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TNF-alpha mediated apoptosis plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations.

Joussen AM, Doehmen S, Le ML, Koizumi K, Radetzky S, Krohne TU, Poulaki V, Semkova I, Kociok N - Mol. Vis. (2009)

Bottom Line: Long-term studies demonstrated that pericyte loss and endothelial cell loss were reduced in comparison to wild-type diabetic controls.Our study identifies an important role for TNF-alpha in the pathogenesis of signature diabetic retinopathy pathologies and demonstrates that etanercept can inhibit retinal cell death and long-term complication of diabetes.Taken together, our results suggest that etanercept could prove beneficial in preventing both early and late vascular diabetic complications.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Heinrich Heine University, Duesseldorf, Germany. JoussenA@googlemail.com

ABSTRACT

Purpose: The pathophysiology of diabetic retinopathy involves leukocyte adhesion to retinal vasculature, early blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell death. We investigated the involvement of tumor necrosis factor alpha (TNF-alpha) in diabetes-related histopathological changes in two relevant rodent models.

Methods: In short-term studies, Long-Evans rats with streptozotocin-induced diabetes were treated with or without the TNF-alpha inhibitor, etanercept. For long-term studies, tumor necrosis factor receptor I (TNF-RI)-deficient mice and TNF-RII-deficient mice, as well as C57/Bl6 wild-type mice, were fed 30% galactose for up to 20 months. The retinal histopathological alterations of hypergalactosemia were analyzed in trypsin digest preparations. Endothelial cell injury and apoptosis in rat retinas were evaluated by propidium iodide, TUNEL, CytoDeath staining, and DNA fragmentation ELISA. Caspase 3 and 8 activity was evaluated by immunoblotting and quantitative enzymatic activity assay.

Results: Etanercept suppressed caspase activation, retinal cell injury, and apoptosis in short-term diabetic rats. Pericyte and endothelial cell loss were also reduced in long-term hypergalactosemic mice. Long-term studies demonstrated that pericyte loss and endothelial cell loss were reduced in comparison to wild-type diabetic controls.

Conclusions: Our study identifies an important role for TNF-alpha in the pathogenesis of signature diabetic retinopathy pathologies and demonstrates that etanercept can inhibit retinal cell death and long-term complication of diabetes. Taken together, our results suggest that etanercept could prove beneficial in preventing both early and late vascular diabetic complications.

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Related in: MedlinePlus

Caspase 8 enzymatic activity in rat retinas. A: The level of protein expression of caspase 8 in the rat retina was evaluated by western blotting. Diabetes increased the cleaved form of caspase 8 (activated form), whereas treatment with etanercept reduced the diabetes-induced cleavage of caspase 8. B: The enzymatic activity of caspase 8 in the rat retina was evaluated by a colorimetric analysis. Diabetes increased the activity of caspase 8 as measured by the conversion of its specific substrate. Treatment with etanercept reduced the diabetes-induced caspase 8 activation and therefore the conversion of its substrate and correlates with the reduction in the cleaved form of the caspase as measured by the western blot shown.
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f5: Caspase 8 enzymatic activity in rat retinas. A: The level of protein expression of caspase 8 in the rat retina was evaluated by western blotting. Diabetes increased the cleaved form of caspase 8 (activated form), whereas treatment with etanercept reduced the diabetes-induced cleavage of caspase 8. B: The enzymatic activity of caspase 8 in the rat retina was evaluated by a colorimetric analysis. Diabetes increased the activity of caspase 8 as measured by the conversion of its specific substrate. Treatment with etanercept reduced the diabetes-induced caspase 8 activation and therefore the conversion of its substrate and correlates with the reduction in the cleaved form of the caspase as measured by the western blot shown.

Mentions: The increase in M30 staining observed in diabetic retinas suggested the involvement of caspases in mediating apoptotic cell death. During apoptosis, caspases are activated in a hierarchical manner to cleave structural and functional proteins needed for cell survival. Cleaved products for both caspase 8 and caspase 3 were found by western blot analysis in the retinal lysates of rats after two weeks of diabetes, proving the activation of these caspases in the diabetic retina in the rat (Figure 5 and Figure 6). Administration of the soluble TNF-α inhibitor in the diabetic rats significantly reduced the cleaved forms for both caspase 8 (14 kDa band; Figure 5) and caspase 3 (17 kDa band; Figure 6), an observation that correlates with the reduced levels of apoptotic cells in these rats as described above. Furthermore, the ability of these caspases to cleave their specific substrates was tested directly in the retinal lysates. Caspase 3 was found to cleave its substrate efficiently as early as 1 min after incubation. In comparison to nondiabetic animals, the substrate conversion rate was 2.16-fold higher in the diabetic retinas (322.69±99.71 versus 149.73±54.51 arbitrary units; n=3; p<0.001). Treatment of diabetic animals with etanercept reduced the conversion rate to 262.01±74.9 (n=3; p=0.061).


