HDL regulates TGFß-receptor lipid raft partitioning, restoring contractile features of cholesterol-loaded vascular smooth muscle cells

Abstract

AbstractBackgroundCholesterol-loading of mouse aortic vascular smooth muscle cells (mVSMCs) downregulatesmiR-143/145, a master regulator of the contractile state downstream of TGFβ signaling.In vitro,this results in transitioning from a contractile mVSMC to a macrophage-like state. This process likely occursin vivobased on studies in mouse and human atherosclerotic plaques.ObjectivesTo test whether cholesterol-loading reduces VSMC TGFβ signaling and if cholesterol efflux will restore signaling and the contractile statein vitroandin vivo.MethodsHuman coronary artery (h)VSMCs were cholesterol-loaded, then treated with HDL (to promote cholesterol efflux). Forin vivostudies, partial conditional deletion ofTgfβr2in lineage-traced VSMC mice was induced. Mice wild-type for VSMCTgfβr2or partially deficient (Tgfβr2+/-) were made hypercholesterolemic to establish atherosclerosis. Mice were then treated with apoA1 (which forms HDL).ResultsCholesterol-loading of hVSMCs downregulated TGFβ signaling and contractile gene expression; macrophage markers were induced. TGFβ signaling positively regulatedmiR-143/145expression, increasingActa2expression and suppressing KLF4. Cholesterol-loading localized TGFβ receptors into lipid rafts, with consequent TGFβ signaling downregulation. Notably, in cholesterol-loaded hVSMCs HDL particles displaced receptors from lipid rafts and increased TGFβ signaling, resulting in enhancedmiR-145expression and decreased KLF4-dependent macrophage features. ApoA1 infusion intoTgfβr2+/-mice restoredActa2expression and decreased macrophage-marker expression in plaque VSMCs, with evidence of increased TGFβ signaling.ConclusionsCholesterol suppresses TGFβ signaling and the contractile state in hVSMC through partitioning of TGFβ receptors into lipid rafts. These changes can be reversed by promotion of cholesterol efflux, consistent with evidencein vivo.Condensed abstractMany cells identified as macrophage-like in human and mouse atherosclerotic plaques are thought to be of VSMC origin. We identified cholesterol-mediated downregulation of TGFβ signalingin vitroin human (h)VSMCs by localization of TGFβ receptors in membrane lipid rafts, which was reversed by HDL-mediated cholesterol efflux. This restored VSMC contractile marker (Acta2) and suppressed macrophage marker (CD68) expression by promoting TGFβ enhancement ofmiR-145expression.In vivo, administration of apoA1 (which forms HDL) to atherosclerotic mice also promoted VSMCActa2expression and reduced CD68 expression. Because macrophage-like VSMC are thought to have adverse properties, our studies not only show mechanistically how cholesterol causes their transition, but also suggest that efflux-competent HDL particles may have a therapeutic role by restoring a more favorable phenotypic state of VSMC in atherosclerotic plaques.