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Intervention with an erythropoietin-derived peptide protects against neuroglial and vascular degeneration during diabetic retinopathy.

McVicar CM, Hamilton R, Colhoun LM, Gardiner TA, Brines M, Cerami A, Stitt AW - Diabetes (2011)

Bottom Line: In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001).CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05).In OIR, pHBSP had no effect on preretinal neovascularization at any dose.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vision and Vascular Science, Queen’s University Belfast, Belfast, Northern Ireland, UK.

ABSTRACT

Objective: Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.

Research design and methods: After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 μg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1-30 μg/kg pHBSP or control peptide).

Results: pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.

Conclusions: Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

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pHBSP protects against DNA damage in diabetic retina. pHBSP treatment prevents diabetes-induced ganglion cell death. Cntl, control; Sc, scrambled pHBSP; and Db, diabetic. A: Cell count of terminal deoxynucleotidyl TUNEL-positive cells in the GCL was assessed by counting the number of fluorescent TUNEL-positive cells in the GCL divided by total number of cells present in the GCL. TUNEL of pHBSP-treated rats showed a significant increase in the percentage of cells with DNA strand breaks in the GCL of diabetic rats compared with control (*P < 0.05; ***P < 0.001). Treatment with pHBSP decreased TUNEL positivity by 49% when compared with diabetic rat with scrambled pHBSP (++P < 0.01). Counts were assessed by image analysis in multiple sections. Images were taken at three separate points (three fields at 300 µm2 each) on the central retina at ×40 magnification and presented as the average nuclei in the GCL. B: Cell count of TUNEL-positive cells in the entire retina was assessed by counting the number of fluorescent TUNEL-positive cells divided by total number of cells present. The diabetic retina treated with the scrambled peptide displayed TUNEL-positive cells. This was reduced to control levels of ∼10% with the pHBSP peptide (**P < 0.01). Data are mean ± SEM; n = 6 per group. Counts were assessed by image analysis in multiple sections. Images were taken at three separate points (three fields at 300 μm2 each) on the central retina at ×40 magnification and presented as the average nuclei in the GCL. pHBSP treatment prevents diabetes-induced ganglion cell death because there are more TUNEL-positive cells in the diabetic with scrambled pHBSP–treated animals relative to the control and scrambled pHBSP in the GCL and in the entire retina. Retinal nuclei were also counterstained with PI. This is evident in the images showing TUNEL positivity in control and Sc (C), control and pHBSP (D), diabetic and Sc (E), and diabetic and pHBSP (F). (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: pHBSP protects against DNA damage in diabetic retina. pHBSP treatment prevents diabetes-induced ganglion cell death. Cntl, control; Sc, scrambled pHBSP; and Db, diabetic. A: Cell count of terminal deoxynucleotidyl TUNEL-positive cells in the GCL was assessed by counting the number of fluorescent TUNEL-positive cells in the GCL divided by total number of cells present in the GCL. TUNEL of pHBSP-treated rats showed a significant increase in the percentage of cells with DNA strand breaks in the GCL of diabetic rats compared with control (*P < 0.05; ***P < 0.001). Treatment with pHBSP decreased TUNEL positivity by 49% when compared with diabetic rat with scrambled pHBSP (++P < 0.01). Counts were assessed by image analysis in multiple sections. Images were taken at three separate points (three fields at 300 µm2 each) on the central retina at ×40 magnification and presented as the average nuclei in the GCL. B: Cell count of TUNEL-positive cells in the entire retina was assessed by counting the number of fluorescent TUNEL-positive cells divided by total number of cells present. The diabetic retina treated with the scrambled peptide displayed TUNEL-positive cells. This was reduced to control levels of ∼10% with the pHBSP peptide (**P < 0.01). Data are mean ± SEM; n = 6 per group. Counts were assessed by image analysis in multiple sections. Images were taken at three separate points (three fields at 300 μm2 each) on the central retina at ×40 magnification and presented as the average nuclei in the GCL. pHBSP treatment prevents diabetes-induced ganglion cell death because there are more TUNEL-positive cells in the diabetic with scrambled pHBSP–treated animals relative to the control and scrambled pHBSP in the GCL and in the entire retina. Retinal nuclei were also counterstained with PI. This is evident in the images showing TUNEL positivity in control and Sc (C), control and pHBSP (D), diabetic and Sc (E), and diabetic and pHBSP (F). (A high-quality digital representation of this figure is available in the online issue.)

Mentions: As has been previously reported in rats of comparable diabetes duration (29), there was a significant increase in TUNEL-positive cells in the retina (P < 0.001) (Fig. 3A and B). TUNEL-positive cells, indicating DNA strand breaks, were apparent in the GCL, INL, and ONL. Treatment with pHBSP decreased TUNEL positivity in the GCL by 49% when compared with diabetic controls (P < 0.01) (Fig. 3B). In terms of overall diabetes-related TUNEL positivity in the retina, pHBSP provided significant protection when compared with diabetic with scrambled peptide (P < 0.005) (Fig. 3C). Caspase-3–positive cells were observed in the INL mainly, but there were no differences observed between the groups (Supplementary Fig. 2). And 8-OHdG–positive cells were found only in the diabetic retinae on the border of the ONL both in those that received the scrambled peptide and to a lesser extent in those that received the active pHBSP (Supplementary Fig. 3).


