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Improvement of retinal vascular injury in diabetic rats by statins is associated with the inhibition of mitochondrial reactive oxygen species pathway mediated by peroxisome proliferator-activated receptor gamma coactivator 1alpha.

Zheng Z, Chen H, Wang H, Ke B, Zheng B, Li Q, Li P, Su L, Gu Q, Xu X - Diabetes (2010)

Bottom Line: The aim of this study was to clarify the beneficial effects and mechanism of action of simvastatin against diabetes-induced retinal vascular damage.Simvastatin significantly upregulated PGC-1alpha (P < 0.01), subsequently decreased Deltapsim (P < 0.05) and ROS generation (P < 0.01), inhibited PARP activation (P < 0.01), and further reduced VEGF expression (P < 0.01) and p38 MAPK activity (P < 0.01).Those changes were associated with the decrease of retinal vascular permeability, retinal capillary cells apoptosis, and formation of acellular capillaries.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, First People’s Hospital of Shanghai Affiliated to Shanghai Jiaotong University, Shanghai, China.

ABSTRACT

Objective: Mitochondrial reactive oxygen species (ROS) plays a key role in diabetic retinopathy (DR) pathogenesis. However, whether simvastatin decreases diabetes-induced mitochondrial ROS production remains uncertain. The aim of this study was to clarify the beneficial effects and mechanism of action of simvastatin against diabetes-induced retinal vascular damage.

Research design and methods: Diabetic rats and control animals were randomly assigned to receive simvastatin or vehicle for 24 weeks, and bovine retinal capillary endothelial cells (BRECs) were incubated with normal or high glucose with or without simvastatin. Vascular endothelial growth factor (VEGF) and peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) in the rat retinas or BRECs were examined by Western blotting and real-time RT-PCR, and poly (ADP-ribose) polymerase (PARP), and p38 MAPK were examined by Western blotting. Mitochondrial membrane potential (Deltapsim) and ROS production were assayed using the potentiometric dye 5,5',6,6'- Tetrachloro1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) or CM-H(2)DCFDA fluorescent probes.

Results: Simvastatin significantly upregulated PGC-1alpha (P < 0.01), subsequently decreased Deltapsim (P < 0.05) and ROS generation (P < 0.01), inhibited PARP activation (P < 0.01), and further reduced VEGF expression (P < 0.01) and p38 MAPK activity (P < 0.01). Those changes were associated with the decrease of retinal vascular permeability, retinal capillary cells apoptosis, and formation of acellular capillaries.

Conclusions: Simvastatin decreases diabetes-induced mitochondrial ROS production and exerts protective effects against early retinal vascular damage in diabetic rats in association with the inhibition of mitochondrial ROS/PARP pathway mediated by PGC-1alpha. The understanding of the mechanisms of action of statins has important implications in the prevention and treatment of mitochondrial oxidative stress-related illness such as DR.

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Protein expression and apoptosis of cell analysis in retinas and BRECs. A: Representative Western blot analysis of poly(ADP-ribosyl)ated protein expression in retinas in control, DM, and DM + S. Equal protein loading was confirmed by detection of β-actin. Quantification of signal intensity by poly(ADP-ribosyl)ated protein densitometry in the retinas obtained from all three groups. B: Apoptosis cell in normal glucose (NG), high glucose (HG), HG + S, HG + S + mevalonate (HG + S + M), and HG + PJ-34. C: Representative Western blot analysis of poly(ADP-ribosyl)ated, VEGF, p38 MAPK, and p-p38 MAPK protein expression in BRECs in the five groups. Bars indicate SD. A representative experiment of the three is shown (**P < 0.01 vs. control; ##P < 0.01 vs. DM; n = 8; **P < 0.01 vs. NG; ##P < 0.01 vs. HG; $$P < 0.01 vs. HG + S; n = 9). (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: Protein expression and apoptosis of cell analysis in retinas and BRECs. A: Representative Western blot analysis of poly(ADP-ribosyl)ated protein expression in retinas in control, DM, and DM + S. Equal protein loading was confirmed by detection of β-actin. Quantification of signal intensity by poly(ADP-ribosyl)ated protein densitometry in the retinas obtained from all three groups. B: Apoptosis cell in normal glucose (NG), high glucose (HG), HG + S, HG + S + mevalonate (HG + S + M), and HG + PJ-34. C: Representative Western blot analysis of poly(ADP-ribosyl)ated, VEGF, p38 MAPK, and p-p38 MAPK protein expression in BRECs in the five groups. Bars indicate SD. A representative experiment of the three is shown (**P < 0.01 vs. control; ##P < 0.01 vs. DM; n = 8; **P < 0.01 vs. NG; ##P < 0.01 vs. HG; $$P < 0.01 vs. HG + S; n = 9). (A high-quality digital representation of this figure is available in the online issue.)

