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In vivo and in vitro studies of arterial calcifications in diabetes

The association to osteoprotegerin and the influence of hormonal and metabolic factors

Ping Liu Olesen

Abstract

Introduction: Diabetes is associated with an increased prevalence of cardiovascular disease and mortality. Medial calcification appears to be a strong independent predictor of cardiovascular mortality in diabetes. Evidence from clinical observations, animal models and molecular studies suggest that medial calcification in diabetes is an active process, which share same features with osteogenesis. Factors that regulate bone cell differentiation and mineralization are also found in calcified vessel wall.
Hypothesis and Aims: We hypothesize that arterial media calcifications are the consequences of a series of diffuse matrix alterations in the arterial wall caused by metabolic and hormonal disorder of diabetes. Bone related protein (OPG) may occur in altered amount in the arterial wall in diabetes and during the calcification. High insulin level may modulate the expression of OPG and, also influence the progress of calcification. Aims of the study: 1. To analyze the level of OPG in arterial tissue from diabetic individuals. 2. To estimate the acute influence of metabolic and hormonal factors on the production of OPG from vascular cells. 3. To measure the influence of metabolic and hormonal factors in vitro-induced calcification in vascular smooth muscle cells (VSMCs).
Material and methods: Aortic samples from 21 diabetic and 42 non-diabetic individuals have been extracted and analyzed for OPG and calcium content from separated areas (plaques intima, plaques medium, normal intima, and normal media). The production of OPG from human VSMCs was measured in conditioned medium by ELISA. In vitro induced calcifications in VSMCs were evaluated by calcium content in the cell layer. Histochemistry and RT-PCR were used to demonstrate the expression of bone-related protein in calcified cells. Secreted hyaluronic acid was also measured.
Results: The content of OPG was increased in tunica media from diabetic samples. No differences between diabetic and non-diabetic subjects were observed in tunica intima. Human VSMCs produce approximately 30 times more OPG than human umbilical vein endothelial cells. The production of OPG was decreased after insulin treatment in VSMCs, whereas TNF-α or IL-1β promotes synthesis of OPG, time and dose dependent effectswere observed both in protein and mRNA levels. Using β-glycerophosphate was able to induce calcification in VSMCs; the progress of calcification is time dependent. Increased activity of alkaline phosphatase ( ALP, marker for osteoblast early differentiation), up-regulated bone sialoprotein (BSP, major non-collagenous protein in the extracellular matrix of bone) and high level of hyaluronic acid were found in calcified cells. High dose of insulin (1000μU/ml) significantly accelerate calcification, whereas 200μU/ml insulin showed only a trend toward augmented levels in later stage. No alterations in OPG levels were observed in early calcification stage; but a declined OPG was found in late stage accompanied by strong calcification. We found that serum stimulates cells to produce OPG and decelerates calcification in VSMCs.
Conclusion: Increased OPG amount in the tunica media in diabetes may be part of generalized matrix alterations; the calcification in VSMCs is, at least partially a cell-mediated process with upregulated bone proteins, which can be accelerated with insulin at high concentrations. Altered OPG levels during the calcification are compatible with the idea, that OPG is a potent regulator of vascular calcification,which may be important for the elucidation of diabetic macroangiopathy.

Ping Liu Olesen