Hyperglycemia affects Matrix Metalloproteinase Secretion in Human Umbilical Vein Endothelial Cells in vitro

Abstract

Goal: The aim of the current thesis was to study the effect of hyperglycemia (HG) on MMP secretion from human primary endothelial cells. Background: Diabetes Mellitus is characterized by abnormal high levels of glucose in the blood. Endothelial dysfunction is a serious complication of sustained hyperglycemia. Diabetic complications are main reasons for mortality in diabetes. The matrix metalloproteinases (MMPs) and their corresponding inhibitors are the main regulators of the turnover of extracellular matrix (ECM) components, and have been suggested to play an important role in vascular remodelling taking place in diabetes. MMP secretion from endothelial cells may become altered in HG conditions and result in dysregulation of ECM turnover, which may affect vascular functions with relevance to diabetes. HG conditions may also affect intracellular signalling through pathways sensing changes in cellular glucose, such as the hexosamine pathway Methods: Primary human umbilical vein endothelial cells (HUVEC) were cultured in vitro as a model system of the human endothelium. HUVEC were exposed to HG (25 mM) to imitate the diabetic hyperglycemic state and the effect on secretion of MMP-2 and MMP-9 was quantified. MMP secretion was analyzed by gelatine zymography. HUVEC were cultured on traditional plastic wells and on semipermeable filter inserts. The effect of blocking the hexosamine pathway using the inhibitor DON, on MMP secretion was also investigated. The O-GlcNAcylated proteins were analyzed by Western blotting. Bands obtained after gelatine zymography were measured using the programme Quantity-One (Bio-Rad). Results: Results obtained suggest that there is a weak trend of reduction in MMP-2 secretion in HUVEC grown on plastic. However, variable results were discovered in HUVEC grown on filters. The filter experiments showed that MMP-2 was mostly secreted to the apical side of polarized HUVEC. Finally, exposing HUVEC to DON caused a decrease in MMP-2 secretion both on plastic and on filter. MMP-9 secretion was not detected in any of the current experiments. Conclusions: The present work showed that HG influences MMP secretion, indicating a reduction in MMP-2 secretion in HUVEC grown on plastic. However, results were not definite and further investigations are needed to study the effects of hyperglycemia on MMP-2 secretion in cultures of primary human endothelial cells and to further investigate the potential role of MMPs as a therapeutic target in diabetes.