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