Genetic analysis of heparan sulfate in vascular biology

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Endothelial cells express heparan sulfate proteoglycans that bind to plasma and extracellular matrix proteins 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 genetically altering endothelial HS in mice. The specific genes that will be studied include two N-deacetylase/N-sulfotransferase isozymes (NDST1 and NDST2) that initiate the modification reactions, the uronosyl 2-O-sulfotransferase (HS2OST) and glucosaminyl 6-O-sulfotransferase-1 (HS6OST1), which generate the binding sites for ligands on the chains. Systemic inactivation of NDST1 and HS2OST in mice have shown that they are essential for early development, making it difficult to assess their role in physiology in adult animals. Therefore, our plan consists of examining mutant mice with selective endothelial-cell null mutations in these genes, using the Cre-loxP recombination system. Mutant alleles of NDST1 have already been made, and targeting of HS2OST and HS6OST1 are in different stages of development. Targeted deletion of these genes in endothelial cells and leukocytes will be accomplished by breeding to Tie2Cre mice. For each mutant, the degree of penetrance of the mutations will be assessed in isolated cells, and the proteoglycan and glycosaminoglycan composition will be analyzed. Bone marrow transplantation and adoptive transfer experiments will allow us to study heparan sulfate in both the endothelium and in leukocytes. To examine the role of heparan sulfate in hemostasis, blood coagulation, thrombus formation and fibrinolysis will be examined, with particular emphasis on heparin-binding proteinases (Protein C and tissue plasminogen activator) and serpins (antithrombin, tissue factor pathway inhibitor, and plasminogen activator inhibitor-I). Initial experiments indicate that the bleeding time is altered in NDST1 deficient mice, which may reflect alterations in one or more of these factors. Inflammatory responses appear to be depressed in the mutant as well, in models of thioglycollate-induced peritonitis, oxazolone-induced allergic contact dermatitis, and excisional wound healing. To study these systems further, the effect of altering heparan sulfate on P- and Lselectin and neutrophil chemokines KC and MIP-2 will be examined. The long range goal of these studies is to determine the role of heparan sulfate in vascular biology, with the purpose of defining potential targets for pharmaceutical intervention.