Project description:Signal-induced proliferation-associated gene 1 (Sipa1) is known as a specific Rap1 GTPase-activating protein that negatively regulates Rap1 signaling. Although Sipa1 has been extensively studied in cancer research, its role in the wound healing response after myocardial infarction (MI) remains unexplored. To investigate the role of endogenous Sipa1 in MI, we performed permanent left anterior descending artery ligation in both Sipa1 knockout mice and their control littermates. Bone marrow transplantation, flow cytometry, cell sorting, and transcriptomic analysis were conducted to identify the cellular source of Sipa1 in the infarcted heart. The role of cardiac fibroblast-derived Sipa1 during MI was examined using Sipa1 deletion approaches, specifically in cardiac fibroblasts, in vivo and in vitro. Mice deficient in Sipa1 exhibited improved post-MI survival and cardiac function, along with attenuated expression of inflammatory mediators and diminished accumulation of Ly6Chigh monocytes and CCR2+ macrophages in the infarcted heart. Although Sipa1 was broadly expressed in the heart, cardiac fibroblasts were responsible for the Sipa1-induced deleterious phenotype as demonstrated by cardiac fibroblast-specific Sipa1 conditional knockout mice, which averted excessive inflammation and adverse cardiac remodeling following MI. Mechanistically, Sipa1 promotes the production of CCL2, CCL7 and granulocyte/macrophage colony-stimulating factor in the cardiac fibroblasts early after MI via a non-canonical RasGRP2-Ras-JNK signaling pathway, irrespective of canonical Rap1, thereby facilitating the accumulation and activation of inflammatory monocytes and macrophages. These results identify a previously unknown fibroblast-immune axis characterized by Sipa1, which initiates excessive inflammation and leads to poor outcomes after MI. Targeting Sipa1 offers a potential novel therapeutic strategy to optimize post-MI wound healing response, thereby preventing the development of chronic ischemic heart failure.

Project description:BACKGROUND: Myocardial infarction (MI), one of the leading causes of mortality worldwide, remains a clinical challenge due to limitations in current therapeutic strategies. The cardiac vascular network, as the fundamental architecture sustaining myocardial function, delivers oxygen and nutrients with high precision to the heart tissue. However, the dynamic regulatory mechanisms governing this network post-MI remain incompletely elucidated. Notably, the pivotal role of cardiac endothelial cells in the pathophysiological progression of MI calls for further comprehensive investigation.METHODS: We examined alterations in Signal Peptide, CUB Domain And EGF Like Domain Containing 3 (SCUBE3) protein expression in the myocardium of MI-afflicted mice and human MI heart tissues. Utilizing genetically engineered mouse models in combination with diverse cellular and molecular biology techniques, we systematically dissected the functional role of SCUBE3 in MI and its underlying mechanisms in cardiac endothelial cell metabolism.RESULTS: Proteomic profiling of serum samples from MI patients demonstrated a significant elevation in SCUBE3 protein levels. Immunohistochemical assessment revealed markedly increased SCUBE3 expression localized predominantly within fibroblasts in the infarct zone of cardiac tissues from MI patients. Consistently, in a murine MI model, SCUBE3 expression was also upregulated and primarily distributed in fibroblasts within the infarcted myocardial regions. Employing inducible, fibroblast-specific SCUBE3 overexpression and knockout mouse models, we observed that SCUBE3 overexpression robustly enhanced post-MI angiogenesis, improved microcirculatory network integrity, and attenuated myocardial injury. In contrast, SCUBE3 ablation exacerbated infarct-induced cardiac damage. Subsequent protein tracking and mass spectrometry analyses demonstrated that SCUBE3 selectively binds to and activates vascular endothelial growth factor receptor 1 (VEGFR1) on endothelial cells, thereby mediating downstream biological effects. Transcriptomic sequencing revealed that SCUBE3 significantly upregulates fatty acid metabolism–related pathways. Live-cell dynamic analysis further confirmed that SCUBE3 promotes fatty acid uptake and enhances the metabolic activity of endothelial cells. Metabolomic profiling indicated that SCUBE3 facilitates the conversion of unsaturated fatty acids into phosphosugars and nucleotides, supplying essential substrates and energy to support endothelial cell proliferation. In SCUBE3-overexpressing mice, endothelial cell–specific inducible VEGFR1 knockout completely abrogated SCUBE3-driven fatty acid metabolism and its associated cardioprotective effects. Moreover, beyond its role in angiogenesis SCUBE3 also markedly enhances cardiomyocyte regenerative capacity, highlighting its potential as a novel therapeutic target for myocardial repair. Conclusions: Our findings establish that fibroblast-derived SCUBE3 interacts with VEGFR1 on endothelial cells to promote fatty acid metabolism, thereby providing energy substrates necessary for endothelial proliferation. This study elucidates the critical function of the SCUBE3–VEGFR1 signaling axis in post-MI vascular regeneration through metabolic regulation, presenting a promising avenue for therapeutic intervention in cardiac repair.

