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(Investigative Ophthalmology and Visual Science. 2007;48:3805-3811.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1280
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Oxidative Damage in the Retinal Mitochondria of Diabetic Mice: Possible Protection by Superoxide Dismutase

Mamta Kanwar,1 Pooi-See Chan,1 Timothy S. Kern,2 and Renu A. Kowluru1

1From the Kresge Eye Institute, Wayne State University, Detroit, Michigan; and 2Case Western Reserve University, Cleveland, Ohio.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Superoxide levels are elevated in the retina in patients with diabetes, and cytochrome c is released from the mitochondria. The purpose of this study was to elucidate the mechanism involved in the oxidative damage of retinal mitochondria in diabetes and to determine whether mitochondrial superoxide dismutase (MnSOD) provides protection.

METHODS. Effects of diabetes were investigated on superoxide and GSH levels, electron transport complexes I and III, and membrane permeability in the isolated mitochondria prepared from the retinas of streptozotocin diabetic mice. To investigate the effect of MnSOD, retinal mitochondrial oxidative stress and electron transport complexes were determined in mice overexpressing MnSOD (MnSOD-Tg). Histopathology was evaluated in trypsin-digested retina.

RESULTS. Retinal mitochondria had twofold increase in superoxide levels in nontransgenic (wild-type [WT]) diabetic mice compared with WT nondiabetic mice. In the same retina, diabetes decreased mitochondrial GSH levels by 40% and complex III activity by approximately 20%, and it increased mitochondrial membrane permeability (swelling) by more than twofold; however, complex I activity was not affected. Overexpression of MnSOD inhibited diabetes-induced increases in mitochondrial superoxide levels and membrane permeability and the decrease in complex III activity. GSH values, however, were not statistically different in WT and MnSOD-Tg diabetic mice. In contrast to the diabetes-induced increase in the number of degenerate (acellular) capillaries in WT diabetic mice, the numbers of acellular capillaries in MnSOD-Tg nondiabetic and diabetic mice were similar to those in WT nondiabetic mice.

CONCLUSIONS. Retinal mitochondria experience increased oxidative damage in diabetes, and complex III is one of the sources of increased superoxide. MnSOD protects the retina from diabetes-induced abnormalities in the mitochondria and prevents vascular histopathology, strongly implicating the role for MnSOD in the pathogenesis of retinopathy in diabetes.

Diabetic retinopathy, the leading cause of acquired blindness in young adults, is a multifactorial disease. Although sustained hyperglycemia has been implicated in its pathogenesis,1 the exact mechanism of its development remains ambiguous. The retina and its capillary cells experience increased oxidative stress in diabetes; superoxide levels are elevated, and antioxidant defense systems are impaired.2 3 4 5 6 7 In the pathogenesis of diabetic retinopathy, capillary cells undergo accelerated apoptosis before any histopathology is seen in the retinal vasculature.8 9 10

Mitochondria are the major source of superoxide production and are subjected to direct attack of reactive oxygen species (ROS).11 Mitochondria generate superoxide through a series of electron carriers arranged spatially according to their redox potentials, but a small amount of electrons leak and form singlet oxygen that is quickly dismutated to hydrogen peroxide by the mitochondrial superoxide dismutase (MnSOD).12 Complex I (dinitrophenylhydrazine DNPH-coenzyme Q reductase) releases superoxide into the matrix, and complex III (coenzyme Q cytochrome c reductase) releases superoxide to both sides of the inner membrane.13 Mitochondrial dysfunction itself can lead to increased production of ROS, which can increase oxidative stress if the defense mechanisms of the cell are overwhelmed.14 ROS generated by mitochondria are considered to be responsible for the activation of major independent, but interrelated, pathogenic mechanisms for diabetic complications as modeled in endothelial cells exposed to hyperglycemia in vitro.15 16 We have shown that in diabetes, retinal mitochondria experience dysfunction: cytochrome c is released into the cytosol, Bax is translocated into the mitochondria, and the activity of MnSOD and its mRNA levels are decreased.7 Further, the overexpression of MnSOD provides protection to the retina and its vascular cells from diabetes-induced increases in oxidative stress, nitrative stress, and DNA damage.7 17

