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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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TUNEL staining of retinal sections. Paraffin sections of eyes from nondiabetic rats (A), nondiabetic rats treated with etanercept (B), diabetic rats (C), and diabetic rats treated with etanercept (D) were assessed by TUNEL. Sections were subsequently counterstained with hematoxylin. Insert shows an overview of a diabetic retina with the ganglion cell layer (above), the inner nuclear layer (middle, light blue) and the outer nuclear layer (below, dark blue). Numbers of positive endothelial cells were counted in each of 12 sections and expressed as percent of total endothelial cells. Treatment with etanercept reduced diabetes-induced endothelial cell apoptosis, as detected by TUNEL staining.
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f3: TUNEL staining of retinal sections. Paraffin sections of eyes from nondiabetic rats (A), nondiabetic rats treated with etanercept (B), diabetic rats (C), and diabetic rats treated with etanercept (D) were assessed by TUNEL. Sections were subsequently counterstained with hematoxylin. Insert shows an overview of a diabetic retina with the ganglion cell layer (above), the inner nuclear layer (middle, light blue) and the outer nuclear layer (below, dark blue). Numbers of positive endothelial cells were counted in each of 12 sections and expressed as percent of total endothelial cells. Treatment with etanercept reduced diabetes-induced endothelial cell apoptosis, as detected by TUNEL staining.

Mentions: To localize the apoptotic cell death within the retina, we stained formalin-fixed, paraffin-embedded retinal sections by using TUNEL assay (Figure 3) and M30 CytoDeath antibody (Figure 4). TUNEL staining labels fragmented DNA that is localized in the nucleus, whereas M30 stains caspase-cleaved cytokeratins that are located in the cytoplasm. Almost no TUNEL positivity or M30 staining was detected anywhere in the retina in nondiabetic animals with or without etanercept treatment (Figure 3 and Figure 4). In two-week diabetic animals, TUNEL-positive and M30-positive cells were located predominantly in the vascular endothelium of the retinal vessels as well as in the inner nuclear layer. Few positive cells were found in the ganglion layer. Treatment with etanercept was able to reduce TUNEL and M30 staining throughout the retina in diabetic animals (Figure 4).


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)

TUNEL staining of retinal sections. Paraffin sections of eyes from nondiabetic rats (A), nondiabetic rats treated with etanercept (B), diabetic rats (C), and diabetic rats treated with etanercept (D) were assessed by TUNEL. Sections were subsequently counterstained with hematoxylin. Insert shows an overview of a diabetic retina with the ganglion cell layer (above), the inner nuclear layer (middle, light blue) and the outer nuclear layer (below, dark blue). Numbers of positive endothelial cells were counted in each of 12 sections and expressed as percent of total endothelial cells. Treatment with etanercept reduced diabetes-induced endothelial cell apoptosis, as detected by TUNEL staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: TUNEL staining of retinal sections. Paraffin sections of eyes from nondiabetic rats (A), nondiabetic rats treated with etanercept (B), diabetic rats (C), and diabetic rats treated with etanercept (D) were assessed by TUNEL. Sections were subsequently counterstained with hematoxylin. Insert shows an overview of a diabetic retina with the ganglion cell layer (above), the inner nuclear layer (middle, light blue) and the outer nuclear layer (below, dark blue). Numbers of positive endothelial cells were counted in each of 12 sections and expressed as percent of total endothelial cells. Treatment with etanercept reduced diabetes-induced endothelial cell apoptosis, as detected by TUNEL staining.
Mentions: To localize the apoptotic cell death within the retina, we stained formalin-fixed, paraffin-embedded retinal sections by using TUNEL assay (Figure 3) and M30 CytoDeath antibody (Figure 4). TUNEL staining labels fragmented DNA that is localized in the nucleus, whereas M30 stains caspase-cleaved cytokeratins that are located in the cytoplasm. Almost no TUNEL positivity or M30 staining was detected anywhere in the retina in nondiabetic animals with or without etanercept treatment (Figure 3 and Figure 4). In two-week diabetic animals, TUNEL-positive and M30-positive cells were located predominantly in the vascular endothelium of the retinal vessels as well as in the inner nuclear layer. Few positive cells were found in the ganglion layer. Treatment with etanercept was able to reduce TUNEL and M30 staining throughout the retina in diabetic animals (Figure 4).

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