Hypoglycemia Induces Endoplasmic Reticulum (ER) Stress in Retinal Capillary Pericytes and Endothelial Cells

Abstract

Purpose:: In diabetic retinopathy (DR), the selective destruction of retinal capillary pericytes followed by the subsequent destruction of endothelial cells leads to the formation of acellular capillaries which result in retinal nonperfusion and hypoxia. While studies indicate that retinal capillary changes are linked to hyperglycemia, both the DCCT and UKPDS Studies report that the establishment of tight hyperglycemic control is accompanied by an initial increase in the progression of DR. Since the establishment of tight control is associated with increased incidences of hypoglycemia and because ER stress which induces unfolded protein response (UPR) has been linked to changes in diabetes, the purpose of this study was to investigate whether hypoglycemia and fluctuations of glucose levels induce ER stress in retinal capillary cells.

Methods:: Rat retinal pericyte (TR-rPCT) and endothelial (TR-iBRB) cell lines were cultured in DMEM containing 5 mM glucose. After reaching 80% confluence, the cells were cultured for 24 hours in similar DMEM containing various levels of glucose to simulate hyperglycemia to hypoglycemic conditions. Cell viability was assessed with a colorimetric MTS 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, and ATF4 -- and the general apoptotic biomarker procaspase-3 and 12.

Results:: Hyperglycemia is associated with increased TUNEL staining for apoptosis in pericytes but not endothelial cells. In contrast, TUNEL staining indicative of apoptosis occurs with both pericytes and endothelial cells under hypoglycemic conditions. ER stress linked UPR is not induced in either pericytes or endothelial cells grown under either hyperglycemic conditions or under conditions where glucose levels drop from hyperglycemic to normal (5 mM) glucose levels. However, ER stress linked UPR is induced in both pericytes and endothelial cells when glucose levels are decreased to hypoglycemic (< 5 mM) levels.

Conclusions:: The increased progression of DR associated with initial tight control may be linked to ER stress induced UPR in both retinal capillary and pericyte cells.

Keywords: diabetic retinopathy • vascular cells • apoptosis/cell death