Because the mitochondrial electron transport system can be both the source and a target of excess ROS, this study was undertaken to elucidate the mechanism involved in superoxide-induced oxidative damage of retinal mitochondria in diabetes. With the use of mitochondria from the retina of nondiabetic and diabetic mice, we investigated the effect of diabetes on superoxide and reduced glutathione (GSH) levels, activities of electron transport complexes I and III, and mitochondrial membrane permeability. In addition, the ability of MnSOD overexpression to protect against diabetes-induced retinal mitochondrial oxidative stress and electron transport dysfunction, and capillary degeneration were also determined.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Hemizygous MnSOD transgenic mice with a C57BL/6 background were developed using human ß-actin-MnSOD expression construct 2, as described recently.17 18 Transgenic mice were generated by microinjecting fertilized eggs harvested from female B6C3 (C57BL/6 x C3H) F1 hybrid mice mated with male B6C3 F1 mice. The hemizygous MnSOD-Tg mice were bred with wild-type B6C3 F1 mice to generate experimental animals, and the litters were genotyped by Southern blot analysis. The expression of the MnSOD transgene was found in many tissues, including the retina.17 Both MnSOD-Tg and wild-type (WT) mice were obtained from the same litters. Because the C3H mice could carry the rd1 allele that could affect mitochondrial function, MnSOD Tg mice and their WT littermates mice were also genotyped for the rd1 allele using PCR (rd1 primers: forward, 5'-CAT CCC ACC TGA GCT CAC AGA AAG-3'; reverse, 5'-GCC TAC AAC AGA GGA GCT TCT AGC-3'. A similar rd1 transgene was observed in WT and Tg mice (diabetic and nondiabetic).

A group of MnSOD-Tg mice and their WT littermates (body weight [BW], 18–22 g) were made diabetic by intraperitoneal injection of streptozotocin (55 mg/kg BW) for 5 consecutive days.17 Mice with blood glucose levels higher than 250 mg/dL, 3 days after the last injection of streptozotocin, were included in the diabetes group. Age-matched MnSOD-Tg and WT mice served as controls. All mice were weighed once a week, and their blood glucose levels were monitored every other week. Diabetic mice (WT and MnSOD-Tg) were injected with insulin (0.1–0.2 IU) 1 to 3 times a week to prevent weight loss and ketonuria. The entire mice colony had free access to food (standard laboratory chow) and water. Glycated hemoglobin (GHb) levels were measured by affinity columns (as routinely used in our laboratory4 at 2 months of diabetes and every 3 months thereafter). Mice were killed at approximately 6 months of diabetes (for biochemical measurements) by an overdose of pentobarbital; their retinas were isolated immediately under a dissecting microscope and were kept on ice before use to prepare mitochondria. Another set of mice was killed at 12 to 13 months of diabetes for histopathology, and the eyes were stored in 10% buffered formalin solution. For comparison, age-matched nondiabetic MnSOD-Tg and WT mice were also killed at both time points. Treatment of the animals conformed to the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research.

Preparation of Mitochondria
Mitochondria were prepared from freshly isolated retinas by the centrifugation method.19 Two retinas were pooled and suspended in the mitochondria buffer (25 mM Tris-HCl, pH 7.4, 250 mM sucrose, 2 mM EDTA, 10 µg/mL leupeptin, and 10 µg /mL aprotinin) and were gently homogenized with a glass homogenizer. The homogenate was centrifuged at 750g for 5 minutes at 4°C to remove cell debris and nuclei, and the supernatant was centrifuged again at 10,000g for 15 minutes. The resultant mitochondrial pellet was suspended in the mitochondrial buffer, and protein was determined by the bicinchoninic acid assay (Sigma Chemical, St. Louis, MO).

