Endothelial cells express heparan sulfate proteoglycans that bind to plasma proteins and leukocytes 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 creating mutant mice with altered endothelial heparan sulfate proteoglycans and by examining how these changes affect blood vessel structure, blood coagulation, fibrinolysis and leukocyte adhesion. The fine structure of heparan sulfate depends on the expression of two different N-deacetylase/N-sulfotransferases that N-deacetylate subsets of N-acetylglucosamine residues and simultaneously add negatively charged sulfate to the free amino groups. cDNA clones have been reported for both N-deacetylase/N- sulfotransferases, but their relative contribution to heparan sulfate formation in endothelial cells has not been described. Therefore, our plan consists of generating mutant mice with deletions in one or both genes. Specifically, deletions in endothelial cells will be made by breeding animals containing an allele flanked by loxP recombination sites with mice expressing Cre recombinase under the control of the tek promoter. A second specific aim involves tissue-specific disruption of glypican-1, a GPI-linked heparan sulfate proteoglycan expressed by endothelial cells. If glypican-1 deletion leads to a phenotype, additional mutants will be prepared to assess the function of the heparan sulfate chains versus the core protein.
The third aim focuses on the biological outcomes of the proposed mutations, with emphasis on the role of heparan sulfate and glypican-1 in vascular biology. For each mutant, the proteoglycan and glycosaminoglycan composition will be analyzed in tissues and isolated endothelial cells. Histological examination will reveal if the mutations affect the development of the vasculature. Blood coagulation, thrombus formation and fibrinolysis will be measured to assess how the altered proteoglycan composition affects hemostasis. Finally, the effect of altering heparan sulfate on an inflammatory response will be evaluated to test the hypothesis that endothelial cell heparan sulfate may serve as a counter receptor for P-selectin and L-selectin. Altering N-sulfation of heparan sulfate and glypican-1 expression in mice should provide insights into proteoglycan function in normal tissues and cells and clarify their role in disease.
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