Diabetes-enhanced tumor necrosis factor-alpha production promotes apoptosis and the loss of retinal microvascular cells in type 1 and type 2 models of diabetic retinopathy

Abstract

Retinal microvascular cell loss plays a critical role in the pathogenesis of diabetic retinopathy. To examine this further, type 1 streptozotocin-induced diabetic rats and type 2 Zucker diabetic fatty rats were treated by intravitreal injection of the tumor necrosis factor-specific inhibitor pegsunercept, and the impact was measured by analysis of retinal trypsin digests. For type 2 diabetic rats, the number of endothelial cells and pericytes positive for diabetes-enhanced activated caspase-3 decreased by 81% and 86%, respectively, when treated with pegsunercept (P < 0.05). Similarly, the number of diabetes-enhanced terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive endothelial cells and pericytes decreased by 81% and 67% respectively when treated with pegsunercept (P < 0.05). Diabetes-increased activated caspase-3- and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive microvascular cell numbers were both reduced by 81% and 80%, respectively, in pegsunercept-treated type 1 diabetic rats (P < 0.05). Inhibition of tumor necrosis factor reduced type 1 diabetes-enhanced pericyte ghost formation by 87% and the number of type 2 diabetes-enhanced pericyte ghosts by 62% (P < 0.05). Similarly, increased acellular capillary formation caused by type 1 and type 2 diabetes was reduced by 68% and 67%, respectively, when treated with pegsunercept (P < 0.05). These results demonstrate a previously unrecognized role of tumor necrosis factor-alpha in promoting the early pathogenesis of diabetic retinopathy leading to loss of retinal microvascular cells and demonstrate the potential therapeutic benefit of modulating its activity.

Figure 1

Diabetes increases retinal microvascular cell apoptosis. a: A low power image of a typical retinal trypsin digest of the retinal microvasculature is shown. Vertical lines represent the boundaries of contiguous fields in which the entire retina was examined for the presence of TUNEL- or cleaved caspase-3-positive cells. Rectangles in the mid-retinal area represent the areas examined for acellular capillaries and pericyte ghosts. Original magnification ×20. b: Representative image of a TUNEL-positive retinal microvascular cell in a diabetic specimen. Upper left panel, DAPI staining; lower left panel, TUNEL staining; upper right panel, phase contrast image of the corresponding field; lower right panel, merged TUNEL/DAPI images. Arrow points to a TUNEL-positive microvascular cells. The bar represents 20 μm at original magnification ×400.

Figure 2

TNF inhibition confers protection in both type 1 and type 2 diabetic animals against diabetes-induced microvascular cell apoptosis. a: The mean number of cleaved caspase-3-positive cells was determined in normoglycemic control rats (C), streptozotocin-induced diabetic rats (DM), and streptozotocin-induced diabetic rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 5). b: The mean number of cleaved caspase-3-positive cells was determined in normoglycemic control rats (C), ZDF diabetic rats (DM) and ZDF diabetic rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 5). c: The mean number of TUNEL-positive cells in type 1 diabetic model was determined in normoglycemic control rats (C), streptozotocin-induced diabetic rats (DM) and streptozotocin-induced diabetic rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 6). d: The mean number of TUNEL-positive microvascular cells was determined in normoglycemic control rats (C), ZDF diabetic rats (DM) and ZDF diabetic rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 6). *Statistically significant compared to control (P < 0.05). **Statistically significant as compared with diabetic (P < 0.05).

Figure 3

TNF inhibition reduces pericyte and endothelial cell apoptosis in type 2 diabetic retina. a: Representative image of a cleaved caspase-3-positive pericyte (upper left panel) and endothelial cell (lower left panel), phase contrast (central panel), and PAS staining (right panel). Arrow points to a cleaved caspase-3-positive microvascular cells in a diabetic specimen. b: The mean number of cleaved caspase-3-positive endothelial cells and pericytes was determined in normoglycemic rats (C), Zucker diabetic fatty rats (DM) and Zucker diabetic fatty rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 5). c: The mean number of TUNEL-positive retinal endothelial cells and pericytes was determined in normoglycemic control rats (C), ZDF diabetic rats (DM), and ZDF diabetic rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 6). *Statistically significant compared to control (P < 0.05). **Statistically significant as compared with diabetic (P < 0.05).

Figure 4

TNF inhibition reduces pericyte ghost formation in both type 1 and type 2 diabetic animals. a: RTDs was prepared from a diabetic specimen and stained with PAS-hematoxylin. Arrow points to a pericyte ghost. The bar represents 20 μm at original magnification, ×400. b: The mean number of pericyte ghosts was determined in normoglycemic control rats (C), streptozotocin-induced diabetic rats (DM), and streptozotocin-induced diabetic rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 5). c: The mean number of pericyte ghosts was determined in normoglycemic control rats (C), ZDF diabetic rats (DM) and ZDF diabetic rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 5). *Statistically significant as compared with control (P < 0.05). **Statistically significant as compared with diabetic (P < 0.05).

Figure 5

TNF inhibition reduces acellular capillary in both type 1 and type 2 diabetic animals. a: PAS-hematoxylin-stained RTDs from a diabetic retina. Arrows point to an acellular capillary. The bar represents 20 μm at original magnification, ×400. b: The mean number of acellular capillaries was determined in normoglycemic control rats (C), streptozotocin-induced diabetic rats (DM) and streptozotocin-induced diabetic rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 6). c: The mean number of acellular capillaries was determined in normoglycemic control rats (C), ZDF diabetic rats (DM), and ZDF diabetic rats treated with pegsunercept (DM + Peg). Data represent the mean ± SEM (n = 6). *Statistically significant compared to control (P < 0.05). **Statistically significant compared to diabetic (P < 0.05).