Superoxide
Superoxide levels were quantified in the mitochondria isolated from the retina using lucigenin (bis-N-methylacridinium nitrate).6 20 The rate of superoxide production was measured with a luminescence spectrometer (LS55; Perkin-Elmer). Freshly prepared mitochondria were diluted to 0.5 mg protein/mL in respiratory buffer (5 mM K2PO4, 125 mM KCl, pH 7.25), sonicated, and diluted to 50 µg protein/mL in hypotonic buffer (10 mM MOPS and 0.5 mM EDTA, pH 7.6) supplemented with lucigenin (20 µM). After the addition of 70 µM NADH, luminescence was monitored for 5 minutes with readings recorded every 20 seconds. The assay was run in a total volume of 100 µL containing 2 to 5 µg mitochondrial protein. Blanks included all the components except the sample, and the blank values were subtracted from the sample readings. As a positive control, 100 mM Tiron (4,5-dihydroxy-1,3-benzene-disulfonic acid) was used to scavenge superoxide.

Glutathione
Glutathione levels were measured in the mitochondria isolated from the retina by an enzymatic recycling method using the kit from Cayman Chemical (Ann Arbor, MI). Mitochondrial protein (3–5 µg) was deproteinized by phosphoric acid, and GSH concentration was measured in the resultant supernatant. DTNB (5, 5'-dithiobis-2-nitrobenzoic acid) was used to produce a yellow compound, TMB (5-thio-2-nitrobenzioc acid), which was measured at 410 nm.

Electron Respiratory Chain Complex Activity
For analysis of complex I, isolated mitochondria were suspended in the hypotonic buffer at a concentration of 50 µg/mL. The final assay volume of 100 µL contained 4 to 6 µg protein, 5 µM antimycin A, 2 mM potassium cyanide, and 60 µM ubiquinone-1, and the assay was initiated by the addition of 100 µM NADH. Its consumption was monitored spectrophotometrically at 340 nm.

The activity of complex III was assayed using mitochondrial suspension that was diluted to 2.5 µg/mL protein in the hypotonic buffer.20 The final assay volume of 100 µL had 0.3 to 0.5 µg mitochondrial protein, 40 µM reduced decylubiquinone, and 2 mM KCN; the assay was initiated by the addition of 50 µM cytochrome c, and the reduction of cytochrome c was measured at 550 nm.

Mitochondrial Membrane Permeability
A sudden increase in the permeability of the mitochondrial inner membrane to small ions and molecules resulted in the collapse of mitochondrial membrane potential. The collapse of mitochondrial membrane was quantified by measuring the swelling of the mitochondria using the published spectrophotometric method of Van Remmen et al.21 In an assay volume of 100 µL, mitochondrial protein (5–20 µg protein) was allowed to equilibrate for 30 seconds at 25°C with 3 mM HEPES buffer (pH 7.4) containing 215 mM mannitol, 71 mM sucrose, and 5 mM succinate. The transition was induced by calcium chloride (400 µM). The decrease in absorbance at 540 nm was followed until the absorbance was stabilized. The extent of swelling was calculated as a percentage of swelling with respect to the maximum swelling achieved by exposure to calcium chloride.

Trypsin Digestion
The retina from the formalin-fixed eyes was rinsed overnight with water and was digested with a crude trypsin mixture (3% trypsin 1:250 containing 0.2 M sodium fluoride; Invitrogen-Gibco, Grand Island, NY) for 2 hours at 37°C. After the neuroretinal tissue was gently brushed away, the isolated vascular tree was air dried onto a glass microscope slide and was stained with periodic acid-Schiff and hematoxylin for histologic evaluation. Acellular capillaries, defined as basement membrane tubes lacking cell nuclei and maintaining at least one fourth the normal capillary caliber over their lengths, were counted in multiple mid-retinal fields and standardized to retinal area (per square millimeter).

Because the C3H mouse strain is known to develop spontaneous retinal degeneration, in the histologic analysis of the isolated retinal vasculature, 10% to 15% of the total animals (both WT and MnSOD-Tg) showed variable amounts of this vascular abnormality. These animals were not included in the analysis.