TNF-alpha mediated apoptosis plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations.

Joussen AM, Doehmen S, Le ML, Koizumi K, Radetzky S, Krohne TU, Poulaki V, Semkova I, Kociok N - Mol. Vis. (2009)

Caspase 8 enzymatic activity in rat retinas. A: The level of protein expression of caspase 8 in the rat retina was evaluated by western blotting. Diabetes increased the cleaved form of caspase 8 (activated form), whereas treatment with etanercept reduced the diabetes-induced cleavage of caspase 8. B: The enzymatic activity of caspase 8 in the rat retina was evaluated by a colorimetric analysis. Diabetes increased the activity of caspase 8 as measured by the conversion of its specific substrate. Treatment with etanercept reduced the diabetes-induced caspase 8 activation and therefore the conversion of its substrate and correlates with the reduction in the cleaved form of the caspase as measured by the western blot shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2716944&req=5

f5: Caspase 8 enzymatic activity in rat retinas. A: The level of protein expression of caspase 8 in the rat retina was evaluated by western blotting. Diabetes increased the cleaved form of caspase 8 (activated form), whereas treatment with etanercept reduced the diabetes-induced cleavage of caspase 8. B: The enzymatic activity of caspase 8 in the rat retina was evaluated by a colorimetric analysis. Diabetes increased the activity of caspase 8 as measured by the conversion of its specific substrate. Treatment with etanercept reduced the diabetes-induced caspase 8 activation and therefore the conversion of its substrate and correlates with the reduction in the cleaved form of the caspase as measured by the western blot shown.
Mentions: The increase in M30 staining observed in diabetic retinas suggested the involvement of caspases in mediating apoptotic cell death. During apoptosis, caspases are activated in a hierarchical manner to cleave structural and functional proteins needed for cell survival. Cleaved products for both caspase 8 and caspase 3 were found by western blot analysis in the retinal lysates of rats after two weeks of diabetes, proving the activation of these caspases in the diabetic retina in the rat (Figure 5 and Figure 6). Administration of the soluble TNF-α inhibitor in the diabetic rats significantly reduced the cleaved forms for both caspase 8 (14 kDa band; Figure 5) and caspase 3 (17 kDa band; Figure 6), an observation that correlates with the reduced levels of apoptotic cells in these rats as described above. Furthermore, the ability of these caspases to cleave their specific substrates was tested directly in the retinal lysates. Caspase 3 was found to cleave its substrate efficiently as early as 1 min after incubation. In comparison to nondiabetic animals, the substrate conversion rate was 2.16-fold higher in the diabetic retinas (322.69±99.71 versus 149.73±54.51 arbitrary units; n=3; p<0.001). Treatment of diabetic animals with etanercept reduced the conversion rate to 262.01±74.9 (n=3; p=0.061).

Bottom Line: Long-term studies demonstrated that pericyte loss and endothelial cell loss were reduced in comparison to wild-type diabetic controls.Our study identifies an important role for TNF-alpha in the pathogenesis of signature diabetic retinopathy pathologies and demonstrates that etanercept can inhibit retinal cell death and long-term complication of diabetes.Taken together, our results suggest that etanercept could prove beneficial in preventing both early and late vascular diabetic complications.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Heinrich Heine University, Duesseldorf, Germany. JoussenA@googlemail.com

ABSTRACT

Purpose: The pathophysiology of diabetic retinopathy involves leukocyte adhesion to retinal vasculature, early blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell death. We investigated the involvement of tumor necrosis factor alpha (TNF-alpha) in diabetes-related histopathological changes in two relevant rodent models.

Methods: In short-term studies, Long-Evans rats with streptozotocin-induced diabetes were treated with or without the TNF-alpha inhibitor, etanercept. For long-term studies, tumor necrosis factor receptor I (TNF-RI)-deficient mice and TNF-RII-deficient mice, as well as C57/Bl6 wild-type mice, were fed 30% galactose for up to 20 months. The retinal histopathological alterations of hypergalactosemia were analyzed in trypsin digest preparations. Endothelial cell injury and apoptosis in rat retinas were evaluated by propidium iodide, TUNEL, CytoDeath staining, and DNA fragmentation ELISA. Caspase 3 and 8 activity was evaluated by immunoblotting and quantitative enzymatic activity assay.

Results: Etanercept suppressed caspase activation, retinal cell injury, and apoptosis in short-term diabetic rats. Pericyte and endothelial cell loss were also reduced in long-term hypergalactosemic mice. Long-term studies demonstrated that pericyte loss and endothelial cell loss were reduced in comparison to wild-type diabetic controls.

Conclusions: Our study identifies an important role for TNF-alpha in the pathogenesis of signature diabetic retinopathy pathologies and demonstrates that etanercept can inhibit retinal cell death and long-term complication of diabetes. Taken together, our results suggest that etanercept could prove beneficial in preventing both early and late vascular diabetic complications.

Show MeSH
Related in: MedlinePlus