ENDOTHELIAL MATRIX IN ATHEROGENESIS

刺激正常动脉形成的关键刺激事件 动脉粥样硬化似乎是富含载脂蛋白B的内皮下滞留 动脉基质中的脂蛋白。在这项建议中，我们将研究 已知的作用是贪婪地结合富含载脂蛋白的脂蛋白。允许一种分子 方法，我们已经克隆、测序并表达了人的cdna。 软骨素-6-磺酸转移酶(C6ST)是C6S生物合成的关键酶。 我们记录了在人内皮细胞中的表达，并获得了 描述了基因组克隆的特征。 目的I：调节血管内皮细胞蛋白多糖的组装和功能 体外培养。我们将重点关注四个监管刺激，每一个都会发生变化 蛋白多糖结构，并与动脉粥样硬化的发生有关：剪切力， 低氧、氧化的脂蛋白和细胞因子。蛋白多糖通过以下方式组装 培养的内皮细胞将通过C6：C4硫酸盐比率进行评估， C6ST mRNA和蛋白的表达、C6ST mRNA的转录和稳定性 C6ST的磷酸化及亚细胞分布和表达 启动子构建。蛋白多糖功能将通过亲和力进行评估 共电泳(ACE)凝胶建立与人结合的结构 低密度脂蛋白，以及我们的脂蛋白滞留的细胞培养模型。 目的II：内皮特异性C6ST转基因中的动脉粥样硬化形成。至 直接检查内皮细胞基质变化对 体内动脉粥样硬化的发生，我们建议创建C6ST转基因基因 表达仅限于大血管内皮细胞。C6ST的分布 消息和蛋白质将在显微镜下检查，主动脉 蛋白多糖的评估将如目标一所述。低密度脂蛋白在 体外和体内的主动脉、内皮功能和动脉粥样硬化 与高脂血症载脂蛋白E基因敲除小鼠杂交后病变的发展 然后就会确定了。 目的III：C6ST在人类疾病中的作用。我们将确定 C6ST信息和蛋白在正常和动脉粥样硬化中的分布 人类动脉：筛查经证实的患者的C6ST基因多态性 有病或无病的心导管置入术 C6ST基因座为低C6s病。 总括而言，这些建议的研究将大大加强我们的 了解内皮细胞基质组装，这可能会 导致动脉粥样硬化病变发展的巨大差异 动脉部位之间以及具有相似血脂的个体之间 配置文件。

The key instigating event that provokes a normal artery to become atherosclerotic appears to be the sub-endothelial retention of apoB-rich lipoproteins by arterial matrix. In this proposal, we will examine the role known to avidly bind apoB-rich lipoproteins. To allow a molecular approach, we have cloned, sequenced, and expressed the human cDNA for chondroitin-6-sulfotransferase (C6ST), the key enzyme in C6S biosynthesis. We have documented expression in human endothelial cells and obtained and characterized genomic clones. Aim I: Regulation of endothelial proteoglycan assembly and function in vitro. We will focus on four regulatory stimuli, each of which changes proteoglycan structure and has been linked to atherogenesis: shear stress, hypoxia, oxidized lipoproteins, and cytokines. Proteoglycan assembly by cultured endothelial cells will be assessed by the C6:C4 sulfate ration, expression of C6ST mRNA & protein, C6ST mRNA transcription & stability, C6ST phosphorylation & subcellular distribution, and expression of C6ST promoter constructs. Proteoglycan function will be assessed by affinity co-electrophoresis (ACE) gels to establish binding constructs to human LDL, and by our cell-culture model of lipoprotein retention. Aim II: Atherogenesis in endothelial-specific C6ST transgenics. To directly examine the effects of endothelial matrix variations on atherogenesis in vivo, we propose to create C6ST transgenics with expression limited to large-vessel endothelium. Distribution of C6ST message and protein will be examined microscopically, and aortic proteoglycans will be assessed as described in Aim I. Retention of LDL in aortae ex vivo and in vivo, endothelial function, and atherosclerotic lesion development after crossing to hyperlipidemic apoE knock-out mice will then be determined. Aim III: The role of C6ST in human disease. We will determine the distribution of C6ST message and protein in normal and atherosclerotic human arteries: screen for C6ST polymorphisms in patients proven by cardiac catheterization to be with or without disease; & test linkage of the C6ST locus with a disease of low C6S. Overall, these proposed studied will substantially enhanced our understanding of endothelial matrix assembly, which is likely to contribute to the large variation in atherosclerotic lesion development between arterial sites and amongst individuals with similar plasma lipid profiles.