Statistical Analysis
Each measurement was made in duplicate, and the assay was repeated 3 or more times. Data are expressed as mean ± SD. Statistical analysis was performed using the nonparametric Kruskal-Wallis test followed by Mann-Whitney U test. P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
Hyperglycemia
The entire diabetic mice colony (MnSOD-Tg and WT) had blood glucose values that were more than 3 times higher than their age-matched nondiabetic littermates, and they remained elevated throughout the entire duration of the experiment. In the same MnSOD-Tg and WT diabetic mice, GHb values were elevated 2.5- to 3-fold. MnSOD-Tg diabetic and WT diabetic mice had hyperglycemia of similar severity; glucose and GHb values were similar in these two groups of diabetic mice (Table 1) .


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  		TABLE 1. Effect of Diabetes on the Severity of Hyperglycemia in Wild-Type and MnSOD-Tg Mice

 
MnSOD Expression
Western blot data showed a 50% to 60% increase in the expression of MnSOD in the retina from MnSOD-Tg mice compared with their WT littermates, and this was also confirmed by MnSOD enzyme assay. These results were in agreement with our previous study.17

Superoxide
Superoxide levels were significantly increased in the mitochondria isolated from the retinas of WT diabetic mice, and the values were more than twofold higher in WT diabetic group than in the WT nondiabetic group (P = 0.026). However, the overexpression of MnSOD prevented diabetes-induced increases in the superoxide production in retina; superoxide levels were similar in the mitochondria obtained from nondiabetic and diabetic MnSOD-Tg mice (P = 0.67), and these values were not different from those obtained from the nondiabetic WT group (P = 0.40; Fig. 1 ). Adding Tiron to the assay system inhibited the generation of superoxide, confirming that the assay detected primarily superoxide (data not shown).


Figure 1
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  		FIGURE 1. Superoxide levels in the retinal mitochondria: effect of diabetes and MnSOD overexpression. Superoxide levels were measured in the isolated mitochondria obtained from the retinas of WT and MnSOD-Tg diabetic mice (about 6 months of duration) and age-matched nondiabetic mice using lucigenin. After NADH was added, luminescence was monitored on a luminescence spectrophotometer. Values are mean ± SD of six or more mice in each group. *P < 0.05 compared with WT nondiabetic mice. **P < 0.05 compared with WT diabetic mice.

 
Glutathione
Figure 2 shows that the levels of GSH were decreased by approximately 40% in the mitochondria prepared from the retinas of WT diabetic mice compared with those observed in the mitochondria of WT nondiabetic mice. Overexpression of MnSOD had a marginal effect on the protection of a diabetes-induced decrease in GSH levels; GSH levels remained subnormal by approximately 25% in the mitochondria obtained from MnSOD-Tg diabetic mice compared with MnSOD-Tg nondiabetic mice, and these values obtained from MnSOD-Tg diabetic mice were not statistically different from those obtained from WT diabetic mice (P = 0.49).


Figure 2
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  		FIGURE 2. Mitochondrial GSH levels in the retina in diabetes and the effect of MnSOD overexpression. GSH levels were measured in the isolated mitochondria. The mitochondria was deproteinized by phosphoric acid, and GSH was measured in the supernatant using DTNB. Values are mean ± SD of six mice each in WT diabetic and nondiabetic groups and seven mice each in MnSOD-Tg diabetic and nondiabetic groups. *P < 0.05 compared with WT nondiabetic mice. **P < 0.05 compared with WT diabetic mice.

 
Complex I and Complex III
The activities of complexes I and III were measured in the mitochondria isolated from the retinas of diabetic and nondiabetic WT and MnSOD-Tg mice. Diabetes had no significant effect on the activity of complex I in retinal mitochondria; the values were not statistically different (P = 0.19) in WT diabetic and nondiabetic groups. Overexpression of MnSOD did not affect the activity of complex I; similar values were obtained in diabetic and nondiabetic MnSOD-Tg groups (Fig. 3) .


