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P.F. Kador, K. Ikesugi, M.L. Mulhern, K.–I. Hosoya, T. Terasaki, K. Blessing, T. Shinohara; Glucose Imbalance Induces the Unfolded Protein Response (UPR) in Rat Retinal Capillary Pericyte and Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4235.
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The selective destruction of retinal capillary pericytes, the hallmark of diabetic retinopathy, has been linked to hyperglycemia and the accumulation of polyols. Their destruction is reduced by tight control of hyperglycemia; however, the establishment of tight control is often accompanied by increased incidences of hypoglycemia. The purpose of this study was to investigate the affect of hexose fluctuations on endoplasmic reticulum (ER) stress in retinal pericytes and endothelial cells since ER stress can generate an unfolded protein response (UPR) and UPR–dependent apoptosis.
The conditionally immortalized rat retinal pericyte (TR–rPCT) and endothelial (TR–iBRB) cell lines were cultured in 100 mm dishes coated with collagen type 1 in DMEM medium containing 5 mM glucose. After reaching 80% confluence, the cells were cultured for 24 hrs in similar DMEM media containing various levels of glucose or galactose with/without 10 µM of the aldose reductase inhibitor AL–1576. Cell Viability was assessed with a colorimetric MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay). TUNEL Staining was conducted using a fluorescein in situ cell death detection kit (Roche Diagnostics). Protein blot analysis of the SDS–PAGE of the cell homogenates was conducted with antibodies against three specific enzymes for UPR (GRP78/Bip, CHOP, ATF4) and procaspase–12 and the general apoptotic biomarker procaspase–3.
Hypoglycemia but not hyperglycemia was observed to significantly activate UPR–specific enzymes in pericytes. Strong UPR activation, leading to apoptosis was also observed when pericytes were cultured in glucose concentrations that were reduced from high to low or no glucose. In contrast, both hypoglycemia and hyperglycemia (such as 50–100 mM of glucose) activated the UPR in endothelial cells. This UPR induction was not affected by the aldose reductase inhibitor AL–1576. ROS generation as well as apoptosis was observed in both pericytes and endothelial cells.
UPR activation contributes to cell death in cultured retinal capillary pericytes and endothelial cells. Endothelial cells are more sensitive to UPR activation by hyperglycemic rather than hypoglycemic conditions while the UPR activation in pericytes only occurs with hypoglycemia.
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