ENDOTHELIAL HEPARAN SULFATE IN VASCULAR BIOLOGY

内皮细胞表达结合硫酸乙酰肝素的蛋白多糖 通过碳水化合物蛋白作用于血浆蛋白和白细胞 互动。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的重要作用 生物学和止血。该项目的目标是测试 这个想法是通过创造内皮肝素改变的突变小鼠来实现的 通过研究这些变化是如何影响的 血管结构、凝血、纤溶和 白细胞黏附。硫酸乙酰肝素的精细结构取决于 两种不同的N-脱乙酰酶/N-磺酸转移酶的表达 N-乙酰氨基葡萄糖残基的N-脱乙酰化亚群和 同时将带负电荷的硫酸盐加到游离氨基上 组。已报道了N-脱乙酰基酶/N-脱乙酰基酶的cDNA克隆。 磺基转移酶，但它们对肝素的相对贡献 硫酸盐在内皮细胞中的形成尚未被描述。 因此，我们的计划包括用 一个或两个基因的缺失。具体地说，删除 内皮细胞将通过饲养含有一种 带有loxP重组位点的等位基因与表达Cre的小鼠 Tek启动子控制下的重组酶。第二个具体问题 AIM涉及组织特异性地破坏GPI连锁的Glypcan-1 内皮细胞表达硫酸乙酰肝素蛋白多糖。如果 Glypcan-1缺失导致表型，其他突变体将 准备评估硫酸乙酰肝素链的功能 而不是核心蛋白。第三个目标集中在生物学上 建议突变的结果，重点是 血管生物学中的硫酸乙酰肝素和Glypcan-1。对于每个突变体， 将对蛋白多糖和糖胺多糖的组成进行分析 在组织和分离的内皮细胞中。组织学检查 将揭示这些突变是否会影响 脉管系统。凝血、血栓形成和纤溶 将被测量以评估改变的蛋白多糖组成如何 影响止血。最后，改变硫酸乙酰肝素的效果 将对炎症反应进行评估以检验该假说 内皮细胞硫酸乙酰肝素可以作为一种计数器 P-选择素受体和L-选择素受体。改性N-硫酸盐化 硫酸乙酰肝素和Glypcan-1在小鼠中的表达应提供 对正常组织和细胞中蛋白多糖功能的洞察和 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.