Figure 3
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  		FIGURE 3. Complex I activity in retinal mitochondria. Complex I activity was assayed in the isolated mitochondria by measuring the consumption of NADH at 340 nm in a medium that had 4 to 6 µg mitochondrial protein, antimycin A, potassium cyanide, and ubiquinone-1. Each measurement was made in duplicate in six or more mice in each of the four groups. Control values were obtained from the mitochondria of WT nondiabetic mice and were considered 100%.

 
In the same WT diabetic mice, complex III activity was decreased by approximately 20% in the retinal mitochondria compared with WT nondiabetic mice (P = 0.049). MnSOD overexpression protected the mitochondria from diabetes-induced inhibition of complex III activity; the values obtained from the mitochondria of MnSOD-Tg diabetic mice and nondiabetic mice were comparable and were not different from those obtained from the mitochondria of the WT nondiabetic mice (P = 0.84; Fig. 4 ).


Figure 4
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  		FIGURE 4. Complex III activity in retinal mitochondria and protection by MnSOD. The activity of ubiquinone-cytochrome c reductase of mitochondrial complex III was measured in retinal mitochondria by measuring the reduction of cytochrome c at 550 nm. Control values were obtained from the mitochondria of WT nondiabetic mice, and were considered 100%. Values are represented as mean ± SD of six or more mice in each of the four groups of mice. *P < 0.05 compared with WT normal mice. **P < 0.05 compared with WT diabetic mice.

 
Mitochondria Membrane Permeability
Mitochondrial swelling that represented a sudden increase in the permeability of the mitochondrial inner membrane to small ions and molecules resulting in the collapse of mitochondrial membrane potential was measured in the mitochondria prepared from the retinas of MnSOD-Tg and WT mice. As shown in Figure 5 , mitochondrial membrane permeability transition was increased by more than 2.5-fold in the retinas of WT diabetic mice compared with nondiabetic WT mice. Overexpression of MnSOD prevented diabetes-induced increases in retinal mitochondria permeability; similar values were obtained from the retinas of MnSOD-Tg diabetic and nondiabetic groups (P = 0.202) and were significantly different from those obtained from the WT diabetic group (P = 0.001).


Figure 5
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  		FIGURE 5. Swelling in the retinal mitochondria: effect of diabetes and MnSOD overexpression. Mitochondrial swelling was determined by measuring the collapse of mitochondrial membrane potential spectrophotometrically by measuring the decrease in absorbance at 540 nm induced by calcium chloride. The extent of swelling was calculated as a percentage of swelling with respect to the maximum swelling achieved by exposure to external calcium, and the values obtained from the mitochondria of WT nondiabetic mice were considered as 100%. Results are expressed as mean ± SD of six or more mice in each group. *P < 0.05 compared with WT nondiabetic mice. **P < 0.05 compared with WT diabetic mice.

 
Retinal Histopathology
The number of acellular capillaries counted in the trypsin-digested retinal vasculature at 12 to 13 months of diabetes was elevated by almost 2-fold in the WT diabetic mice compared with WT normal mice (P = 0.047). Diabetes had no effect on the number of retinal acellular capillaries in the MnSOD-Tg group (P = 0.297); the numbers were similar in MnSOD-Tg diabetic and nondiabetic groups and in the WT normal group (P > 0.05; Fig. 6 ).


Figure 6
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  		FIGURE 6. Retinal histopathology and MnSOD overexpression: Trypsin-digested retinal microvasculature was stained with periodic acid-Schiff and hematoxylin. The number of acellular capillaries was counted in multiple midretinal fields and standardized to retinal area (per square millimeter). Results are expressed as mean ± SD of five mice in each WT nondiabetic and diabetic groups and six mice each in MnSOD-Tg non diabetic and diabetic groups. *P < 0.05 compared with WT nondiabetic mice. **P < 0.05 compared with WT diabetic mice. (A) Acellular capillary is marked with a wide arrow.

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
This study is the first to demonstrate conclusively that the source of increased superoxide seen in the retina in diabetes is the mitochondria. We show that the electron transport system of the retina is also impaired and that these diabetes-induced abnormalities are prevented by overexpression of the mitochondrial superoxide scavenging enzyme MnSOD. Further, our exciting histopathology results reveal that MnSOD protects the retina from the diabetes-induced histopathology characteristic of the early stages of diabetic retinopathy.

