Genetic Analysis of Heparan Sulfate in Vascular Biology

项目3的目标是研究硫酸化糖胺多聚糖(肝素、软骨素和 皮肤素硫酸盐)在血管生理学中的作用，特别强调葡聚糖的硫化模式 锁链。在过去的授权期内，我们成功地创造了改变了硫酸乙酰肝素的条件突变 生物合成，并检查伤口修复和肿瘤生长。在这次更新中，我们计划完成我们的研究 改变糖醛酸2-0-硫化和氨基葡萄糖6-O-硫化对硫酸乙酰肝素的影响 然后将这种分析扩展到主要的硫酸半乳糖胺聚糖、软骨素-4-硫酸盐和 硫酸皮肤素。在大多数组织中，软骨素和硫酸皮肤素被归类为结构角色 或作为病理生理学的促成因素，但其他数据表明，这些多糖也可以调节 生长因子的结合和激活。大多数可用数据来自体外结合研究或细胞 培养实验。因此，需要进行基因研究来确定这些因素的相关性 活体观察。因为我们预计糖胺多糖生物合成中的系统性突变将 结果导致胚胎或围产期死亡，我们建议将目标基因的条件等位基因 选择性地改变其在内皮细胞和中性粒细胞中的表达。为达致这些目标，我们建议 制备靶向构建物并建立糖胺多聚糖相关酶缺陷的小鼠系 生物合成，特别是硫酸乙酰肝素6-O-磺酸转移酶(H6st)，软骨素4-O-磺酸转移酶 (C4st2)和皮肤素4-O-磺基转移酶(D4st1)。具有组织特异性的纯合子零动物和突变体 将使用CRE小鼠进行删除。我们的计划是研究改变的后果 血管内皮细胞和中性粒细胞中的糖胺多聚糖，并通过检测突变对 白细胞的募集和渗出以及伤口和肿瘤的生长。拟议中的实验 代表了一种确定肝素、软骨素和皮肤素功能的经验性遗传方法 细胞和组织中的硫酸盐。这些发现可能会产生新的见解，导致药物干预 治疗慢性疾病，如炎症和癌症。此外，该项目还将为 其他研究人员对糖胺聚糖在其他器官系统中的生理功能感兴趣。

The objective of Project 3 is to study the function of sulfated glycosaminoglycans (heparan, chondroitin and dermatan sulfates) in vascular physiology, with particular emphasis on the sulfation pattern of the glycan chains. Over the last grant period, we successfully created conditional mutants altered in heparan sulfate biosynthesis and examined wound repair and tumor growth. In this renewal we plan to finish our studies of heparan sulfate by examining the impact of altering uronic acid 2-0-sulfation and glucosamine 6-O-sulfation and then extend this analysis to the major sulfated galactosaminoglycans, chondroitin-4-sulfate and dermatan sulfate. In most tissues, chondroitin and dermatan sulfate have been relegated to structural roles or as contributory factors to pathophysiology, but other data suggest that these glycans can also mediate growth factor binding and activation. Most of the available data derives from in vitro binding studies or cell culture experiments. Thus, a need exists for genetic studies to determine the relevance of these observations in vivo. Because we anticipate that systemic mutations in glycosaminoglycan biosynthesis will result in embryonic or perinatal lethality, we propose to make conditional alleles of the target genes and to alter their expression selectively in endothelial cells and neutrophils. To achieve these goals, we propose to prepare targeting constructs and create mouse lines defective in enzymes involved in glycosaminoglycan biosynthesis, specifically heparan sulfate 6-O-sulfotransferase (H6st), chondroitin 4-O-sulfotransferase (C4st2), and dermatan 4-O-sulfotransferase (D4st1). Homozygous null animals and mutants with tissuespecific deletions will be made using Cre mice. Our plan is to examine the consequences of altering glycosaminoglycans in endothelial cells and neutrophils and by examining the effect of the mutations on leukocyte recruitment and diapedesis as well as wound and tumor growth. The proposed experiments represent an empirical, genetic approach for determining the function of heparan, chondroitin and dermatan sulfate in cells and tissues. The findings could yield new insights leading to pharmaceutical interventions for treating chronic diseases, such as inflammation and cancer. Additionally, the project will provide tools for other investigators interested in the physiological function of glycosaminoglycans in other organ systems.