Vascular Differentiation and Patterning

描述（由申请人提供）：血管模式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，血管发育遍及胚胎的大部分。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，但血管并没有形成。这一中线无血管区一直维持到双侧背侧主动脉融合形成单个背侧主动脉。我们最近发现，脊索来源的BMP拮抗剂作为负调节血管生长，并负责中线无血管区的发生。这证明了BMP拮抗剂在血管发育中的新的抑制作用。然而，它没有解释BMP拮抗剂如何在有效的血管促进信号如VEGF存在下抑制血管形成。它也仍然不确定中线区域如何变化，以允许成对的背侧骨在发育后期融合。我们的初步数据表明：a）脊索和BMP拮抗剂都可以抑制培养的中胚层中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VEGF受体Vegfr 2蛋白的快速丧失（也称为Flk 1），而Vegfr 2 mRNA水平保持不受影响;和c）当成对的背侧脊肌开始沿中线沿着融合时，脊索中BMP拮抗剂的表达下降。这些结果导致以下假设：1）内皮细胞分化的局部抑制是脊索和BMP拮抗剂在中线处形成无血管区的机制; 2）BMP拮抗剂诱导的分化内皮细胞的抑制是由Vegfr 2蛋白积累的转录后抑制介导的;和3）脊索中BMP拮抗剂的发育调节性下降使得双侧背侧嵴髓在发育后期沿沿着融合。我们将测试这三个假设，通过去除和添加BMP拮抗剂幼稚中胚层，分化的内皮细胞，和配对的背侧骨，并分析改变所产生的内皮细胞分化和血管内皮细胞中的Vegfr 2水平以及背侧骨融合。本文提出的研究将确定一种新的信号机制，调节血管分化和模式，并建立一个合理的治疗成人血管疾病的基础。公共卫生相关性：未来的治疗方法，再生或修复血管损伤或先天性疾病后，将受益于一个清晰的了解机制，调节血管的发展。许多积极的信号机制已被确定，但很少有人知道负调控。该项目旨在首次确定一种调节血管细胞分化和功能的新型抑制机制。

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.