Mitochondria are the major source of superoxide, and these mitochondrial ROS cause damage to mitochondrial components. Thus, mitochondria are at risk for damage under conditions of oxidative stress such as diabetes. MnSOD acts as a first line of defense to protect mitochondria and other cellular components by scavenging superoxide anion in the mitochondrial matrix.22 In the pathogenesis of diabetic complications, superoxide is considered to be a causal link between elevated glucose and the major metabolic abnormalities associated with vascular complications.15 Superoxide levels are elevated in the retinas in diabetes and in aortic endothelial cells in high glucose conditions, and the inhibitors of mitochondrial electron transport chain are shown to prevent such increases.6 7 16 17 We have shown that the overexpression of MnSOD provides protection to the retina from increased oxidative damage experienced in diabetic conditions and to the capillary cells in high glucose conditions.7 17 Here we provide direct evidence that superoxide levels are significantly elevated in the mitochondria isolated from the retinas of diabetic mice and that the overexpression of MnSOD prevents diabetes-induced increases in mitochondrial superoxide. Although diabetes has been reported to increase superoxide levels in the whole retina, and the source, based on the use of chemical inhibitors of electron transport chain, is postulated to be the mitochondria, this study is the first to conclusively demonstrate that the mitochondria is the source of increased superoxide. Moreover, we show that this diabetes-induced increase in mitochondrial superoxide and increase in the number of acellular capillaries in the retina can be inhibited by the overexpression of MnSOD.

Retinal mitochondria become dysfunctional in diabetes when the duration of diabetes is such that capillary cell apoptosis can be observed and is implicated in the accelerated loss of capillary cells.19 In addition, mitochondrial dysfunction can lead to increased production of ROS; if the defense mechanism is overwhelmed, this could further increase oxidative stress.14 GSH, a thiol-containing tripeptide, is one of the major antioxidants that play a central role in the cellular defense against free radicals and hydroperoxides.23 Although most cellular GSH is present in the cytoplasm, subcellular pools of GSH are also found in nuclear and mitochondrial compartments.24 Maintenance of the mitochondrial GSH pool is important for cellular and mitochondrial redox homeostasis and for protection against ROS.25 Our present data suggest that in diabetes, the retinal mitochondria experience a double insult: increased generation of superoxide and compromised antioxidant defenses, including GSH. Overexpression of MnSOD provides minimal protection from diabetes-induced decreases in mitochondrial GSH levels. This is unexpected because our recent study has shown that MnSOD overexpression prevents diabetes-induced decreases in GSH levels in the whole retina.17 The reason for such discordant findings is unclear, but compartmentalization of GSH in different pools26 could account for this.

The mitochondrial electron transport chain system is a major source of superoxide production.13 Superoxide is produced from both complex I and complex III, and MnSOD converts them to H2O2.27 Further, complexes I, III, and IV of the electron respiratory chain are the main mitochondrial targets of hyperglycemia-induced injury.20 28 Complex I releases superoxide into the matrix and complex III releases to both sides of the inner membrane, suggesting extramitochondrial release of superoxide.14 Complex I, a multisubunit enzyme that transfers electrons from NADH to ubiquinone, is the first site of oxidative phosphorylation.29 Figure 3 shows that diabetes does not alter the activity of complex I in the retinal mitochondria, suggesting that complex I might not contribute to the increased superoxide levels seen in the retinal mitochondria in diabetes.

