Endothelial SRSF1 Promotes Ischemia-Induced Angiogenesis via ATF3-KLF2-S1PR1 Pathway

BACKGROUND: Peripheral artery disease is a severe ischemic vascular pathology without effective pharmacological approaches and improving angiogenesis to recover blood perfusion is a promising therapeutic strategy. Endothelial cells are the primary cell type contributing to angiogenesis in response to ischemia. However, the molecular mechanisms regulating ischemia-induced angiogenesis remain elusive. METHODS: We used a discovery-driven approach to identify elevated SRSF1 (serine/arginine splicing factor 1) expression in endothelial cells after ischemia. We used loss- and gain-of-function approaches to explore the role of SRSF1 in angiogenesis both in vivo and in vitro. A mouse model of hindlimb ischemia was used to evaluate ischemia-induced angiogenesis. We also investigated the mechanisms through transcriptome, enhanced crosslinking and immunoprecipitation sequencing, RNA pull-down, and chromatin immunoprecipitation-quantitative polymerase chain reaction analysis. RESULTS: Proteomic analyses identified endogenous SRSF1 accumulated in endothelial cells of the ischemic muscle and responded to hypoxia. Mice deficient in endothelial SRSF1 exhibited impaired blood flow recovery and impaired vasculature formation after hindlimb ischemia. Importantly, overexpression of SRSF1 enhanced blood flow recovery and angiogenesis after hindlimb ischemia. SRSF1 overexpression enhanced the angiogenic functions of human endothelial cells, promoting tube formation, sprouting capability, and cell migration, while SRSF1 knockdown suppressed these functions. Mechanistically, SRSF1 modulated the alternative splicing of ATF3 (activating transcription factor 3) by directly binding to ATF3 pre-mRNA (precursor messenger RNA), and SRSF1 overexpression elevated full-length ATF3 transcript at the expense of truncated ATF3Δzip2 transcript. ATF3 then bound directly to the KLF2 (Krüppel-like factor 2) promoter, suppressed KLF2 expression and downstream S1PR1 (sphingosine-1-phosphate receptor 1) signaling. Through upregulation of full-length ATF3 and downregulating KLF2-S1PR1 signaling, SRSF1 promoted endothelial tube formation and angiogenesis. In addition, alprostadil, the prostaglandin E1 analog, could activate the SRSF1 signaling to improve endothelial angiogenesis in vitro and in vivo. CONCLUSIONS: Our findings identified SRSF1 as a novel regulator of ischemia-induced angiogenesis that enhances endothelial angiogenic functions by regulating the ATF3-KLF2-S1PR1 pathway. These results suggest that modulation of endothelial SRSF1 may represent a promising therapeutic approach for treating ischemic vascular diseases.