Expression of apoptosis regulatory genes by retinal pericytes after rapid glucose reduction.
OBJECTIVE
Retinal capillary pericytes underwent apoptosis in vitro after they had been exposed long-term to high levels of glucose followed by a rapid glucose reduction. The present work was designed to study the expression of bcl-2 family members and apoptosis regulatory genes and to determine the status of oxidative stress induced by high concentrations of glucose in this in vitro apoptosis model.


METHODS
Pericytes were grown in normal or high glucose concentrations (5, 20, 30, and 40 mM) for 10 days and then exposed to a rapid reduction of glucose to 0.5 mM or 5 mM. Pericyte cell death was evaluated by determining the loss of cell viability and the fragmentation of DNA using agarose gel electrophoresis. In parallel, the quantitative reverse transcription-polymerase chain reaction technique was used to determine the expression of bcl-2, bax, p53, and glutathione peroxidase (GSH-Px) genes. The intracellular level of glutathione (GSH) and the DNA fragmentation were determined simultaneously for pericytes treated with or without exogenous GSH monoethylester. Retinal capillary endothelial cells, experiencing the same glucose variation, were studied as a comparison.


RESULTS
For pericytes, downregulation of bcl-2 was observed as early as 24 hours after rapid glucose reduction, whereas DNA fragmentation was not detectable at that time. After 72 hours, a decreased protein ratio of Bcl-2 to Bax was concomitant to evident loss of pericyte viability. During the period of high glucose and the following glucose reduction, p53 expression essentially was unchanged. Decreased levels of GSH induced by high concentrations of glucose (>30 mM) became further depleted when the glucose levels were rapidly reduced. Addition of GSH monoethylester to the medium restored the level of GSH in pericytes and prevented pericyte apoptosis induced by glucose variation. Moreover, the mRNA levels of GSH-Px were significantly elevated. By contrast, with the same glucose reduction endothelial cells did not undergo apoptosis. Their mRNA levels of bcl-2, bax, and GSH-Px essentially were unchanged.


CONCLUSIONS
High levels of glucose and rapid reduction of glucose modulate the expression of bcl-2 family genes in retinal pericytes. Upregulation of GSH-Px and depletion of GSH indicate a reparative process of accelerated elimination of reactive oxygen species following rapid glucose reduction. These findings indicate that the aggravated oxidative stress and the weakened antioxidant defense induced by the combined effects of high levels of glucose and subsequent rapid glucose reduction cause pericyte apoptosis. Prevention of DNA fragmentation of pericytes by exogenous GSH further supports this notion. Because endothelial cells did not show similar pathologic changes, this proposed mechanism seems to be specific to pericytes.
Department of Ophthalmology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102, USA.
W
W
Li
X
X
Liu
Z
Z
He
M
M
Yanoff
B
B
Jian
X
X
Ye
D000818:Animals; D017209:Apoptosis; D002196:Capillaries / Q000166:cytology; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005947:Glucose / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D051028:bcl-2-Associated X Protein
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D005979:Glutathione Peroxidase; D005978:Glutathione; D005947:Glucose