Heparan sulfate proteoglycan interacts with Slit/Robo to regulate angiogenesis

描述(申请人提供)：我们研究的长期目标是了解硫酸乙酰肝素蛋白多糖(HSPGs)在血管生物学和相关人类疾病中的作用及其细胞和分子机制。血管生成是一个形成新血管的过程，对发育和一些常见的人类疾病是必不可少的。HSPGs被认为对血管生成具有重要的调控作用。我们在小鼠(T-NDST1-/-小鼠)体内通过内皮细胞(EC)特异性的消融硫酸乙酰肝素(HS)生物合成基因N-脱乙酰酶/N-磺基转移酶-1(NDST1)来验证这一假说。我们观察到T-NDST1-/-小鼠表现出血管生成缺陷，首次在体内证明EC HS调节生理性血管生成。更有趣的是，我们发现T-NDST1-/-横隔膜的局部血管生成缺陷导致先天性横隔疝，这与轴突引导分子SLIT3缺失(SLIT3-/-)小鼠的发育缺陷相似。这一观察结果提示了EC NDST1缺乏可能损害SLIT3的生物学功能，导致T-NDST1-/-小鼠出现SLIT3样表型的潜在机制。我们的研究进一步发现：1)。SLIT3是一种新的血管生成因子；T-NDST1-/-和NDST3-/-小鼠在横隔膜表现出相似的局限性血管生成缺陷；EC NDST1在体内与SLIT3、Robo1和Robo4基因相互作用；内皮细胞NDST1在体内外促进SLIT3诱导的血管生成；Hs同时绑定了SLIT3和Robo1。这些发现导致了我们的中心假设，即EC HSPG与Sit/Robo相互作用，调节血管生成。为了验证这一假设，我们将追求以下三个具体目标：目标。1.研究SLIT3/Robo通路在血管生成中的作用。我们将研究SLIT3和Robo突变小鼠的血管模式和视网膜血管形成，以确定SLIT3/Robo对发育中血管生成的需求。我们已经观察到SLIT3在体内诱导新生血管，并将进一步确定SLIT3与哪些机器人相互作用来调节血管生成。我们还将确定SLIT3-Robo相互作用对EC功能的影响。目的2.确定EC HS对SLIT3/Robo介导的血管生成的调节作用。我们观察到EC NDST1促进了SLIT3介导的体内血管生成，并将进一步确定ROBO是否受到类似的调控。我们将继续用SLIT3/Robo突变小鼠培育T-NDST1-/-小鼠，以确定EC NDST1是否与SLIT3/Robo存在基因上的相互作用。我们还将确定NDST1缺陷对SLIT3/Robo介导的EC功能和SLIT3/Robo结合的影响。目的3.阐明HS内SLIT3/Robo结合位点的结构特征。我们将生产重组人SLIT3、Robo1和Robo4蛋白，并制备亲和层析柱来分离与SLIT3、Robo1和Robo4结合的HS片段。结合SLIT3/Robo结合的HS片段的结构特征将通过尺寸和组成分析结合HS类似物来推断。公共卫生相关性：血管生成与癌症和组织修复等人类疾病密切相关。因此，我们提出的研究不仅有望显著提高我们对血管生成的理解，而且还可能导致治疗基于血管生成的人类疾病的新的药物干预措施。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the roles and the cellular and molecular mechanisms of heparan sulfate proteoglycans (HSPGs) in vascular biology and related human diseases. Angiogenesis is a process of forming new blood vessels and is essential for development and for some common human diseases. HSPGs have been hypothesized to critically regulate angiogenesis. We tested this hypothesis in vivo by endothelial cell (EC)-specific ablation of the heparan sulfate (HS) biosynthetic gene N-deacetylase/N- sulfotransferase-1 (Ndst1) in mice (T-Ndst1-/- mice). We observed that the T-Ndst1-/- mice display angiogenesis defects, showing the first in vivo evidence that EC HS regulates physiological angiogenesis. More intriguingly, we found that the localized angiogenesis defect in T-Ndst1-/- diaphragm leads to congenital diaphragmatic hernia, which phenocopies the developmental defect in the axon guidance molecule Slit3 null (Slit3-/-) mice. This observation suggests a potential mechanism by which EC Ndst1 deficiency may impair the biological function of Slit3, leading to the Slit3-like phenotype in the T-Ndst1-/- mice. Our studies further found that: 1). Slit3 is a novel angiogenic factor; 2). T-Ndst1-/- and Ndst3-/- mice exhibit similar localized angiogenesis defect in diaphragm; 3). EC Ndst1 interacts genetically with Slit3, Robo1 and Robo4 in vivo; 4). Endothelial Ndst1 facilitates Slit3-induced angiogenesis in vitro and in vivo; and 5). HS binds both Slit3 and Robo1. These findings led to our central hypothesis that EC HSPG interacts with Slit/Robo to regulate angiogenesis. To test this hypothesis, we will pursue the following three specific aims: Aim. 1. Characterize the function of the Slit3/Robo pathway in angiogenesis. We will examine the vascular pattern and retinal vascularization in Slit3 and Robo mutant mice to determine the requirement of Slit3/Robo for developmental angiogenesis. We have observed that Slit3 induces neovascularization in vivo and will further determine the Robos that Slit3 interacts with to regulate angiogenesis. We will also determine the effects of the Slit3-Robo interaction on EC functions. Aim 2. Determine the regulatory role of EC HS on Slit3/Robo-mediated angiogenesis. We have observed that EC Ndst1 facilitates Slit3-mediated angiogenesis in vivo and will further determine whether Robos are similarly regulated. We will continue the breeding of T-Ndst1-/- mice with Slit3/Robo mutant mice to determine if EC Ndst1 interacts genetically with Slit3/Robo. We will also determine the consequences of Ndst1 deficiency on Slit3/Robo-mediated EC function and Slit3/Robo binding. Aim 3. Elucidate the structural features of Slit3/Robo-binding sites within HS. We will produce recombinant human Slit3, Robo1 and Robo4 proteins and prepare affinity columns to isolate Slit3-, Robo1- and Robo4-binding HS fragments. The structural features of Slit3/Robo-binding HS fragments will be deduced by size and composition analyses in conjunction with HS analogs. PUBLIC HEALTH RELEVANCE: Angiogenesis is critically related to human diseases, such as cancer and tissue repair. Therefore, our proposed studies are expected to not only significantly advance our understanding of angiogenesis, but may also lead to novel pharmaceutical interventions for treatment of angiogenesis-based human diseases.