Project description:BACKGROUND: Myocardial infarction (MI), one of the leading causes of mortality worldwide, remains a clinical challenge due to limitations in current therapeutic strategies. The cardiac vascular network, as the fundamental architecture sustaining myocardial function, delivers oxygen and nutrients with high precision to the heart tissue. However, the dynamic regulatory mechanisms governing this network post-MI remain incompletely elucidated. Notably, the pivotal role of cardiac endothelial cells in the pathophysiological progression of MI calls for further comprehensive investigation.METHODS: We examined alterations in Signal Peptide, CUB Domain And EGF Like Domain Containing 3 (SCUBE3) protein expression in the myocardium of MI-afflicted mice and human MI heart tissues. Utilizing genetically engineered mouse models in combination with diverse cellular and molecular biology techniques, we systematically dissected the functional role of SCUBE3 in MI and its underlying mechanisms in cardiac endothelial cell metabolism.RESULTS: Proteomic profiling of serum samples from MI patients demonstrated a significant elevation in SCUBE3 protein levels. Immunohistochemical assessment revealed markedly increased SCUBE3 expression localized predominantly within fibroblasts in the infarct zone of cardiac tissues from MI patients. Consistently, in a murine MI model, SCUBE3 expression was also upregulated and primarily distributed in fibroblasts within the infarcted myocardial regions. Employing inducible, fibroblast-specific SCUBE3 overexpression and knockout mouse models, we observed that SCUBE3 overexpression robustly enhanced post-MI angiogenesis, improved microcirculatory network integrity, and attenuated myocardial injury. In contrast, SCUBE3 ablation exacerbated infarct-induced cardiac damage. Subsequent protein tracking and mass spectrometry analyses demonstrated that SCUBE3 selectively binds to and activates vascular endothelial growth factor receptor 1 (VEGFR1) on endothelial cells, thereby mediating downstream biological effects. Transcriptomic sequencing revealed that SCUBE3 significantly upregulates fatty acid metabolism–related pathways. Live-cell dynamic analysis further confirmed that SCUBE3 promotes fatty acid uptake and enhances the metabolic activity of endothelial cells. Metabolomic profiling indicated that SCUBE3 facilitates the conversion of unsaturated fatty acids into phosphosugars and nucleotides, supplying essential substrates and energy to support endothelial cell proliferation. In SCUBE3-overexpressing mice, endothelial cell–specific inducible VEGFR1 knockout completely abrogated SCUBE3-driven fatty acid metabolism and its associated cardioprotective effects. Moreover, beyond its role in angiogenesis SCUBE3 also markedly enhances cardiomyocyte regenerative capacity, highlighting its potential as a novel therapeutic target for myocardial repair.

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RNA next-generation sequencing of cardiac fibroblasts isolated from young and old mice 7 days post-MI

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