MARKERS AND MECHANISMS OF VASCULAR DISEASE IN DIABETES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our primary goal is to define the roles of dyslipidemia and oxidative stress in accelerating macrovascular disease (atherosclerosis) in diabetes. Microvascular disease (nephropathy and retinopathy) will be studied as secondary end-points. The place of our project in the broad scope of the program is shown in Figure 1. We will continue (and expand) our longitudinal and cross-sectional studies of the DCCT/EDIC cohort of Type 1 diabetic patients. This cohort is the largest and best-characterized group of Type 1 diabetic patients in the world. Though still relatively young, it has provided exciting data during the first funding period of the Program Project (1996-present). To obtain a higher "hard" event rate (myocardial infarction, stroke etc.) and to study the more common form of diabetes, we will add a Type 2 diabetes cohort from the VA Cooperative Trial "Glycemia and Complications in Diabetes, Type 2". This prospective trial commences October 2000, and as in the DCCT, patients will be randomized to intensive and standard glycemic control. The VA trial provides us with the opportunity to study patients during the randomized portion of the study (in the DCCT, randomization finished in 1993). We have obtained interim funding to support our collaboration with the VA trial in its first year. We will also perform basic science studies to investigate hypotheses we have developed over the past four years relating to dyslipidemia, oxidative stress and atherogenesis in diabetes. These studies, in collaboration with the other Projects and Cores and the Coordinating Centers of both studies, will greatly increase our understanding of the complex multi-factorial mechanisms underlying vascular disease in both types of diabetes. Our Specific Aims are: 1. To define the role of dyslipidemia in the vascular complications of Type 1 and Type 2 diabetes. We will perform comprehensive lipoprotein analyses, including Nuclear Magnetic Resonance (NMR) lipoprotein profiles, levels of apolipoproteins, Lipoprotein (a), and studies of lipoprotein-associated enzymes. We hypothesize that dyslipidemia, including both quantitative and qualitative lipoprotein abnormalities which can now be assessed in much greater detail than ever before, mediates endothelial cell injury and activation, contributing to accelerated atherosclerosis in diabetes. We will obtain NMR lipoprotein profiles of selected subjects from the DCCT/EDIC Type 1 diabetes cohort ("Type 1 Cohort") on two occasions, completing a total of seven determinations over the entire DCCT/EDIC study duration (1983-2006). We will also obtain NMR analyses prospectively on two occasions in the VA Cooperative Trial Type 2 diabetes cohort ("Type 2 Cohort") during 2000-2006. We will measure apolipoproteins A1, B, E, and Lp(a). We will determine activities of the following lipoprotein-related enzymes: paraoxonase (PON), Lecithin Cholesterol Acyl Transferase (LCAT), and Platelet Activating Factor Acetyl Hydrolase (PAFAH). The data will be related to lipoprotein subclass distribution, and cross-sectionally and prospectively to vascular complication status, randomization groups, and interventions. 2. To define the roles of oxidative stress, inflammation, and homocysteine in the vascular complications of Type 1 and Type 2 diabetes. We hypothesize that oxidative stress, inflammatory processes, and elevated homocysteine levels mediate endothelial injury and accelerated atherosclerosis in diabetes. In both cohorts, we will determine products of free radical oxidation and antioxidant reserves in plasma to assess oxidative stress, C-reactive protein and serum amyloid A as measures of inflammatory processes, and plasma homocysteine levels.3. To determine the effect of lipoprotein associated enzymes on lipoprotein subclass distribution. We hypothesize that lipoprotein subclass distribution in serum is affected by LCAT, CETP, and PAFAH activity. Complementing studies of NMR determined lipoprotein subclass distribution and enzyme activities in vivo (Specific Aim 1); we will incubate serum with purified enzymes in vitro and determine effects on lipoprotein subclass distribution by NMR. 4. To determine functional characteristics of LDL, HDL, and their subclasses from Type 1 and Type 2 diabetic patients and control subjects in studies using human aortic endothelial cells (HAEC) and human red blood cell (RBC) membranes. We hypothesize that altered interactions of lipoproteins with vascular cells, related to dyslipidemia and oxidative stress, contribute to endothelial injury and accelerated atherosclerosis in diabetes. Using well- characterized lipoproteins from Type 1 and Type 2 diabetic subjects and from non-diabetic control subjects, we will determine: (i) effects of large, medium, and small LDL on HAEC expression of proteins mediating fibrinolysis, vascular tone, and adhesion molecules, and on matrix binding and intracellular calcium flux.(ii) ability of total HDL, and of large (cardioprotective) HDL2 and small (non-protective) HDL3 to:(a) mitigate oxidized-LDL induced HAEC adhesion molecule expression, tPA suppression, PAI-1 and NO production. (b) mitigate TNF- induced HAEC adhesion molecule expression(c) protect LDL and RBC membranes from in vitro oxidation(d) remove pre-formed lipid peroxides from oxidized RBC membranes