Transforming growth factor-beta signaling via ALK1 and ALK5 regulates distinct functional pathways in vein graft intimal hyperplasia

Abstract

29Rationale: Transforming growth factor-beta (TGFβ) is tightly regulated at multiple levels, with regulation at 30 the receptor level now recognized as a key determinant of the cellular response to this pleiotropic cytokine. 31TGFβ promotes saphenous vein graft neointima formation after coronary artery bypass graft (CABG) surgery, 32 inducing smooth muscle cell (SMC) hyperplasia and fibrosis by signaling via activin receptor-like kinase 33 5(ALK5). However, the role of the alternate TGFβ receptor ALK1 remains completely unknown. 34 Objective: To define the receptor pathways activated by TGFβ in SMCs and their mechanistic importance 35 during CABG neointima formation. 36 Methods and results: Radioligand co-IP assays revealed direct interactions between TGFβ, ALK5 and ALK1 37 in primary saphenous vein graft SMC (HSVSMC) from patients undergoing CABG. Knockdown and 38 pharmacological inhibition of ALK5 or ALK1 in HSVSMC significantly attenuated TGFβ-induced 39 phosphorylation of receptor-regulated (R)-Smads 2/3 and 1/5, respectively. Microarray profiling followed by 40 qRT-PCR validation showed that TGFβ induced distinct transcriptional networks downstream of ALK5 or 41 ALK1, associated with HSVSMC contractility and migration, respectively and confirmed using migration 42 assays as well as qRT-PCR and western blot assays of contractile SMC markers. scRNAseq analysis of 43 TGFβ-treated HSVSMC identified distinct subgroups of cells showing ALK5 or ALK1 transcriptional 44 responses, while RNA velocity analyses indicated divergence in differentiation towards ALK5 or ALK1-45 dominant lineages. ALK1, ALK5 and their downstream effectors pSmad1/5 and pSmad2/3 were localized to 46 αSMA+ neointimal SMCs in remodelled mouse vein grafts. Pharmacological inhibition or genetic ablation of 47 Smad1/5 substantially reducing neointima formation following acute vascular injury. Notably, expression and 48 activation of ALK1, ALK5 and their respective downstream R-Smads was already evident in hyperplastic 49 saphenous veins prior to grafting. 50 Conclusions: Whilst canonical TGFβ signaling via ALK5 promotes a contractile HSVSMC phenotype, 51 transactivation of ALK1 by TGFβ induces neointima formation by driving cell migration. Restoring the 52 balance between ALK1 and ALK5 in HSVSMC may represent a novel therapeutic strategy for vein graft 53 failure. 54

Targeting Smad-Mediated TGFß Pathway in Coronary Artery Bypass Graft

“…A substantial reduction in neointima was observed in LDN193189-treated mice compared to control. 106 However, wire injured femoral arteries of Smad3-null mice displayed large neointimal hyperplasia compared with wild-type mice, suggesting that that Smad3 is protective, 105 contrary to the previous hypothesis that it mediates restenosis. 107 However, overexpression of Smad7 inhibited angiotensin II and TGFβ-induced expression of CTGF and procollagen, as well as the production of fibronectin in VSMCs.…”

“…Similarly, treatment with ALK1 kinase inhibitor KO2288/KO and the ALK5 kinase inhibitor SB525334/SB (Figure 3) downregulated Smad1/5 and Smad2 respectively. 106 Specifically, while ALK5 signaling was activated in both intact and injured vessels, ALK1 signaling was only found to be active in injured vessels. Thus, it appears that ALK-1 and Smad1/5 activation are the pathological hallmark of the SMC response to acute vascular injury, promoting neointima formation and inward remodeling.…”

“…111 ALK1 and its signaling components were also found to be highly expressed in preimplantation saphenous veins showing substantial pre-existing neointima formation, indicating its role in graft restenosis. 106 ALK1’s role in regulating cell proliferation and migration and the modulation of extracellular matrix (ECM) protein expression strongly indicates an important function in cardiovascular remodeling and suggests that the ALK1/smad1/5/8 pathway is potentially a powerful therapeutic target. 71,109,113 Induction of the phosphorylation of Smad1/5 and Smad2 by the binding of TGFβ to ALK1 and ALK5 respectively has been demonstrated in human saphenous vein SMCs using ALK inhibitors and siRNA approaches.…”

“…Thus, it appears that ALK-1 and Smad1/5 activation are the pathological hallmark of the SMC response to acute vascular injury, promoting neointima formation and inward remodeling. 106…”