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VEGF外，血管形态发生的主要调节因子，还有一类先前被认为是轴突引导线索的分子（ephrins，信号蛋白，狭缝蛋白和网状蛋白），是血管模式形成所必需的。最近发现了一种新的信号级联，通过VEGF通路和Sema3E -Plexin-D1通路之间的相互作用调节血管生成。结果表明，在视网膜中，VEGF直接控制活跃发芽血管前部内皮细胞Plexin-D1的表达。虽然视网膜神经元分泌的Sema3E分布均匀，但VEGF通过调节Plexin-D1的表达，在时间和空间上调控Sema3E-Plexin-D1信号。Sema3E-Plexin-D1信号随后负向调节vegf诱导的Dll4-Notch信号通路的活性，Dll4-Notch信号通路控制尖端和柄细胞命运决定的平衡。因此，Sema3E-Plexin-D1信号对于控制视网膜血管网络的拓扑结构是非常必要的。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发现的基础上，旨在填补理解血管生成中这种新型反馈机制的细胞自主机制的知识空白。我的第一个目标是研究Sema3E-Plexin-D1信号在发育性血管生成过程中的体内功能。我推测，通过调节VEGF活性，Sema3E-Plexin-D1信号决定了茎尖/茎部细胞转换的时间，这对于连续血管网络的形成至关重要。我将利用视网膜外植体和分化胚胎干细胞分析结合延时成像研究这种负反馈机制是如何转化为尖端/柄细胞行为的。我将追踪缺乏Sema3E-Plexin-D1信号的细胞行为，并分析其对血管拓扑的影响。我的第二个目标是了解Sema3E-Plexin-D1信号在血管生成过程中如何调节VEGF活性的分子机制。我假设Sema3E-Plexin-D1通过直接调节VEGFR2运输或VEGF诱导的Erk磷酸化来调节VEGF活性和下游Dll4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检验这两个假设。总之，我的研究旨在进一步了解控制血管生成过程和神经血管相互作用的基本分子和细胞机制。它们还将为心血管疾病、涉及血管生成的疾病和血管生成依赖性肿瘤的治疗提供新的潜在治疗策略。

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.