Genetic Analysis of Heparan Sulfate in Vascular Biology

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The objective of Project 3 is to study the function of sulfated glycosaminoglycans (heparan, chondroitin and dermatan sulfates) in vascular physiology, with particular emphasis on the sulfation pattern of the glycan chains. Over the last grant period, we successfully created conditional mutants altered in heparan sulfate biosynthesis and examined wound repair and tumor growth. In this renewal we plan to finish our studies of heparan sulfate by examining the impact of altering uronic acid 2-0-sulfation and glucosamine 6-O-sulfation and then extend this analysis to the major sulfated galactosaminoglycans, chondroitin-4-sulfate and dermatan sulfate. In most tissues, chondroitin and dermatan sulfate have been relegated to structural roles or as contributory factors to pathophysiology, but other data suggest that these glycans can also mediate growth factor binding and activation. Most of the available data derives from in vitro binding studies or cell culture experiments. Thus, a need exists for genetic studies to determine the relevance of these observations in vivo. Because we anticipate that systemic mutations in glycosaminoglycan biosynthesis will result in embryonic or perinatal lethality, we propose to make conditional alleles of the target genes and to alter their expression selectively in endothelial cells and neutrophils. To achieve these goals, we propose to prepare targeting constructs and create mouse lines defective in enzymes involved in glycosaminoglycan biosynthesis, specifically heparan sulfate 6-O-sulfotransferase (H6st), chondroitin 4-O-sulfotransferase (C4st2), and dermatan 4-O-sulfotransferase (D4st1). Homozygous null animals and mutants with tissuespecific deletions will be made using Cre mice. Our plan is to examine the consequences of altering glycosaminoglycans in endothelial cells and neutrophils and by examining the effect of the mutations on leukocyte recruitment and diapedesis as well as wound and tumor growth. The proposed experiments represent an empirical, genetic approach for determining the function of heparan, chondroitin and dermatan sulfate in cells and tissues. The findings could yield new insights leading to pharmaceutical interventions for treating chronic diseases, such as inflammation and cancer. Additionally, the project will provide tools for other investigators interested in the physiological function of glycosaminoglycans in other organ systems.