Mentions: PARP activity was demonstrated using a mAb to detect poly(ADP-ribosyl)ated proteins, which is the product of the enzyme. As illustrated in Fig. 3A, a marked increase was observed in poly(ADP-ribosyl)ation of the proteins obtained from the retinal extract of 6-month diabetic rats as compared with the nondiabetic controls, and it was significantly inhibited by simvastatin, which was accompanied by upregulation of VEGF (Fig. 1A and B) and activation of p38MAPK (Fig. 2F). Moreover, the effects were similar to that with PJ-34 (data not shown). In vitro, PJ-34 also significantly inhibited VEGF expression and activation of p38 MAPK and prevented the apoptosis of BRECs incubated in HG, as did simvastatin (Fig. 3B and C). In addition, we found that PARP antisense oligonucleotides downregulate VEGF expression (data not shown) and inhibited the activation of p38 MAPK in BRECs (22). These results suggest that the effect of simvastatin on retinopathy in diabetic rats may be associated with the inhibition of PARP activity.


Improvement of retinal vascular injury in diabetic rats by statins is associated with the inhibition of mitochondrial reactive oxygen species pathway mediated by peroxisome proliferator-activated receptor gamma coactivator 1alpha.

Zheng Z, Chen H, Wang H, Ke B, Zheng B, Li Q, Li P, Su L, Gu Q, Xu X - Diabetes (2010)

Protein expression and apoptosis of cell analysis in retinas and BRECs. A: Representative Western blot analysis of poly(ADP-ribosyl)ated protein expression in retinas in control, DM, and DM + S. Equal protein loading was confirmed by detection of β-actin. Quantification of signal intensity by poly(ADP-ribosyl)ated protein densitometry in the retinas obtained from all three groups. B: Apoptosis cell in normal glucose (NG), high glucose (HG), HG + S, HG + S + mevalonate (HG + S + M), and HG + PJ-34. C: Representative Western blot analysis of poly(ADP-ribosyl)ated, VEGF, p38 MAPK, and p-p38 MAPK protein expression in BRECs in the five groups. Bars indicate SD. A representative experiment of the three is shown (**P < 0.01 vs. control; ##P < 0.01 vs. DM; n = 8; **P < 0.01 vs. NG; ##P < 0.01 vs. HG; $$P < 0.01 vs. HG + S; n = 9). (A high-quality digital representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 3: Protein expression and apoptosis of cell analysis in retinas and BRECs. A: Representative Western blot analysis of poly(ADP-ribosyl)ated protein expression in retinas in control, DM, and DM + S. Equal protein loading was confirmed by detection of β-actin. Quantification of signal intensity by poly(ADP-ribosyl)ated protein densitometry in the retinas obtained from all three groups. B: Apoptosis cell in normal glucose (NG), high glucose (HG), HG + S, HG + S + mevalonate (HG + S + M), and HG + PJ-34. C: Representative Western blot analysis of poly(ADP-ribosyl)ated, VEGF, p38 MAPK, and p-p38 MAPK protein expression in BRECs in the five groups. Bars indicate SD. A representative experiment of the three is shown (**P < 0.01 vs. control; ##P < 0.01 vs. DM; n = 8; **P < 0.01 vs. NG; ##P < 0.01 vs. HG; $$P < 0.01 vs. HG + S; n = 9). (A high-quality digital representation of this figure is available in the online issue.)
Mentions: PARP activity was demonstrated using a mAb to detect poly(ADP-ribosyl)ated proteins, which is the product of the enzyme. As illustrated in Fig. 3A, a marked increase was observed in poly(ADP-ribosyl)ation of the proteins obtained from the retinal extract of 6-month diabetic rats as compared with the nondiabetic controls, and it was significantly inhibited by simvastatin, which was accompanied by upregulation of VEGF (Fig. 1A and B) and activation of p38MAPK (Fig. 2F). Moreover, the effects were similar to that with PJ-34 (data not shown). In vitro, PJ-34 also significantly inhibited VEGF expression and activation of p38 MAPK and prevented the apoptosis of BRECs incubated in HG, as did simvastatin (Fig. 3B and C). In addition, we found that PARP antisense oligonucleotides downregulate VEGF expression (data not shown) and inhibited the activation of p38 MAPK in BRECs (22). These results suggest that the effect of simvastatin on retinopathy in diabetic rats may be associated with the inhibition of PARP activity.

