Profilin-1 phosphorylation in neovascularization

缺血性心脏病，包括心肌梗死（MI），是导致 的发病率和死亡率。新血管形成，即新血管的形成， 心肌梗死后心脏修复和再生的基础。新生血管化的进展， 血管生成, 也就是说，血管内皮细胞（EC）发芽和生长， 血管发生，也就是，新生血管 通过从循环中募集的血管干细胞（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 揭示了血管化EC和VSC迁移响应VEGF的新的调节机制， 通过肌动蛋白结合profilin-1（Pfn-1）的磷酸化。血管谱系特异性磷酸化敲入- 小鼠中死亡的Pfn-1 Y129 F突变体表明Pfn-1磷酸化对于缺血诱导的 后肢的新血管形成Pfn-1磷酸化缺陷抑制主动脉EC出芽 和伤口诱导的新血管形成，并且还抑制VSC归巢到缺血后肢， 提示Pfn-1磷酸化在血管生成和血管发生中的关键作用。机械论 研究表明，Pfn-1磷酸化增加Pfn-1与G-肌动蛋白的结合，并促进肌动蛋白 聚合和细胞迁移。有趣的是，Pfn-1磷酸化直接由VEGFR 2/Src诱导， 独立于经典的PI 3 K介导的多步信号级联。此外，Pfn-1磷酸化是 在人MI患者的梗塞心脏组织的毛细血管中强烈且优先诱导。基于 基于这些发现，我假设Pfn-1磷酸化代表了一个新的、关键的调节节点 心肌梗死（MI）后组织修复和再生过程中的新血管形成。测试 基于这一假设，我提出了两个具体的研究目标：1）确定Pfn-1磷酸化的作用 心肌梗死后组织修复和再生过程中的新血管形成。我们将确定Pfn-1的作用 磷酸化在EC介导的血管生成中以及在VSC归巢到缺血性心脏和缺血性心脏中起作用。 小鼠心肌梗死模型中的血管发生。我们还将测试实验性疗法， 用于治疗MI的磷酸化刺激肽。2)为了明确VEGF的调控机制， 诱导Pfn-1磷酸化。我将确定Pfn-1的VEGFR 2/Src的结构域和具体位点 磷酸化我将通过识别潜在的蛋白质结合伴侣来确定调控机制 和酪氨酸磷酸酶。这些研究将提供重要的洞察机制控制 心肌梗死后新血管形成，并可能导致新的治疗方法的发展，用于治疗 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.