Background: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to vascular repair and remodeling but also to pathologies including intimal hyperplasia. The mTORC1 inhibitor rapamycin is an effective drug-eluting stent agent that promotes VSMC differentiation. TGFβ also promotes VSMC differentiation through SMAD transcription factors. We investigated unexpected convergence between these pathways in VSMC plasticity.
Methods: We assessed the interactions between rapamycin and the Transforming Growth Factor-β Receptor 1 (ALK5) signaling in human coronary artery SMCs (hCASMCs) and in vascular remodeling using inducible SMC–specific knockout mice (ALK5iKO).
Results: Genome-wide histone H3K27 acetylation analysis unexpectedly identified SMAD binding elements (SBE) as the motif most enriched after rapamycin treatment of hCASMCs. We found that rapamycin treatment promotes rapid phosphorylation of SMAD2/3 (pSMAD2/3). Signaling studies revealed this phosphorylation required ALK5 activity but not TGF-β ligand, and ALK5 and SMAD2/3 were both required for rapamycin-induced differentiation. We determined that rapamycin relieves FKBP12 inhibition of ALK5 to promote ligand-independent Smad signaling, and FKBP12 knockdown was sufficient to induce contractile genes. Notably, rapamycin treatment induced an interaction between TET2 and SMAD2/3, and these SMADs were required for differentiation-associated chromatin remodeling, including DNA hydroxymethylation and histone acetylation at contractile genes, suggesting that SMADs and TET2 function in concert at SBE- and CArG-containing promoter regions. We also found that mTORC1 inhibition with Raptor knockdown induces TET2 expression but fails to induce chromatin remodeling and SMC differentiation in the absence of SMAD2/3. In vivo, ALK5iKO mice exhibited severe intimal hyperplasia after carotid artery injury compared to controls and were entirely resistant to the therapeutic effect of rapamycin, despite persistent inhibition of mTORC1. Consistent with the in vitro findings, rapamycin treatment elevated pSMAD3 staining post-injury in the medial layer of control but not ALK5iKO mice.
Conclusions: We report the unexpected observation that rapamycin requires FKBP12/ALK5/SMAD2/3 signaling to promote TET2-dependent chromatin remodeling and VSMC differentiation. Understanding these mechanisms may provide new avenues to modulate VSMC phenotype and may hold therapeutic promise for cardiovascular diseases.