instmctions): (Project 1, Hascall) Glycosaminoglycans In Diabetic Vascular Pathologies - Our studies show that cells dividing in hyperglycemia initiate synthesis of hyaluronan (HA) Into Intracellular compartments (endoplasmic reticulum, golgi, transport vesicles), which Initiates autophagy, upregulation of cyclin D3, and cyclin D3- medlated formation of a monocyte-adhesive matrix, and that this process occurs in glomeruli of streptozotocin treated diabetic rats within 1 week. Our new data show that this process Is also likely occurring in vascular endothelial cells and associated connective tissue cells In the diabetic kidney. Further, our experiments show that 4-methylumbelliferone-beta-xyloside and heparin Inhibit this process in distinctly different ways. The xyloside prevents Intracellular HA synthesis by diverting the UDP-sugar substrates from the cytosol into the golgi by increasing chondroitin sulfate synthesis ~10 fold, and this prevents synthesis of a monocyte-adhesive HA matrix. Heparin blocks Intracellular HA synthesis, but initiates excessive HA synthesis at the plasma membrane with formation of a monocyte-adhesive HA matrix. Daily treatment of diabetic rats with heparin prevents diabetic nephropathy and proteinurea. However, analyses of glomeruli in sections from 6 week diabetic rats with and without heparin treatment reveal striking differences: 1) extensive HA matrix with embedded macrophages and autophagic mesangial cells in untreated diabetic glomeruli, and 2) minimal HA matrix, no mesangial autophagy, but with at least as many embedded macrophages, in heparin treated diabetic glomeruli. Overall, our results support the following mechanisms: 1) hyperglycemia diverts monocytes to a pro-Inflammatory phenotype, and they become macrophages that promote inflammatory pathologies when they are recruited into glomeruli and vascular tissues and encounter the HA matrix. 2) Heparin prevents the 'pro-inflammatory'response, and the 'pro-repair'monocytes become macrophages that initiate an efficient phagocytotic mechanism to remove the matrix without extensive inflammation. Alms 1 and 2 will explore these mechanisms In vascular enothelial cells and monocytes.
Aim 3 will explore these mechanisms In diabetic rats treated with the xyloside and with heparin.

Public Health Relevance

Diabetic pathological complications are a major, costly and increasing problem in our society. They are characterized by inflammatory processes, but the underlying mechanisms remain elusive, and the role of the extracellular matrix is generally overlooked. Our studies demonstrate that monocyte-adhesive HA matrices are generated by cells exposed to various stresses, and that the dialogue of inflammatory cells with this matrix i.s central tn most, if nnt all inflammatnrv includinn those in riiahfitic nathnlnnie.g;-

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National Heart, Lung, and Blood Institute (NHLBI)
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Cleveland Clinic Lerner
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