ENDOTHELIAL MATRIX IN ATHEROGENESIS

促使正常动脉变细的关键因素 动脉粥样硬化似乎是富含载脂蛋白B的内皮下滞留， 脂蛋白类在本建议中，我们将研究 已知具有与富含载脂蛋白B的脂蛋白结合的作用。让一个分子 方法，我们已经克隆，测序，并表达了人类cDNA， 软骨素-6-磺基转移酶（C6 ST）是C6 S生物合成的关键酶。 我们已经记录了在人内皮细胞中的表达， 特征基因组克隆。 目的I：调节内皮细胞蛋白多糖的组装和功能， 体外我们将重点关注四种调节刺激， 蛋白聚糖结构并与动脉粥样硬化形成有关：剪切应力， 缺氧、氧化脂蛋白和细胞因子。蛋白聚糖组装 通过C6：C4硫酸盐比率评估培养的内皮细胞， C6 ST mRNA和蛋白表达，C6 ST mRNA转录和稳定性， C6 ST的磷酸化和亚细胞分布以及C6 ST的表达 启动子构建体。将通过亲和力评估蛋白聚糖功能 共电泳（ACE）凝胶，以建立与人抗体的结合构建体。 低密度脂蛋白，并通过我们的细胞培养模型的脂蛋白保留。 目的II：内皮特异性C6 ST转基因动物的动脉粥样硬化发生。到 直接检测内皮基质变异对 在体内动脉粥样硬化形成中，我们建议创建C6 ST转基因， 表达局限于大血管内皮。C6 ST的分布 将在显微镜下检查信息和蛋白质， 将如目的I中所述评估蛋白聚糖。LDL的保留 离体和体内血管内皮功能与动脉粥样硬化 与高脂血症apoE基因敲除小鼠杂交后病变发展 然后将被确定。 目的III：C6 ST在人类疾病中的作用。康贝特人将以 正常人和动脉粥样硬化患者C6 ST信息和蛋白的分布 人类动脉：筛查C6 ST多态性患者， 有无疾病的心导管检查; C6 ST位点与低C6 S疾病有关。 总的来说，这些拟议的研究将大大提高我们的 了解内皮基质组装，这可能会 导致动脉粥样硬化病变发展的巨大变化 动脉部位之间和具有相似血脂的个体之间 数据区.

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.