Vascular Differentiation and Patterning

描述(申请人提供)：血管构型的负调控是血管生物学中最不了解的过程之一。在高等脊椎动物中，血管在胚胎的大部分部位发育。但在脊索周围中线的一个区域，尽管存在多个血管形成的阳性信号，但血管没有形成。这个中线无血管区一直保持到两侧背主动脉融合形成单一的背主动脉。我们最近发现，脊索来源的BMP拮抗剂作为血管生长的负调控因子，对中线无血管区的形成负责。这表明了BMP拮抗剂在血管发育中的一个新的抑制作用。然而，它没有解释BMP拮抗剂是如何在存在强大的促进血管形成的信号(如血管内皮生长因子)的情况下抑制血管形成的。中线区域如何改变以允许成对的背主动脉在以后的发育中融合也仍然不确定。我们的初步数据显示：a)脊索和BMP拮抗剂都可以抑制培养的中胚层内皮细胞分化标志物的表达；b)BMP拮抗剂处理分化的内皮细胞导致血管内皮生长因子受体VEGFR2蛋白(也称为Flk1)的快速丢失，而VEGFR2的mRNA表达保持不变；以及c)随着成对的背主动脉开始沿中线融合，BMP拮抗剂在脊索的表达下降。这些结果导致假设：1)局部抑制内皮细胞分化是脊索和BMP拮抗剂在中线形成无血管区的一个机制；2)BMP拮抗剂诱导的对分化内皮细胞的抑制是通过转录后抑制VEGFR2蛋白积累而介导的；以及3)BMP拮抗剂在脊索发育受控的下降允许双侧背主动脉在发育后期沿着中线融合。我们将通过在原始中胚层、分化内皮细胞和成对的背主动脉中移除和添加BMP拮抗剂来检验这三个假说，并分析由此产生的内皮细胞分化和内皮细胞中VEGFR2水平的变化以及背主动脉融合。本文提出的研究将确定一种新的信号机制，调节血管分化和图案化，并为成人血管疾病的合理治疗奠定基础。与公共卫生相关：对于损伤或先天性疾病后血管再生或修复的未来治疗方法，将受益于对调节血管发育的机制的清楚了解。已经确定了许多积极的信号机制，但对负面调控知之甚少。该项目旨在首次确定一种调节血管细胞分化和功能的新的抑制机制。

DESCRIPTION (provided by applicant): The negative regulation of vascular patterning is one of the least understood processes in vascular biology. In higher vertebrates, blood vessels develop throughout most of the embryo. But in one region at the midline surrounding the notochord, despite the presence of multiple positive signals for vessel formation, blood vessels do not form. This midline avascular zone is maintained until the bilateral dorsal aortae fuse to form a single dorsal aorta. We recently found that notochord-derived BMP antagonists serve as negative regulators of vascular growth and are responsible for midline avascular zone genesis. This demonstrates a novel inhibitory role of BMP antagonists in vessel development. However, it does not explain how BMP antagonists inhibit vessel formation in the presence of potent vessel promoting signals, such as VEGF. It also remains uncertain how the midline region changes to permit the paired dorsal aortae to fuse later in development. Our preliminary data show that: a) both notochord and BMP antagonists can suppress the expression of endothelial cell differentiation markers in cultured mesoderm; b) BMP antagonist-treatment of differentiated endothelial cells results in a rapid loss of VEGF receptor Vegfr2 protein (also known as Flk1), while Vegfr2 mRNA levels remain unaffected; and c) the expression of BMP antagonists in the notochord declines as the paired dorsal aortae begin to fuse along the midline. These results lead to the hypotheses that: 1) local suppression of endothelial cell differentiation is a mechanism of avascular zone formation by notochord and BMP antagonists at the midline; 2) BMP antagonist- induced inhibition of differentiated endothelial cells is mediated by post-transcriptional suppression of Vegfr2 protein accumulation; and 3) a developmentally regulated decline of BMP antagonists in the notochord allows the bilateral dorsal aortae to fuse along the midline later in development. We will test these three hypotheses by removing and adding BMP antagonists to naive mesoderm, differentiated endothelial cells, and paired dorsal aortae, and analyzing alteration in resulting endothelial cell differentiation and Vegfr2 levels in endothelial cells as well as dorsal aortae fusion. The studies proposed here will identify a novel signaling mechanism that regulates vessel differentiation and patterning, and build a foundation for rational therapeutics of vascular disorder in adults. PUBLIC HEALTH RELEVANCE: Future therapeutic approaches to regeneration or repair of blood vessels after injury or congenital disease would benefit from a clear understanding of the mechanisms that regulate vessel development. Many positive signaling mechanisms have been identified, but little is known about negative regulation. This project aims to determine, for the first time, a novel inhibitory mechanism that regulates vascular cell differentiation and function.