The molecular mechanism of Semaphorin 3E-Plexin-D1 signaling in shaping vascular topology during development

心血管系统的正常功能取决于其在发育过程中的正确模式。除了血管形态发生的主要调节因子VEGF之外，血管图案化还需要一类先前被认为是轴突引导线索的分子（肝配蛋白、脑信号蛋白、狭缝和netrins）。最近发现了一种通过VEGF途径和Semaphorin 3E（Sema 3E）-丛蛋白-D1途径之间的相互作用来调节血管生成的新型信号级联。研究表明，在视网膜中，VEGF直接控制活跃出芽血管前端的内皮细胞中丛蛋白-D1的表达。虽然视网膜神经元分泌的Sema 3E是均匀分布的，但Sema 3E-Plexin-D1信号转导通过VEGF调节Plexin-D1表达而在时间和空间上受到VEGF的调节。然后，Sema 3E-Plexin-D1信号传导负调节VEGF诱导的Dll 4-Notch信号传导途径的活性，其控制顶端和茎细胞命运决定的平衡。因此，Sema 3E-Plexin-D1信号传导对于控制视网膜血管网络的拓扑结构是高度需要的。这种功能在胚胎发育中很重要，但在缺血性视网膜病变等病理条件下也很重要。我的建议建立在这些最新的发现，旨在填补知识的差距差距在理解这种新的反馈机制在血管生成的细胞自主机制。我的第一个目的是研究Sema 3E-Plexin-D1信号在发育血管生成过程中的体内功能。我推测，通过调节VEGF活性，Sema 3E-Plexin-D1信号传导决定了尖端/柄细胞转换的时间，这对于连续的血管网络形成至关重要。我将研究如何将这种负反馈机制转化为尖端/茎细胞的行为，使用视网膜外植体和分化的胚胎干细胞分析结合时间推移成像。我将跟踪细胞行为的Sema 3E-Plexin-D1信号的情况下，并分析其对血管拓扑结构的后果。我的第二个目标是了解Sema 3E-Plexin-D1信号在血管生成过程中如何调节VEGF活性的分子机制。我推测Sema 3E-Plexin-D1通过直接调节VEGFR 2运输或VEGF诱导的Erk磷酸化来调节VEGF活性和下游Dll 4表达。我将使用生物化学和细胞生物学分析以及体内视网膜血管系统来测试这两个假设。总之，我的研究旨在进一步了解基本的分子和细胞机制，管理血管生成过程和神经血管的相互作用。他们还将为治疗心血管疾病、涉及血管生成的疾病和血管生成依赖性肿瘤的潜在治疗策略提供新的思路。

The proper function of the cardiovascular system is dependent on its correct patterning during development. In addition to VEGF, a major regulator of vascular morphogenesis, also a class of molecules previously recognized as axon guidance cues (ephrins, semaphorins, slits, and netrins), is required for vascular patterning. A novel signaling cascade regulating angiogenesis via a reciprocal interaction between the VEGF pathway and the Semaphorin 3E (Sema3E)-Plexin-D1 pathway, was recently discovered. It was shown that in the retina, VEGF directly controls the expression of Plexin-D1 in endothelial cells at the front of actively sprouting blood vessels. Although Sema3E secreted by retinal neurons is evenly distributed, Sema3E-Plexin-D1 signaling is temporally and spatially regulated by VEGF through its regulation of Plexin-D1 expression. Sema3E-Plexin-D1 signaling then negatively regulates the activity of the VEGF-induced Dll4-Notch signaling pathway, which controls the balance of tip and stalk cell fate decisions. Thus, Sema3E-Plexin-D1 signaling is highly required for controling the topology of the retinal vascular network. This function is important in embryonic development, but also in pathlogical conditions like ischemic retinopathy. My proposal builds upon these recent findings and aims to fill the gap of knowledge in understanding the cell autonomous mechanism of this novel feedback mechanism in angiogenesis. My first aim is to examine the in vivo function of Sema3E-Plexin-D1 signaling during developmental angiogenesis. I hypothesize, that by regulating VEGF activity, Sema3E-Plexin-D1 signaling determines the timing of tip/stalk cell switching, which is critical for continuous vascular network formation. I will investigate how this negative feedback mechanism is translated into tip/stalk cell behavior using retinal explant and differentiated embryonic stem cell analysis in combination with time lapse imaging. I will trace cellular behavior in the absence of Sema3E-Plexin-D1 signaling and analyze its consequences on vascular topology. My second aim is to understand the molecular mechanisms of how Sema3E-Plexin-D1 signaling regulates VEGF activity during angiogenesis. I hypothesize that Sema3E-Plexin-D1 modulates VEGF activity and downstream Dll4 expression by directly regulating either VEGFR2 trafficking or VEGF-induced Erk phosphorylation. I will test these two hypotheses using biochemistry and cell biology assays as well as the in vivo retinal vasculature system. Together, my studies aim at furthering the understanding of the fundamental molecular and cellular mechanisms governing angiogenic processes and neurovascular interactions. They will also shed new light on potential therapeutic strategies for the treatment of cardiovascular disease, diseases involving angiogenesis and angiogenesis-dependent tumors.