Background: Diabetes Mellitus (DM) is associated with many different macro- and microvascular complications. Proteoglycans (PGs) are important constituents of the plasma membrane and of the basement membrane supporting the endothelial cell layer. Quantitative and qualitative DM associated modifications of the PGs are being increasingly reported and are proposed to play important roles in the pathogenesis of the long term diabetic complications. PG changes in the endothelium may affect important functions such as turnover of lipoproteins, filtration properties, and regulation of chemokines during inflammation, which are all relevant in DM. Hyperglycemia leads to an accelerated intracellular flux of glucose in many cells and as a consequence, generation of methylglyoxal (MGO) will rise. Plasma levels of MGO have shown to be elevated in diabetic patients. A prolonged chronic low inflammatory state is observed in DM together with an increase in proinflammatory cytokines like interleukin-1á (IL-1á) and tumor necrosis factor-á (TNF-á). The aim of this study was to increase the understanding of the molecular mechanisms leading to DM related alterations in PG metabolism by studying de novo biosynthesis and secretion of PGs by cultured primary human endothelial cells.
Methods: Primary human umbilical vein endothelial cells (HUVEC) were established and cultured in vitro. To mimic the metabolic environment of DM in vivo the HUVEC were cultured in medium with different concentrations of MGO (0.1, 0.25, 0.5, 1.0mM) for 24 and 48 hours. They were also cultured for 24 hours in medium containing the proinflammatory cytokines IL-1á (2 ng/ml) and TNF-á (7 ng/ml). The cells were labeled with [35S]-sulfate 24 hours before harvesting. The labeled material was purified by Sephadex G-50 fine chromatography and DEAE-ion exchange chromatography, and analyzed by SDS-PAGE. Results: The major findings in the present study revealed increased biosynthesis and secretion of [35S]-PGs by HUVEC when exposed to low concentrations of MGO in 24 and 48 hours of incubation. In higher concentrations of MGO (1.0mM) the biosynthesis and secretion of [35S]-PGs decreased. Exposing HUVEC to the proinflammatory cytokines IL-1á and TNF-á led to increased biosynthesis and secretion of [35S]-PGs with no change in the ratio of heparan sulfate/chondroitin sulfate glycosaminoglycans.
Conclusions: The results presented in our study suggest that hyperglycemic conditions, using MGO, and inflammatory conditions, using IL-1á and TNF-á, both lead to altered [35S]-PG expression by HUVEC in vitro.