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Endothelial SRSF1 directly regulates ATF3-KLF2 pathway to promote ischemia-induced angiogenesis

ABSTRACT: 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 (ECs) are the primary cell type contributing to angiogenesis in response to ischemia. However, the molecular mechanisms regulating ischemia-induced angiogenesis remain elusive. We used a discovery-driven approach to identify elevated serine/arginine splicing factor 1 (SRSF1) expression in endothelial cells after ischemia. Proteomic analyses identified endogenous SRSF1 accumulated in ECs of the ischemic muscle and responded to hypoxia. Mice deficient in endothelial SRSF1 exhibited impaired blood flow recovery and impaired new capillary formation following HLI. Importantly, overexpression of SRSF1 enhanced blood flow recovery and angiogenesis after HLI. SRSF1 overexpression facilitated, while SRSF1 knockdown suppressed, the angiogenic function of human ECs. To explore the mechanism the mechanisms contributing to SRSF1-promoted endothelial angiogenesis,we conducted transcriptome RNA-seq searching for downstream molecules and verified it through RNA-pull down and chromatin immunoprecipitation qPCR analysis. Mechanistically, SRSF1 modulated ATF3 alternative splicing, wherein SRSF1 directly bound to ATF3 pre-mRNA and elevated full-length ATF3 transcript at the expense of truncated ATF3Δzip2 transcript, and in turn, ATF3 directly bound to the KLF2 promoter and suppressed KLF2-S1PR1 signaling, through which mediated the SRSF1-promoted endothelial tube formation and angiogenesis. Additionally, alprostadil, the prostaglandin E1 analog, could activate the SRSF1 signaling to improve endothelial angiogenesis in vitro and in vivo. Our findings identified SRSF1 as a novel regulator of ischemia-induced angiogenesis that enhances endothelial angiogenic functions by regulating the ATF3-KLF2/SIPR1 pathway. These results suggest that modulation of endothelial SRSF1 may represent a promising therapeutic approach for treating ischemic vascular diseases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE266589 | GEO | 2025/10/27

REPOSITORIES: GEO

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Project description: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 (ECs) are the primary cell type contributing to angiogenesis in response to ischemia. However, the molecular mechanisms regulating ischemia-induced angiogenesis remain elusive. We used a discovery-driven approach to identify elevated 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 (ECs) are the primary cell type contributing to angiogenesis in response to ischemia. However, the molecular mechanisms regulating ischemia-induced angiogenesis remain elusive. We used a discovery-driven approach to identify elevated serine/arginine splicing factor 1 (SRSF1) expression in endothelial cells after ischemia. Proteomic analyses identified endogenous SRSF1 accumulated in ECs of the ischemic muscle and responded to hypoxia. Mice deficient in endothelial SRSF1 exhibited impaired blood flow recovery and impaired new capillary formation following HLI. Importantly, overexpression of SRSF1 enhanced blood flow recovery and angiogenesis after HLI. SRSF1 overexpression facilitated, while SRSF1 knockdown suppressed, the angiogenic function of human ECs. To explore the mechanism the mechanisms contributing to SRSF1-promoted endothelial angiogenesis,we conducted transcriptome RNA-seq searching for downstream molecules and verified it through RNA-pull down and chromatin immunoprecipitation qPCR analysis. We performed enhanced crosslinking and immunoprecipitation (eCLIP)-seq to profile the global SRSF1-binding sites on RNAs in HUVECs. Mechanistically, SRSF1 modulated ATF3 alternative splicing, wherein SRSF1 directly bound to ATF3 pre-mRNA and elevated full-length ATF3 transcript at the expense of truncated ATF3Δzip2 transcript, and in turn, ATF3 directly bound to the KLF2 promoter and suppressed KLF2-S1PR1 signaling, through which mediated the SRSF1-promoted endothelial tube formation and angiogenesis. Additionally, alprostadil, the prostaglandin E1 analog, could activate the SRSF1 signaling to improve endothelial angiogenesis in vitro and in vivo. Our findings identified SRSF1 as a novel regulator of ischemia-induced angiogenesis that enhances endothelial angiogenic functions by regulating the ATF3-KLF2/SIPR1 pathway. These results suggest that modulation of endothelial SRSF1 may represent a promising therapeutic approach for treating ischemic vascular diseases.

Project description:Purpose: To clarify cellular and molecular mechanisms underlying impaired post-ischemic adaptive vascular responses and to evaluate reconstituted HDL (rHDL)-ApoA-I nanotherapy to rescue the defect in type 2 diabetic (T2DM) mouse model of hindlimb ischemia (HLI). Methods: Mice with beta-cell specific over-expression of insulin-like growth factor-2 in atherosclerotic background (IGF-II/ LDLR-/-ApoB100/100) with T2DM features were used in the study with C57BL/6J mice fed with a regular chow-diet (R36, Lactamin) serving as controls. Mice aged between 16 to 20 weeks were used in the study . Mice were anaesthetized with 1.5-2% isoflurane/air mixture during the surgical procedure. Hair was removed from the operation site and cleaned with sterile water. Single incision was made just above medial thigh and superficial femoral artery was separated from femoral vein and nerve. HLI was induced by unilateral ligation of femoral artery distal to the origin of the profunda femoral artery. The skin was closed with interrupted silk sutures. Mice were were administered with reconstituted ApoA-I nanoparticles with phosphatidyl serine core (rHDL) intravenously starting 2 days post-HLI with doses every 2 days until scarification with Saline injections serving as controls. Adductors muscle aortic endothelial cells (AECs) and Ischemic muscle Macrophages and Endothelial Cells (ECs) were isolated using a combination of magnetic assisted cell sorting and flow Cytometry using Macrophage (F4/80) and EC (CD-31) specific markers. Total RNA isolated from FACS sorted cells were used for RNA seq library preparation. Results: Cell-specific gene expression, flow cytometry and immunohistochemistry revealed a persistent Macrophage and Endothelial cell inflammation and a reduced anti-inflammatory M2-macrophage activation associated with impaired collateral remodelling and sprouting angiogenesis in T2DM mice. Furthermore, reconstituted HDL (rHDL)- ApoA-I was found to rescue impaired collateral remodelling and angiogenesis in T2DM mice through modulating EC and macrophage phenotypes. Conclusions: Our results suggest that an impaired collateral remodelling and sprouting angiogenesis in T2DM mice after HLI is associated with a persistent EC and macrophage inflammation and a reduced activation of anti-inflammatory M2-macrophage. Skewing the macrophage phenotype towards the pro-arteriogenic and pro-angiogenic anti-inflammatory M2-macrophages using reconstituted HDL (rHDL)-ApoA-I nanotherapy may serve as a potential therapeutic modality for T2DM PAD.

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Endothelial SRSF1 directly regulates ATF3-KLF2 pathway to promote ischemia-induced angiogenesis

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