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

Abstract

BackgroundCholesterol-loading of mouse aortic vascular smooth muscle cells (mVSMCs) downregulates miR-143/145, a master regulator of the contractile state downstream of TGF{beta} signaling. In vitro, this results in transitioning from a contractile mVSMC to a macrophage-like state. This process likely occurs in vivo based on studies in mouse and human atherosclerotic plaques. ObjectivesTo test whether cholesterol-loading reduces VSMC TGF{beta} signaling and if cholesterol efflux will restore signaling and the contractile state in vitro and in vivo. MethodsHuman coronary artery (h)VSMCs were cholesterol-loaded, then treated with HDL (to promote cholesterol efflux). For in vivo studies, partial conditional deletion of Tgf{beta}r2 in lineage-traced VSMC mice was induced. Mice wild-type for VSMC Tgf{beta}r2 or partially deficient (Tgf{beta}r2+/-) were made hypercholesterolemic to establish atherosclerosis. Mice were then treated with apoA1 (which forms HDL). ResultsCholesterol-loading of hVSMCs downregulated TGF{beta} signaling and contractile gene expression; macrophage markers were induced. TGF{beta} signaling positively regulated miR-143/145 expression, increasing Acta2 expression and suppressing KLF4. Cholesterol-loading localized TGF{beta} receptors into lipid rafts, with consequent TGF{beta} signaling downregulation. Notably, in cholesterol-loaded hVSMCs HDL particles displaced receptors from lipid rafts and increased TGF{beta} signaling, resulting in enhanced miR-145 expression and decreased KLF4-dependent macrophage features. ApoA1 infusion into Tgf{beta}r2+/- mice restored Acta2 expression and decreased macrophage-marker expression in plaque VSMCs, with evidence of increased TGF{beta} signaling. ConclusionsCholesterol suppresses TGF{beta} signaling and the contractile state in hVSMC through partitioning of TGF{beta} receptors into lipid rafts. These changes can be reversed by promotion of cholesterol efflux, consistent with evidence in 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 TGF{beta} signaling in vitro in human (h)VSMCs by localization of TGF{beta} 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 TGF{beta} enhancement of miR-145 expression. In vivo, administration of apoA1 (which forms HDL) to atherosclerotic mice also promoted VSMC Acta2 expression 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.