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S. Dhar, S. Roy, S. Cherian, S. Roy; High Glucose Induced Altered Extracellular Matrix Triggers Apoptosis in Microvascular Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):952.
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To determine whether high glucose–induced excess extracellular matrix triggers apoptosis in microvascular endothelial cells.
Rat microvascular endothelial cells (RMECs) were grown in normal (5mM) glucose medium, high (30mM) glucose medium or medium containing mannitol (30mM) used as control for osmotic effects. After 7 days in culture, the cells were detached and removed, while retaining their extracellular matrix in the respective tissue culture dishes. RMECs grown in normal medium were plated onto the retained extracellular matrices and allowed to grow and replicate at 37°C for 3 days before being assayed for apoptosis. The method used for identifying apoptotic cells was based on the differential uptake of fluorescent dyes by apoptotic cells. To investigate the effect of different matrices on cell growth and apoptosis, cells plated in each well was exposed to ethidium bromide/acridine orange. Images were recorded using a fluorescent microscope equipped with a digital camera to document the number of apoptotic cells. In this study, cells exposed to 0.5mM H202 were used as positive controls for apoptosis.
RMECs grown on extracellular matrix, previously synthesized by RMECs exposed to high glucose conditions, showed significant increase in the number of apoptotic cells compared to those grown on extracellular matrix synthesized by control cells (2.92±1.12% vs 0.83±0.41%, P=0.003). RMECs plated on matrix secreted by cells grown in mannitol showed no difference in the number of apoptotic cells compared to cells grown on extracellular matrix synthesized by control cells. RMECs exposed to 0.5mM H202, as a positive control, showed an increase in apoptosis (87% of total cells).
Cells grown on high glucose matrix exhibited increased apoptosis compared to cells grown on normal matrix. This finding indicates that the altered extracellular matrix may induce apoptosis in microvascular endothelial cells. In diabetic retinopathy, the increased amount of extracellular matrix components present in the retinal vascular basement membrane may induce apoptosis and contribute to subsequent vascular cell loss, a prominent vascular lesion in retinas of diabetic individuals.
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