ENDOTHELIAL HEPARAN SULFATE IN VASCULAR BIOLOGY

内皮细胞表达硫酸乙酰肝素蛋白聚糖， 通过碳水化合物-蛋白质转化为血浆蛋白和白细胞 交互.这些相互作用导致了这样的假设， 内皮细胞蛋白多糖在血管内皮细胞中起重要作用， 生物学和止血。本项目的目标是测试 这个想法是通过制造具有改变的内皮乙酰肝素的突变小鼠， 硫酸蛋白聚糖并通过检查这些变化如何影响 血管结构、血液凝固、纤维蛋白溶解和 白细胞粘附 硫酸乙酰肝素的精细结构取决于 两种不同的N-脱乙酰基酶/N-磺基转移酶的表达 N-乙酰葡糖胺残基的N-脱乙酰化亚类， 同时将带负电荷的硫酸盐加入游离氨基中， 组已经报道了N-脱乙酰基酶/N-脱乙酰基酶的cDNA克隆。 磺基转移酶，但它们对乙酰肝素的相对贡献 内皮细胞中的硫酸盐形成尚未被描述。 因此，我们的计划包括产生突变小鼠， 一个或两个基因缺失。具体而言， 内皮细胞将通过培育含有 侧接loxP重组位点的等位基因与表达Cre的小鼠 在TEK启动子控制下的重组酶。第二特定 目的涉及组织特异性破坏磷脂酰肌醇蛋白聚糖-1，一种与GPI相关的 内皮细胞表达硫酸乙酰肝素蛋白多糖。如果 磷脂酰肌醇蛋白聚糖-1缺失导致表型，另外的突变体将 准备评估硫酸乙酰肝素链的功能 与核心蛋白质的对比。第三个目标是生物学 建议的突变的结果，重点是 硫酸乙酰肝素和磷脂酰肌醇蛋白聚糖-1。对于每个突变体， 分析蛋白聚糖和糖胺聚糖组成 在组织和分离的内皮细胞中。组织学检查 将揭示突变是否会影响 脉管系统凝血、血栓形成和纤溶 以评估改变的蛋白聚糖组成 影响止血。最后，改变硫酸乙酰肝素 对炎症反应的影响将被评估以检验假设 内皮细胞硫酸乙酰肝素可以作为一种对抗剂 P-选择素和L-选择素受体。改变的N-硫酸化 小鼠中硫酸乙酰肝素和磷脂酰肌醇蛋白聚糖-1表达应该提供 了解蛋白聚糖在正常组织和细胞中的功能， 阐明它们在疾病中的作用。

Endothelial cells express heparan sulfate proteoglycans that bind to plasma proteins and leukocytes through carbohydrate-protein interactions. These interactions have led to the hypothesis that endothelial cell proteoglycans play important roles in vascular biology and hemostasis. The objective of this project is to test this idea by creating mutant mice with altered endothelial heparan sulfate proteoglycans and by examining how these changes affect blood vessel structure, blood coagulation, fibrinolysis and leukocyte adhesion. The fine structure of heparan sulfate depends on the expression of two different N-deacetylase/N-sulfotransferases that N-deacetylate subsets of N-acetylglucosamine residues and simultaneously add negatively charged sulfate to the free amino groups. cDNA clones have been reported for both N-deacetylase/N- sulfotransferases, but their relative contribution to heparan sulfate formation in endothelial cells has not been described. Therefore, our plan consists of generating mutant mice with deletions in one or both genes. Specifically, deletions in endothelial cells will be made by breeding animals containing an allele flanked by loxP recombination sites with mice expressing Cre recombinase under the control of the tek promoter. A second specific aim involves tissue-specific disruption of glypican-1, a GPI-linked heparan sulfate proteoglycan expressed by endothelial cells. If glypican-1 deletion leads to a phenotype, additional mutants will be prepared to assess the function of the heparan sulfate chains versus the core protein. The third aim focuses on the biological outcomes of the proposed mutations, with emphasis on the role of heparan sulfate and glypican-1 in vascular biology. For each mutant, the proteoglycan and glycosaminoglycan composition will be analyzed in tissues and isolated endothelial cells. Histological examination will reveal if the mutations affect the development of the vasculature. Blood coagulation, thrombus formation and fibrinolysis will be measured to assess how the altered proteoglycan composition affects hemostasis. Finally, the effect of altering heparan sulfate on an inflammatory response will be evaluated to test the hypothesis that endothelial cell heparan sulfate may serve as a counter receptor for P-selectin and L-selectin. Altering N-sulfation of heparan sulfate and glypican-1 expression in mice should provide insights into proteoglycan function in normal tissues and cells and clarify their role in disease.