Intervention with an erythropoietin-derived peptide protects against neuroglial and vascular degeneration during diabetic retinopathy.

McVicar CM, Hamilton R, Colhoun LM, Gardiner TA, Brines M, Cerami A, Stitt AW - Diabetes (2011)

pHBSP protects against DNA damage in diabetic retina. pHBSP treatment prevents diabetes-induced ganglion cell death. Cntl, control; Sc, scrambled pHBSP; and Db, diabetic. A: Cell count of terminal deoxynucleotidyl TUNEL-positive cells in the GCL was assessed by counting the number of fluorescent TUNEL-positive cells in the GCL divided by total number of cells present in the GCL. TUNEL of pHBSP-treated rats showed a significant increase in the percentage of cells with DNA strand breaks in the GCL of diabetic rats compared with control (*P < 0.05; ***P < 0.001). Treatment with pHBSP decreased TUNEL positivity by 49% when compared with diabetic rat with scrambled pHBSP (++P < 0.01). Counts were assessed by image analysis in multiple sections. Images were taken at three separate points (three fields at 300 µm2 each) on the central retina at ×40 magnification and presented as the average nuclei in the GCL. B: Cell count of TUNEL-positive cells in the entire retina was assessed by counting the number of fluorescent TUNEL-positive cells divided by total number of cells present. The diabetic retina treated with the scrambled peptide displayed TUNEL-positive cells. This was reduced to control levels of ∼10% with the pHBSP peptide (**P < 0.01). Data are mean ± SEM; n = 6 per group. Counts were assessed by image analysis in multiple sections. Images were taken at three separate points (three fields at 300 μm2 each) on the central retina at ×40 magnification and presented as the average nuclei in the GCL. pHBSP treatment prevents diabetes-induced ganglion cell death because there are more TUNEL-positive cells in the diabetic with scrambled pHBSP–treated animals relative to the control and scrambled pHBSP in the GCL and in the entire retina. Retinal nuclei were also counterstained with PI. This is evident in the images showing TUNEL positivity in control and Sc (C), control and pHBSP (D), diabetic and Sc (E), and diabetic and pHBSP (F). (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: pHBSP protects against DNA damage in diabetic retina. pHBSP treatment prevents diabetes-induced ganglion cell death. Cntl, control; Sc, scrambled pHBSP; and Db, diabetic. A: Cell count of terminal deoxynucleotidyl TUNEL-positive cells in the GCL was assessed by counting the number of fluorescent TUNEL-positive cells in the GCL divided by total number of cells present in the GCL. TUNEL of pHBSP-treated rats showed a significant increase in the percentage of cells with DNA strand breaks in the GCL of diabetic rats compared with control (*P < 0.05; ***P < 0.001). Treatment with pHBSP decreased TUNEL positivity by 49% when compared with diabetic rat with scrambled pHBSP (++P < 0.01). Counts were assessed by image analysis in multiple sections. Images were taken at three separate points (three fields at 300 µm2 each) on the central retina at ×40 magnification and presented as the average nuclei in the GCL. B: Cell count of TUNEL-positive cells in the entire retina was assessed by counting the number of fluorescent TUNEL-positive cells divided by total number of cells present. The diabetic retina treated with the scrambled peptide displayed TUNEL-positive cells. This was reduced to control levels of ∼10% with the pHBSP peptide (**P < 0.01). Data are mean ± SEM; n = 6 per group. Counts were assessed by image analysis in multiple sections. Images were taken at three separate points (three fields at 300 μm2 each) on the central retina at ×40 magnification and presented as the average nuclei in the GCL. pHBSP treatment prevents diabetes-induced ganglion cell death because there are more TUNEL-positive cells in the diabetic with scrambled pHBSP–treated animals relative to the control and scrambled pHBSP in the GCL and in the entire retina. Retinal nuclei were also counterstained with PI. This is evident in the images showing TUNEL positivity in control and Sc (C), control and pHBSP (D), diabetic and Sc (E), and diabetic and pHBSP (F). (A high-quality digital representation of this figure is available in the online issue.)
Mentions: As has been previously reported in rats of comparable diabetes duration (29), there was a significant increase in TUNEL-positive cells in the retina (P < 0.001) (Fig. 3A and B). TUNEL-positive cells, indicating DNA strand breaks, were apparent in the GCL, INL, and ONL. Treatment with pHBSP decreased TUNEL positivity in the GCL by 49% when compared with diabetic controls (P < 0.01) (Fig. 3B). In terms of overall diabetes-related TUNEL positivity in the retina, pHBSP provided significant protection when compared with diabetic with scrambled peptide (P < 0.005) (Fig. 3C). Caspase-3–positive cells were observed in the INL mainly, but there were no differences observed between the groups (Supplementary Fig. 2). And 8-OHdG–positive cells were found only in the diabetic retinae on the border of the ONL both in those that received the scrambled peptide and to a lesser extent in those that received the active pHBSP (Supplementary Fig. 3).

Bottom Line: In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001).CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05).In OIR, pHBSP had no effect on preretinal neovascularization at any dose.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vision and Vascular Science, Queen’s University Belfast, Belfast, Northern Ireland, UK.

ABSTRACT

Objective: Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.

Research design and methods: After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 μg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1-30 μg/kg pHBSP or control peptide).

Results: pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.

Conclusions: Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

Show MeSH
Related in: MedlinePlus