Background: Vascular smooth muscle cells (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 TGFβ1 (ALK5) signaling in human coronary artery SMCs and in vascular remodeling using inducible SMC-specific knockout mice (ALK5iKO). Results: Genome-wide histone H3K27 acetylation analysis unexpectedly identified SMAD binding elements as the motif most enriched after rapamycin treatment of hCASMCs. Treatment with rapamycin promoted rapid phosphorylation of SMAD2/3. Extensive signaling studies revealed that this phosphorylation required ALK5 activity but not TGF-β ligand. Surprisingly, ALK5 and SMAD2/3 were 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 H3K27 acetylation at contractile genes, suggesting that SMADs and TET2 function in concert at SBE- and CArG-containing promoter regions. Furthermore, 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 in vitro findings, treatment with rapamycin elevated pSmad3 staining post-injury in the medial layer of control but not ALK5iKO mice. Conclusions: We report the surprising observation that rapamycin requires FKBP12/ALK5/SMAD2/3 signaling in order to promote TET2-dependent chromatin remodeling and SMC differentiation. Understanding these mechanisms may reveal new therapeutic strategies for treating vascular diseases.

Abstract 1147: Crosstalk Between Alk5 And Mtorc1 Signaling Promotes VSMC Differentiation And The Therapeutic Effect Of Rapamycin