Profilin-1 phosphorylation in neovascularization

项目摘要包括心肌梗死(MI)在内的缺血性心脏病是主要原因 世界范围内的发病率和死亡率。新生血管，新血管的形成，是 心肌梗死后心脏修复和再生的基础。新生血管是通过血管生成进行的， 即血管内皮细胞(EC)萌芽和生长，以及血管生成，即新生血管 由循环中招募的血管干细胞(VSC)产生。这两个迁移过程都是 主要由缺血诱导的血管内皮生长因子(VEGF)刺激。我们的初步数据 血管内皮生长因子介导的血管内皮细胞和血管干细胞迁移的新调控机制 通过肌动蛋白结合Profilin-1(PFN-1)的磷酸化。血管谱系特异性的磷酸化敲入- 小鼠死亡的PFN-1Y129F突变体表明PFN-1的磷酸化在缺血诱导中起关键作用 后肢血管新生。PFN-1磷酸化缺陷抑制内皮细胞在主动脉中的萌发 和创伤诱导的新生血管，并抑制VSC归巢到缺血的后肢， 提示PFN-1的磷酸化在血管生成和血管生成中都起着关键作用。机械论 研究表明，PFN-1的磷酸化增加了PFN-1与G-肌动蛋白的结合，并促进了肌动蛋白 聚合和细胞迁移。有趣的是，PFN-1的磷酸化直接由VEGFR2/Src诱导， 独立于经典的PI3K介导的多步信号级联。此外，PFN-1的磷酸化是 在心肌梗死患者的心肌梗死组织毛细血管中强健且优先诱导。基座 根据这些发现，我假设PFN-1的磷酸化代表了一个新的、关键的调节节点 在心肌梗死(MI)后组织修复和再生过程中的新生血管。为了测试 在这一假说中，我建议研究两个特定的目的：1)确定pfn-1磷酸化的作用 在心肌梗死后组织修复和再生过程中的新生血管。我们将确定PFN-1的作用 血管内皮细胞介导的血管生成以及VSC归巢到缺血心脏及其后续的磷酸化 小鼠心肌梗死模型中的血管生成。我们还将测试实验性疗法， 用于治疗心肌梗死的磷酸化刺激肽。2)确定血管内皮生长因子的调控机制- 诱导PFN-1磷酸化。我将确定pfn-1的VEGFR2/Src的结构域(S)和特定位点(S) 磷酸化。我将通过确定潜在的蛋白质结合伙伴来确定调控机制(S) 和酪氨酸磷酸酶(S)。这些研究将为深入了解生物多样性的调控机制提供重要的启示。 心肌梗死后新生血管，并可能导致新的治疗方法的发展 人类的缺血性心脏病。

PROJECT SUMMARY Ischemic heart disease, including myocardial infarction (MI), is the leading cause of morbidity and mortality worldwide. Neovascularization, the formation of new blood vessels, is fundamental to cardiac repair and regeneration after MI. Neovascularization proceeds by angiogenesis, i.e., vascular endothelial cells (EC) sprouting and outgrowth, and by vasculogenesis, i.e., de novo vessel generation by recruited vascular stem cells (VSC) from circulation. Both of these migratory processes are primarily stimulated by ischemia-inducible vascular endothelial growth factor (VEGF). Our preliminary data reveal a new regulatory mechanism for vascularized EC and VSC migration in response to VEGF, mediated by phosphorylation of actin-binding profilin-1 (Pfn-1). Vascular lineage-specific knock-in of phosphorylation- dead Pfn-1Y129F mutant in mice demonstrates that Pfn-1 phosphorylation is critical for ischemia-induced neovascularization in the hindlimb. Deficiency in Pfn-1 phosphorylation inhibits EC sprouting in the aorta and wound-induced neovascularization, and also suppresses VSC homing to the ischemic hindlimb, suggesting a critical role of Pfn-1 phosphorylation in both angiogenesis and vasculogenesis. Mechanistic studies show that Pfn-1 phosphorylation increases Pfn-1 binding to G-actin, and promotes actin polymerization and cell migration. Interestingly, Pfn-1 phosphorylation is directly induced by VEGFR2/Src, independent of classic PI3K-mediated multistep signal cascades. Furthermore, Pfn-1 phosphorylation is robustly and preferentially induced in the capillaries of infarcted cardiac tissue in human MI patients. Based on these findings, I hypothesize that Pfn-1 phosphorylation represents a novel, critical regulatory node in neovascularization during tissue repair and regeneration after myocardial infarction (MI). To test this hypothesis, I propose to investigate two specific aims: 1) To determine the role of Pfn-1 phosphorylation in neovascularization during tissue repair and regeneration after MI. We will determine the role of Pfn-1 phosphorylation in EC-mediated angiogenesis as well as in VSC homing to the ischemic heart and sequent vasculogenesis in a murine myocardial infarction model. We will also test experimental therapy with a phosphorylation-stimulatory peptide for treating MI. 2) To determine the regulatory mechanism of VEGF- induced Pfn-1 phosphorylation. I will determine the domain(s) and specific site(s) of VEGFR2/Src for Pfn-1 phosphorylation. I will determine the regulatory mechanism by identifying potential protein-binding partner(s) and tyrosine phosphatase(s). These studies will provide important insight into the mechanisms controlling post-MI neovascularization, and may lead to the development of novel therapeutic approaches for treating ischemic heart disease in humans.