Activation of the cytosolic calcium-independent phospholipase A2 β isoform contributes to TRPC6 externalization via release of arachidonic acid

Linda M Graham,Priya Putta,Michael A Rosenbaum,Pinaki Chaudhuri,Rocio Guardia-Wolff,Andrew H Smith

doi:10.1016/j.jbc.2021.101180

Abstract

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A2 (PLA2), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA2 in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA2 enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA2 involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA2α, cPLA2γ, iPLA2β, or iPLA2γ. Downregulation of the β isoform of iPLA2 blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.

Highlights

Endothelial cell (EC) healing is crucial for successful vascular interventions [1,2,3]
LysoPC is one of the most potent antimigratory lysophospholipids, and our previous studies have shown that it inhibits EC migration at least in part by activating TRPC6, which leads to a cascade of events resulting in a prolonged increase in [Ca2+]i that activates calpains and inhibits
We postulate that lysoPC activates phospholipase A2 (PLA2) causing release of arachidonic acid (AA), which in turn opens arachidonate-regulated calcium channels leading to the localized increase in calcium

Summary

Introduction

Endothelial cell (EC) healing is crucial for successful vascular interventions [1,2,3]. In a mouse arterial injury model, a high-cholesterol diet significantly impairs endothelial healing in WT mice but is not inhibitory in TRPC6 null mice [6] This suggests that blocking lipid oxidation product(s)-induced TRPC6 activation could promote more rapid EC healing leading to improved outcomes after vascular interventions. LysoPC can activate phospholipase A2 (PLA2) to release arachidonic acid (AA) from EC membranes [12, 13] This AA can activate arachidonate-regulated calcium channels in the plasma membrane [14], and the subsequent Ca2+ entry can provide the local increase in [Ca2+]i required to externalize TRPC6 channels. The purpose of this study is to identify PLA2 subgroup(s) and the specific isoform(s) that contribute to lysoPC-induced TRPC6 externalization and inhibition of EC migration. Inhibiting iPLA2β blocks lysoPC-induced AA release from EC membranes, blocks TRPC6 externalization, and preserves EC migration

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Conclusion