Vascular Differentiation and Patterning

描述（申请人提供）：血管模式的负调控是血管生物学中最不为人所知的过程之一。在高等脊椎动物中，血管在胚胎的大部分发育。但在脊索周围中线的一个区域，尽管存在多种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维持到双侧背主动脉融合形成单个背主动脉。我们最近发现脊索来源的BMP拮抗剂作为血管生长的负调节因子，并负责中线无血管区形成。这证明了BMP拮抗剂在血管发育中的新抑制作用。然而，它并不能解释BMP拮抗剂如何在有效的血管促进信号（如VEGF）存在的情况下抑制血管形成。在发育后期，中线区域如何改变以使成对的背主动脉融合仍不确定。我们的初步数据表明：a)脊索和BMP拮抗剂均能抑制培养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VEGF受体Vegfr2蛋白（也称为Flk1）的快速丢失，而Vegfr2 mRNA水平不受影响；c)脊索中BMP拮抗剂的表达随着成对的背主动脉开始沿中线融合而下降。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带的机制；2) BMP拮抗剂诱导分化内皮细胞的抑制是通过抑制Vegfr2蛋白的转录后积累介导的；3)脊索中BMP拮抗剂的发育调控下降，使双侧背主动脉在发育后期沿中线融合。我们将通过在未成熟的中胚层、分化的内皮细胞和配对的背主动脉中去除和添加BMP拮抗剂来验证这三个假设，并分析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调节血管分化和模式的信号机制，并为成人血管疾病的合理治疗奠定基础。公共卫生相关性：对血管发育调节机制的清晰理解将有利于损伤或先天性疾病后血管再生或修复的未来治疗方法。许多积极的信号机制已被确定，但对消极调节知之甚少。本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.