Genetic analysis of heparan sulfate in vascular biology

内皮细胞表达硫酸乙酰肝素蛋白聚糖，其通过碳水化合物-蛋白质相互作用与血浆和细胞外基质蛋白结合。这些相互作用导致了内皮细胞蛋白聚糖在血管生物学和止血中发挥重要作用的假设。本项目的目的是通过基因改变内皮HS来测试这一想法 对小鼠将被研究的特定基因包括两个N-脱乙酰基酶/N-磺基转移酶同工酶（NDST 1和NDST 2），启动修饰反应，糖醛酸基2-O-磺基转移酶（HS 2 OST）和葡糖胺基6-O-磺基转移酶-1（HS 6 OST 1），其产生链上配体的结合位点。NDST 1和HS 2 OST在小鼠中的系统性失活表明，它们对早期发育至关重要，因此难以评估它们在成年动物中的生理作用。因此，我们的计划包括检查这些基因中具有选择性内皮细胞无效突变的突变小鼠，使用Cre-loxP重组系统。NDST 1的突变等位基因已经产生，HS 2 OST和HS 6 OST 1的靶向处于不同的开发阶段。这些基因的靶向缺失， 内皮细胞和白细胞的培养将通过繁殖成Tie 2Cre小鼠来完成。对于每种突变体，将在分离的细胞中评估突变的突变程度，并分析蛋白聚糖和糖胺聚糖的组成。骨髓移植和过继转移实验将使我们能够研究内皮细胞和白细胞中的硫酸乙酰肝素。为了检查硫酸乙酰肝素在止血、凝血、血栓形成和纤维蛋白溶解中的作用，将检查肝素结合蛋白酶（蛋白C和组织纤溶酶原激活物）和丝氨酸蛋白酶抑制剂（抗凝血酶、组织因子途径抑制剂和纤溶酶原激活物-I）。 最初的实验表明，出血时间在NDST 1缺陷小鼠中改变，这可能反映了这些因素中的一个或多个的改变。在巯基乙酸盐诱导的腹膜炎、恶唑酮诱导的过敏性接触性皮炎和切除伤口愈合模型中，突变体的炎症反应似乎也受到抑制。为了进一步研究这些系统，将检查改变硫酸乙酰肝素对P-和L-选择素以及中性粒细胞趋化因子KC和MIP-2的影响。这些研究的长期目标是 确定硫酸乙酰肝素在血管生物学中的作用，目的是确定药物干预的潜在靶点。

Endothelial cells express heparan sulfate proteoglycans that bind to plasma and extracellular matrix proteins 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 genetically altering endothelial HS in mice. The specific genes that will be studied include two N-deacetylase/N-sulfotransferase isozymes (NDST1 and NDST2) that initiate the modification reactions, the uronosyl 2-O-sulfotransferase (HS2OST) and glucosaminyl 6-O-sulfotransferase-1 (HS6OST1), which generate the binding sites for ligands on the chains. Systemic inactivation of NDST1 and HS2OST in mice have shown that they are essential for early development, making it difficult to assess their role in physiology in adult animals. Therefore, our plan consists of examining mutant mice with selective endothelial-cell null mutations in these genes, using the Cre-loxP recombination system. Mutant alleles of NDST1 have already been made, and targeting of HS2OST and HS6OST1 are in different stages of development. Targeted deletion of these genes in endothelial cells and leukocytes will be accomplished by breeding to Tie2Cre mice. For each mutant, the degree of penetrance of the mutations will be assessed in isolated cells, and the proteoglycan and glycosaminoglycan composition will be analyzed. Bone marrow transplantation and adoptive transfer experiments will allow us to study heparan sulfate in both the endothelium and in leukocytes. To examine the role of heparan sulfate in hemostasis, blood coagulation, thrombus formation and fibrinolysis will be examined, with particular emphasis on heparin-binding proteinases (Protein C and tissue plasminogen activator) and serpins (antithrombin, tissue factor pathway inhibitor, and plasminogen activator inhibitor-I). Initial experiments indicate that the bleeding time is altered in NDST1 deficient mice, which may reflect alterations in one or more of these factors. Inflammatory responses appear to be depressed in the mutant as well, in models of thioglycollate-induced peritonitis, oxazolone-induced allergic contact dermatitis, and excisional wound healing. To study these systems further, the effect of altering heparan sulfate on P- and Lselectin and neutrophil chemokines KC and MIP-2 will be examined. The long range goal of these studies is to determine the role of heparan sulfate in vascular biology, with the purpose of defining potential targets for pharmaceutical intervention.