Bottom Line: The aim of this study was to clarify the beneficial effects and mechanism of action of simvastatin against diabetes-induced retinal vascular damage.Simvastatin significantly upregulated PGC-1alpha (P < 0.01), subsequently decreased Deltapsim (P < 0.05) and ROS generation (P < 0.01), inhibited PARP activation (P < 0.01), and further reduced VEGF expression (P < 0.01) and p38 MAPK activity (P < 0.01).Those changes were associated with the decrease of retinal vascular permeability, retinal capillary cells apoptosis, and formation of acellular capillaries.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, First People’s Hospital of Shanghai Affiliated to Shanghai Jiaotong University, Shanghai, China.

ABSTRACT

Objective: Mitochondrial reactive oxygen species (ROS) plays a key role in diabetic retinopathy (DR) pathogenesis. However, whether simvastatin decreases diabetes-induced mitochondrial ROS production remains uncertain. The aim of this study was to clarify the beneficial effects and mechanism of action of simvastatin against diabetes-induced retinal vascular damage.

Research design and methods: Diabetic rats and control animals were randomly assigned to receive simvastatin or vehicle for 24 weeks, and bovine retinal capillary endothelial cells (BRECs) were incubated with normal or high glucose with or without simvastatin. Vascular endothelial growth factor (VEGF) and peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) in the rat retinas or BRECs were examined by Western blotting and real-time RT-PCR, and poly (ADP-ribose) polymerase (PARP), and p38 MAPK were examined by Western blotting. Mitochondrial membrane potential (Deltapsim) and ROS production were assayed using the potentiometric dye 5,5',6,6'- Tetrachloro1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) or CM-H(2)DCFDA fluorescent probes.

Results: Simvastatin significantly upregulated PGC-1alpha (P < 0.01), subsequently decreased Deltapsim (P < 0.05) and ROS generation (P < 0.01), inhibited PARP activation (P < 0.01), and further reduced VEGF expression (P < 0.01) and p38 MAPK activity (P < 0.01). Those changes were associated with the decrease of retinal vascular permeability, retinal capillary cells apoptosis, and formation of acellular capillaries.

Conclusions: Simvastatin decreases diabetes-induced mitochondrial ROS production and exerts protective effects against early retinal vascular damage in diabetic rats in association with the inhibition of mitochondrial ROS/PARP pathway mediated by PGC-1alpha. The understanding of the mechanisms of action of statins has important implications in the prevention and treatment of mitochondrial oxidative stress-related illness such as DR.

Show MeSH
Related in: MedlinePlus