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.
