Metabolic memory in diabetes - from in vitro oddity to in vivo problem: role of apoptosis

Abstract

Retinal capillary cells undergo apoptosis before pathology characteristic of retinopathy can be observed, and the appearance of apoptotic capillary cell can predict the development of pathology. The purpose of this study is to investigate the effect of reversal of hyperglycemia on retinal capillary cell apoptosis, and identify the apoptosis encoding genes. Streptozotocin-diabetic rats were maintained either in poor glycemic control (PC, glycated hemoglobin, GHb >11%) or in good glycemic control (GC, GHb <6%) for 12 months, or allowed to be in PC for 6 months followed by GC for 6 additional months (PC-GC). Capillary cell apoptosis was determined in the trypsin-digested retinal microvasculature by TUNEL staining, and the genes encoding apoptosis were identified by Oligo GEArray rat apoptosis microarray that profiles 113 genes. Six months of good glycemic control that followed 6 months of poor control failed to attenuate the number of TUNEL-positive capillary cells in the retinal microvasculature. Twenty-three retinal genes, mainly from TNF ligand and receptor, caspase, Bcl-2 and death domain subfamilies that were upregulated by least a two-fold in PC rats remain upregulated after reversal of hyperglycemia. Thus, the continued activation of apoptosis plays a major role in the resistance of retinopathy to halt after re-institution of good glycemic control, and the regulation apoptosis machinery could help retard the progression of diabetic retinopathy.

Fig. 1

TUNEL-positive capillary cells in retinal trypsin digest: trypsin-digested microvessels were stained to detect TUNEL-positive cells using a kit from Roche Molecular Biochemicals. TUNEL-positive cells were identified in a masked fashion by scanning the trypsin-digested retinal microvessels under a Zeiss Axiophot photomicroscope. PC, poor control; PC–GC, poor control for 6 months followed by good control for 6months; GC, good control for the entire 12months; *p < 0.05 compared to normal and #p < 0.05 compared to PC.

Fig. 2

Gene quantification by Q-RTPCR. The Q-RTPCR assay was conducted in 50 ng cDNA template using the ABI-7500 sequence detection system. Each sample was analyzed in triplicate, and B2 M was validated as an appropriate housekeeping gene. Data for the target genes were normalized with B2 M and the fold change in gene expression relative to normal was calculated using the ddCt method. Values are represented as fold change relative to those obtained from normal rat retina. Graph (a) has representative genes from TNF receptor and ligand family and (b) genes from caspase and BCl2 families and NFkB. Black bars represent rats in poor control for 12 months, and grey bars are from rats in poor control for 6 months followed by good control for 6 additional months.