The objecfive is to study the funcfion of sulfated glycosaminoglycans (GAGs) in infiammation, with particular emphasis on the sulfafion pattern ofthe heparan, chondroitin and dermatan sulfate. We have successfully created conditional mouse mutants altered in heparan sulfate biosynthesis and showed that altering overall sulfation diminishes infiammatory responses in the context of neutrophil rolling and infiltrafion. In contrast, altering heparan sulfate in leukocytes does not affect these processes and has only mild effects on adaptive immune responses. In this proposal, we plan to complete our studies of endothelial heparan sulfate by examining the impact of altered uronic acid 2-0-sulfafion and glucosamine 6-O-sulfation using mice with conditional alleles in these enzymes, and then extend this analysis to chondroitin-4-sulfate and dermatan sulfate. Because we anticipate that systemic mutations in chondroitin/dermatan sulfate biosynthesis will result in embryonic or perinatal lethality, we are preparing conditional alleles ofthe target genes and will alter their expression selectively in endothelial cells and myeloid cells. Specifically, we propose to: (1) Create mouse lines with conditional alleles of chondroifin polymerase-1, chondroifin 4-0-sulfotransferase-1, chondroitin 4-0-sulfotransferase-2 , and dermatan 4-0-sulfotransferase and in collaboration with Project 1 a conditional targeting construct to express Hyal4, an endo-N-acetylgalactosaminidase that can degrade chondroifin;(2) Examine the role of GAGs in leukocyte rolling, firm adhesion, and diapedesis in vitro using isolated endothelial cells derived from the mutants;(3) In collaboration with Projects 2 and 4, examine the in vivo consequences of altering endothelial GAGs on inflammafion induced chemically, sterilely and by bacterial infecfion;and (4) Examine the consequences of altering endothelial glycosaminoglycans on vascular permeability. By focusing on inflammatory reacfions that take place in the vasculature, the proposed studies build on a strong base of preliminary data, which together validate GAGs as potential targets for treafing chronic inflammation and ischemic disease. Addifionally, the project will provide model organisms for other invesfigators interested in the physiological function of glycosaminoglycans in other organ systems.

Public Health Relevance

Inflammation is the body's response to injury and infection. The central hypothesis of Project 3 is that glycosaminoglycans affects one or more steps in the inflammatory response and that their function can be ascertained by geneticaly manipulating their biosynthesis in endothelial cells, neutrophils, and macrophages.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Special Emphasis Panel (ZHL1-CSR-H)
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University of California San Diego
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