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

Abstract Background Cholesterol-loading of mouse aortic vascular smooth muscle cells (mVSMCs) downregulates miR-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 occurs in vivo based on studies in mouse and human atherosclerotic plaques. Objectives To test whether cholesterol-loading reduces VSMC TGFβ signaling and if cholesterol efflux will restore signaling and the contractile state in vitro and in vivo . Methods Human coronary artery (h)VSMCs were cholesterol-loaded, then treated with HDL (to promote cholesterol efflux). For in vivo studies, partial conditional deletion of Tgfβr2 in lineage-traced VSMC mice was induced. Mice wild-type for VSMC Tgfβr2 or partially deficient ( Tgfβr2+/- ) were made hypercholesterolemic to establish atherosclerosis. Mice were then treated with apoA1 (which forms HDL). Results Cholesterol-loading of hVSMCs downregulated TGFβ signaling and contractile gene expression; macrophage markers were induced. TGFβ signaling positively regulated miR-143/145 expression, increasing Acta2 expression 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 enhanced miR-145 expression and decreased KLF4-dependent macrophage features. ApoA1 infusion into Tgfβr2+/- mice restored Acta2 expression and decreased macrophage-marker expression in plaque VSMCs, with evidence of increased TGFβ signaling. Conclusions Cholesterol 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 evidence in vivo . Condensed abstract Many 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 signaling in vitro in 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 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.