Decreased heparan sulfate proteoglycans (HSPG) may be a central contributor to the nephropathy, retinopathy, neuropathy and early atherosclerosis of diabetes mellitus (DM). Decreased HSPG has been shown (including our own work on diabetic dyslipidemia) specifically to involve decreased perlecan, the most abundant basement-membrane HSPG. The objective of this proposal is to support the role of perlecan in DM complications, specifically dyslipidemia and atherosclerosis, and to define mechanisms for its decrease. Three hypotheses are proposed, corresponding to each aim of the proposal. Hypothesis 1. Decreased expression of perlecan, independent of diabetes, will impair the clearance of remnant apoB48 lipoprotein and will aggravate the impaired clearance of such lipoproteins resulting from DM, leading to increased atherosclerosis. Heterozygous perlecan gene-knock-out (PKO) mice and control mice will be studied in the presence and absence of streptozotocin-induced DM. The lipoprotein phenotype will be extensively characterized and kinetic studies will define its mechanism. Similar studies will be performed with PKO mice in the apoE-null and human apoB transgenic backgrounds. Hypothesis 2. DM causes a generalized decrease in perlecan independent of organ or cell type that will lead to increased atherosclerosis. The propensity of different mouse strains to exhibit such a decrease will correlate with their described propensity to develop increased arteriosclerosis with diabetes. Analysis of perlecan protein levels will be conducted in tissues from diabetic and non-diabetic mice from the C57BL/6 as well as the BALB/c strains, both on a """"""""chow"""""""" diet and on a high-cholesterol diet (to assess possible interactions of diabetes and diet on perlecan levels). Quantitative arteriosclerosis studies will be conducted in WT and PKO mice both in the C57BL/6 and BALB/c backgrounds and in the apoE-null and human apoB transgenic contexts. In addition, cultured cells representative of tissues affected by complications of diabetes will be exposed to high glucose levels. These experiments will provide an appropriate model for the molecular studies in aim 3 as well as providing the basis for future studies of molecular pathologies related to diabetes. Hypothesis 3. The decrease in perlecan core protein in diabetes mellitus is a result of reduced GAG incorporation into PG. Changes in glycosaminoglycan (GAG) chain size and charge affect proteoglycan function and, likely, synthetic rates and stability. A systematic study of the synthesis and intracellular processing of the perlecan core protein and of the sequence of enzymes involved in GAG synthesis and modification will be performed. This will include characterizing the GAG chains present in HSPG.
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