Abstract
The autogenous arteriovenous fistula (AVF) remains the preferred vascular access for hemodialysis, despite its high rate of maturation failure, primarily due to occlusive stenosis driven by fibrotic remodeling and neointimal hyperplasia. Currently, no therapies effectively improve AVF outcomes, largely due to limited understanding of the cellular and molecular mechanisms underlying venous remodeling. To address this knowledge gap, we conducted single-nucleus (sn) RNA/ATAC-seq analysis using a translational aortocaval fistula model in mice. This analysis showed significant heterogeneity among venous cells in the AVF outflow tract and identified unique venous smooth muscle cells (vSMC) reprogramming from a quiescent to a previously uncharacterized, proliferative and matrix organizing phenotype that retains a contractile state. The presence of these cells was further validated using SMC lineage tracing and multiplex RNA in situ hybridization. Additionally, snATAC-seq demonstrated a significant increase in the motif activity of ATF3 in fibrotic vSMC within AVF compared to controls. Notably, ATF3 is positively regulated by MRTFA, a key co-transcription factor that activates multiple gene programs. MRTFA expression was markedly upregulated in vSMC following AVF creation. Both SMC-specific knockout and pharmacological inhibition of MRTFA/B in mice significantly reduced AVF wall thickness, neointimal formation, and fibrosis while enhancing AVF flow. Consistently, MRTFA and its target genes (COL1A1, COL8A1, PDGFRB) were upregulated in neointimal vSMC in human AVF. Single-cell RNA-seq analysis of human AVF mirrored the contractile-to-fibrotic vSMC reprogramming observed in mouse AVF. Furthermore, CosMx single-cell spatial omics identified distinct localization patterns of multiple vSMC clusters, with fibrotic and stressed vSMC clusters predominantly situated in the neointima, while contractile vSMC clusters were primarily localized in the medial layer of human AVF. Collectively, these studies, for the first time, integrate multiple advanced single-cell omics approaches to define the occurrence and regulation of vSMC reprogramming during AVF remodeling and its contribution to AVF pathology. They also suggest that targeting vSMC-MRTF could provide a novel therapeutic strategy to prevent adverse AVF remodeling, reduce failure rates, and improve AVF patency.