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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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Mitochondrial membrane potential (Δψm) and ROS production (the mitochondria suspensions were diluted to 1 mg protein/ml) in retina or BRECs. A and B: Δ ψm in retina in the control, DM, and DM + S groups (A) or in BRECs in NG, HG, HG + S, HG + PJ-34, and HG + S+ PGC-1α RNAi (B) was examined with the molecular probe JC-1. C and D: ROS production in the retinas of the three groups (C) or in BRECs of the five groups was identified with the fluorescent probe CM-H2DCFDA in BRECs in serum-free DMEM containing HG (D). E: Effects of simvastatin (0, 0.5, 1.0, 5.0, 10.0 μmol/l) on cell apoptosis, ROS, poly(ADP-ribosyl)ated protein, VEGF, and p-p38MAPK in BRECs in serum-free DMEM containing HG. Bars indicate SD. A representative experiment of the three is shown (**P < 0.01 vs. control, #P < 0.05 vs. DM, ##P < 0.01 vs. DM, n = 8; **P < 0.01 vs. NG, ##P < 0.01 vs. HG, $P < 0.05 vs. HG + S, $$P < 0.01 vs. HG + S, n = 9).
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Figure 4: Mitochondrial membrane potential (Δψm) and ROS production (the mitochondria suspensions were diluted to 1 mg protein/ml) in retina or BRECs. A and B: Δ ψm in retina in the control, DM, and DM + S groups (A) or in BRECs in NG, HG, HG + S, HG + PJ-34, and HG + S+ PGC-1α RNAi (B) was examined with the molecular probe JC-1. C and D: ROS production in the retinas of the three groups (C) or in BRECs of the five groups was identified with the fluorescent probe CM-H2DCFDA in BRECs in serum-free DMEM containing HG (D). E: Effects of simvastatin (0, 0.5, 1.0, 5.0, 10.0 μmol/l) on cell apoptosis, ROS, poly(ADP-ribosyl)ated protein, VEGF, and p-p38MAPK in BRECs in serum-free DMEM containing HG. Bars indicate SD. A representative experiment of the three is shown (**P < 0.01 vs. control, #P < 0.05 vs. DM, ##P < 0.01 vs. DM, n = 8; **P < 0.01 vs. NG, ##P < 0.01 vs. HG, $P < 0.05 vs. HG + S, $$P < 0.01 vs. HG + S, n = 9).

Mentions: Our study revealed that the production of Δψm and ROS was significantly increased in the retinas of diabetic rats compared with nondiabetic rats (P < 0.01), and simvastatin treatment reduced these effects (Fig. 4A and C). Similarly, we found that in vitro exposure of BRECs to HG increased Δψm, ROS synthesis, and PARP activity, upregulated VEGF, increased p38 MAPK activity, and concomitantly induced the apoptosis of the cells; however, these changes were significantly inhibited by simvastatin (Figs. 3B and C, Fig. 4B and D). Moreover, we observed dosage-dependent effects of simvastatin on cell apoptosis, ROS generation, PARP activity, VEGF, and p-p38MAPK in BRECs in serum-free DMEM containing HG (Fig. 4E). As described in our previous study (23), localization of ROS production was investigated in cultured cells double-labeled with CM-H2DCFDA to detect ROS production and MitoTracker Red CM-H2XRos (MTR; Molecular Probes) to visualize mitochondria. The results of this study indicated that mitochondria are the major source of ROS production after exposure to HG (data not shown).


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)

Mitochondrial membrane potential (Δψm) and ROS production (the mitochondria suspensions were diluted to 1 mg protein/ml) in retina or BRECs. A and B: Δ ψm in retina in the control, DM, and DM + S groups (A) or in BRECs in NG, HG, HG + S, HG + PJ-34, and HG + S+ PGC-1α RNAi (B) was examined with the molecular probe JC-1. C and D: ROS production in the retinas of the three groups (C) or in BRECs of the five groups was identified with the fluorescent probe CM-H2DCFDA in BRECs in serum-free DMEM containing HG (D). E: Effects of simvastatin (0, 0.5, 1.0, 5.0, 10.0 μmol/l) on cell apoptosis, ROS, poly(ADP-ribosyl)ated protein, VEGF, and p-p38MAPK in BRECs in serum-free DMEM containing HG. Bars indicate SD. A representative experiment of the three is shown (**P < 0.01 vs. control, #P < 0.05 vs. DM, ##P < 0.01 vs. DM, n = 8; **P < 0.01 vs. NG, ##P < 0.01 vs. HG, $P < 0.05 vs. HG + S, $$P < 0.01 vs. HG + S, n = 9).
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Figure 4: Mitochondrial membrane potential (Δψm) and ROS production (the mitochondria suspensions were diluted to 1 mg protein/ml) in retina or BRECs. A and B: Δ ψm in retina in the control, DM, and DM + S groups (A) or in BRECs in NG, HG, HG + S, HG + PJ-34, and HG + S+ PGC-1α RNAi (B) was examined with the molecular probe JC-1. C and D: ROS production in the retinas of the three groups (C) or in BRECs of the five groups was identified with the fluorescent probe CM-H2DCFDA in BRECs in serum-free DMEM containing HG (D). E: Effects of simvastatin (0, 0.5, 1.0, 5.0, 10.0 μmol/l) on cell apoptosis, ROS, poly(ADP-ribosyl)ated protein, VEGF, and p-p38MAPK in BRECs in serum-free DMEM containing HG. Bars indicate SD. A representative experiment of the three is shown (**P < 0.01 vs. control, #P < 0.05 vs. DM, ##P < 0.01 vs. DM, n = 8; **P < 0.01 vs. NG, ##P < 0.01 vs. HG, $P < 0.05 vs. HG + S, $$P < 0.01 vs. HG + S, n = 9).
Mentions: Our study revealed that the production of Δψm and ROS was significantly increased in the retinas of diabetic rats compared with nondiabetic rats (P < 0.01), and simvastatin treatment reduced these effects (Fig. 4A and C). Similarly, we found that in vitro exposure of BRECs to HG increased Δψm, ROS synthesis, and PARP activity, upregulated VEGF, increased p38 MAPK activity, and concomitantly induced the apoptosis of the cells; however, these changes were significantly inhibited by simvastatin (Figs. 3B and C, Fig. 4B and D). Moreover, we observed dosage-dependent effects of simvastatin on cell apoptosis, ROS generation, PARP activity, VEGF, and p-p38MAPK in BRECs in serum-free DMEM containing HG (Fig. 4E). As described in our previous study (23), localization of ROS production was investigated in cultured cells double-labeled with CM-H2DCFDA to detect ROS production and MitoTracker Red CM-H2XRos (MTR; Molecular Probes) to visualize mitochondria. The results of this study indicated that mitochondria are the major source of ROS production after exposure to HG (data not shown).

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