CULTURED RETINAL CAPILLARY PERICYTES DIE BY APOPTOSIS AFTER AN ABRUPTFLUCTUATION FROM HIGH TO LOW GLUCOSE-LEVELS - A COMPARATIVE-STUDY WITH RETINAL CAPILLARY ENDOTHELIAL-CELLS

A number of clinical observations concerning cases of glycemic fluctua tion have prompted us to study whether or not a rapid change in blood glucose concentration can aggravate retinal microvascular pathology du ring the early stage of diabetic retinopathy. We conducted a comparati ve study of retinal capillary pericytes and endothelial cells in vitro . Both types of cells, either in single culture or in co-culture, were initially incubated in medium with high glucose (20-40 mmol/l), follo wed by a rapid reduction of glucose to 3.5, 1, or 0.5 mmol/l. This typ e of reduction of extracellular glucose resulted in depletion of intra cellular glucose, occurring much faster in pericytes than in endotheli al cells. The abrupt reduction in glucose caused pericyte cell shrinka ge and nuclear condensation associated with DNA fragmentation, followe d by loss of cell viability. All of these pericyte changes are apoptos is-like characteristics. This apoptotic process was prevented by the a ddition of cycloheximide, a protein synthesis inhibitor, or by platele t-derived growth factor BB, which is a known competent factor for peri cyte growth. In analysis of signalling pathways during the abrupt fluc tuation of glucose, the occurrence of pericyte apoptosis was an intrac ellular calcium-dependent, protein kinase C and protein kinase A media ted, and poly (ADP-ribose) synthetase-dependent process. Interestingly , a larger degree of DNA fragmentation was observed with a higher magn itude and a longer duration of pre-existing hyperglycaemia. These resu lts suggest that the magnitude and duration of pre-existing hyperglyca emia prime the apoptotic responsiveness of pericytes. Retinal capillar y endothelial cells, after an identical glucose fluctuation treatment did not undergo an apoptotic process.