Linda M (2012) Hypercholesterolemia and oxidative stress inhibit endothelial cell healing after arterial injury. J Vasc Surg 55:489-96
Rosenbaum, Michael A; Miyazaki, Keiko; Colles, Scott M et al. (2010) Antioxidant therapy reverses impaired graft healing in hypercholesterolemic rabbits. J Vasc Surg 51:184-93
Miyazaki, Keiko; Colles, Scott M; Graham, Linda M (2008) Impaired graft healing due to hypercholesterolemia is prevented by dietary supplementation with alpha-tocopherol. J Vasc Surg 48:986-93
Chaudhuri, Pinaki; Colles, Scott M; Bhat, Manjunatha et al. (2008) Elucidation of a TRPC6-TRPC5 channel cascade that restricts endothelial cell movement. Mol Biol Cell 19:3203-11
Patel, Rajendra; Cardneau, Jeffry D; Colles, Scott M et al. (2006) Synthetic smooth muscle cell phenotype is associated with increased nicotinamide adenine dinucleotide phosphate oxidase activity: effect on collagen secretion. J Vasc Surg 43:364-71
Chaudhuri, Pinaki; Colles, Scott M; Fox, Paul L et al. (2005) Protein kinase Cdelta-dependent phosphorylation of syndecan-4 regulates cell migration. Circ Res 97:674-81

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