Abstract
BackgroundInflammatory smooth muscle cells (iSMCs) generated from adventitial stem/progenitor cells (AdSPCs) have been recognised as a new player in cardiovascular disease, and microRNA-214-3p (miR-214-3p) has been implicated in mature vascular SMC functions and neointimal hyperplasia. Here, we attempted to elucidate the functional involvements of miR-214-3p in iSMC differentiation from AdSPCs and unravel the therapeutic potential of miR-214-3p signalling in AdSPCs for injury-induced neointimal hyperplasia.MethodsThe role of miR-214-3p in iSMC differentiation from AdSPCs was evaluated by multiple biochemistry assays. The target of miR-214-3p was identified through binding site mutation and reporter activity analysis. A murine model of injury-induced arterial remodelling and stem cell transplantation was conducted to study the therapeutic potential of miR-214-3p. RT-qPCR analysis was performed to examine the gene expression in healthy and diseased human arteries.ResultsmiR-214-3p prevented iSMC differentiation/generation from AdSPCs by restoring sonic hedgehog-glioma-associated oncogene 1 (Shh-GLI1) signalling. Suppressor of fused (Sufu) was identified as a functional target of miR-214-3p during iSMC generation from AdSPCs. Mechanistic studies revealed that miR-214-3p over-expression or Sufu inhibition can promote nuclear accumulation of GLI1 protein in AdSPCs, and the consensus sequence (GACCACCCA) for GLI1 binding within smooth muscle alpha-actin (SMαA) and serum response factor (SRF) gene promoters is required for their respective regulation by miR-214-3p and Sufu. Additionally, Sufu upregulates multiple inflammatory gene expression (IFNγ, IL-6, MCP-1 and S100A4) in iSMCs. In vivo, transfection of miR-214-3p into the injured vessels resulted in the decreased expression level of Sufu, reduced iSMC generation and inhibited neointimal hyperplasia. Importantly, perivascular transplantation of AdSPCs increased neointimal hyperplasia, whereas transplantation of AdSPCs over-expressing miR-214-3p prevented this. Finally, decreased expression of miR-214-3p but increased expression of Sufu was observed in diseased human arteries.ConclusionsWe present a previously unexplored role for miR-214-3p in iSMC differentiation and neointima iSMC hyperplasia and provide new insights into the therapeutic effects of miR-214-3p in vascular disease.
Highlights
Smooth muscle cell (SMC) accumulation and phenotypic transition are critical steps for vascular remodelling in response to vascular injury
Recent groundbreaking efforts, using SMC-restricted lineage-tracking techniques, have provided definitive evidence to support the conventional concept that medium SMCs are the main cellular origins ofintimal SMCs upon injury [1,2,3], other studies show that vascular stem/progenitor cells (SPCs), SPCs located in the adventitial layer, represent an additional source of theintimal SMCs in the atheroma [4, 5]
To study the potential involvement of miRNAs in SMC differentiation from adventitial stem/progenitor cells (AdSPCs), we first detected if the expression levels of miR-22, miR34a and miR-214, the three top modulated miRNAs during SMC differentiation from embryonic stem cells [20], were altered in this process and found that miR-214-3p is the most upregulated miRNA among them in this SMC differentiation model (Figure S3A). miR-214-3p gain/loss-of-function experiments were conducted in AdSPCs to confirm a role for miR-214-3p in SMC differentiation from AdSPCs
Summary
Smooth muscle cell (SMC) accumulation and phenotypic transition are critical steps for vascular remodelling in response to vascular injury. Investigations into the mechanisms controlling iSMC differentiation from SPCs are essential for us to fully understand the pathogenesis of vascular diseases and remodelling and are of great clinical importance. MicroRNAs (miRNAs) are endogenous, highly conserved single short strands of non-coding RNA. They have been reported as important regulators for stem cell differentiation, cardiovascular development and cardiac regeneration and are emerging as novel therapeutics as well as clinical biomarkers for cardiovascular diseases, including restenosis after vascular injury or stent implantation [17] and atherosclerosis. Inflammatory smooth muscle cells (iSMCs) generated from adventitial stem/progenitor cells (AdSPCs) have been recognised as a new player in cardiovascular disease, and microRNA-214-3p (miR-214-3p) has been implicated in mature vascular SMC functions and neointimal hyperplasia. We attempted to elucidate the functional involvements of miR-214-3p in iSMC differentiation from AdSPCs and unravel the therapeutic potential of miR-214-3p signalling in AdSPCs for injury-induced neointimal hyperplasia
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