Profilin-1 phosphorylation in neovascularization

描述(由申请人提供)：包括心肌梗死(MI)在内的缺血性心脏病是全球发病率和死亡率的主要原因。新生血管的形成是心肌梗死后心脏修复和再生的基础。新生血管通过“血管生成”，即血管内皮细胞(EC)萌芽和生长，以及“血管生成”，即从循环中招募的血管干细胞(VSC)生成新生血管。这两个迁移过程主要是由缺血诱导的血管内皮生长因子(VEGF)刺激的。我们的初步数据揭示了一种新的调节血管内皮细胞和血管干细胞迁移的机制，该机制是通过肌动蛋白结合蛋白-1(PFN-1)的磷酸化来调节血管内皮细胞和血管干细胞对血管内皮生长因子的反应。在小鼠中，血管谱系特异性敲入磷酸化死亡的PFN-1Y129F突变体表明，PFN-1的磷酸化在缺血诱导的后肢新生血管中是关键的。PFN-1磷酸化缺陷抑制了血管内皮细胞在主动脉中的萌发和创伤诱导的新生血管，也抑制了VSC归巢到缺血的后肢，提示PFN-1磷酸化在血管生成和血管生成中都起着关键作用。机制研究表明，PFN-1的磷酸化增加了PFN-1与G-肌动蛋白的结合，并促进了肌动蛋白的聚合和细胞迁移。有趣的是，PFN-1的磷酸化是由VEGFR2/Src直接诱导的，不依赖于经典的PI3K介导的多步信号级联反应。此外，在人类心肌梗塞患者的心肌梗死组织毛细血管中，PFN-1的磷酸化被强烈且优先地诱导。基于这些发现，我推测在心肌梗死(MI)后的组织修复和再生过程中，PFN-1的磷酸化是一个新的、关键的新生血管调节节点。为了验证这一假设，我建议调查两个具体目标：1) 目的：探讨心肌梗死后组织修复和再生过程中PFN-1的磷酸化在新生血管形成中的作用。我们将在小鼠心肌梗死模型中确定PFN-1磷酸化在EC介导的血管生成以及VSC归巢到缺血心脏和随后的血管生成中的作用。我们还将测试一种磷酸化刺激多肽治疗心肌梗死的实验疗法。2)探讨血管内皮生长因子诱导的PFN-1磷酸化的调控机制。我将确定VEGFR2/Src的结构域(S)和特异性位点(S)，以实现pfn-1的磷酸化。我将通过确定潜在的蛋白结合伙伴(S)和酪氨酸磷酸酶(S)来确定调控机制。这些研究将为控制心肌梗死后新生血管的机制提供重要的见解，并可能导致开发新的治疗方法来治疗人类缺血性心脏病。

DESCRIPTION (provided by applicant): 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 wil provide important insight into the mechanisms controling post-MI neovascularization, and may lead to the development of novel therapeutic approaches for treating ischemic heart disease in humans.