Complex III transfers electrons from reduced ubiquinone to cytochrome c and is situated immediately next to the intermembrane space.13 We show that the activity of complex III is decreased in the retina in diabetes, clearly suggesting that increased superoxide experienced in diabetes possibly results from the impaired complex III system. Similar diabetes-induced inhibition of complex III in kidney is reported by others, and modification of the essential arginine residues of cytochrome c1 by methylglyoxal,20 30 or peroxidation of membrane lipid components important for complex III function,31 are postulated as the possible mechanisms. Methylglyoxal levels and lipid peroxides are also elevated in the retina and its capillary cells in diabetes.32 33 34 Our data clearly show that the overexpression of MnSOD protects the mitochondria from diabetes-induced inhibition of complex III. Consistent with our findings, others have shown that ROS-induced loss of complex III activity in bovine heart submitochondria is prevented by the addition of SOD.31 Here we provide evidence of impaired mitochondrial respiratory complex III in diabetes that can be protected by MnSOD. Others have shown an inhibition of hyperglycemia-induced superoxide increase in the retina and in bovine aortic endothelial cells by the inhibitors of complex II6 16 20 28 ; thus, we cannot rule out the role of complex II in diabetes-induced increases in superoxide levels seen in the retina.

Elevated oxidative stress can increase the membrane permeability of mitochondria by opening of nonspecific pores in the inner mitochondrial membrane and can lead to the loss of membrane potential.21 Mitochondrial swelling can cause the release of cytochrome c and the activation of the apoptotic pathway.35 Increased cytochrome c from the mitochondria into the cytosol and Bax translocation into the mitochondria are observed in the diabetic retina when the apoptosis of capillary cells can be detected in the retinal microvasculature.19 Here we provide data showing that mitochondria isolated from the retinas of diabetic animals are swollen and that this swelling can be prevented by the overexpression of MnSOD. This suggests that retinal mitochondria experience an impaired membrane potential in diabetes that allows them to swell and leak and that MnSOD overexpression might protect the mitochondria from releasing proapoptotic proteins and might prevent the retina from diabetes-induced apoptosis and, ultimately, histopathology. In support of this, MnSOD mimetics have been shown to inhibit hyperglycemia-induced increased apoptosis of retinal capillary cells.19

Acellular capillaries represent one of the early features of diabetic retinopathy seen in diabetic rodents.4 8 10 Our study demonstrates that the inhibition of diabetes-induced superoxide accumulation by an overexpression of MnSOD has beneficial effects in inhibiting the development of retinal histopathology. Consistent with this, an overexpression of MnSOD has been shown to provide overall protection to the diabetic heart and to normalize contractility in cardiomyocytes.36 Ours is the first report showing the beneficial effects of MnSOD on the development of retinopathy in diabetes, and it suggests that the therapy designed to scavenge mitochondrial superoxide could help inhibit this major microvascular complication faced by patients with diabetes.

The results presented in this manuscript were obtained without ameliorating the severity of hyperglycemia because the severity of diabetes in MnSOD-Tg and WT mice, which was maintained by regulating blood glucose and body weight, was similar in these two groups of diabetic mice. However, we cannot rule out the potential role of MnSOD in the nonenzymatic glycation of proteins that could contribute to the pathogenesis of diabetic retinopathy.

In conclusion, our study in diabetes demonstrates that retinal mitochondria have elevated superoxide levels, that their membrane permeability is increased, and that complex III is one of the sources of increased superoxide. Overexpression of mitochondrial SOD protects against the development of diabetes-induced abnormalities in the retinal mitochondria and against the development of the early stages of diabetic retinopathy (i.e., acellular capillaries). This strongly implicates the significant role for MnSOD in the pathogenesis of retinopathy in diabetes. Identifying the mechanism of mitochondrial superoxide production in diabetes will help elucidate molecular targets for future therapy.


   Acknowledgements
 
The authors thank Divyesh Sarman and Lamia Atasi for their technical assistance.


   Footnotes
 
Supported in part by grants from the National Institutes of Health, the Juvenile Diabetes Research Foundation, the Thomas Foundation, and Research to Prevent Blindness.

Submitted for publication October 24, 2006; revised February 22 and April 3, 2007; accepted June 12, 2007.

Disclosure: M. Kanwar, None; P.-S. Chan, None; T.S. Kern, None; R.A. Kowluru, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Renu A. Kowluru, Kresge Eye Institute, Wayne State University, 4717 St. Antoine, Detroit, MI 48201; rkowluru{at}med.wayne.edu.


   References
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 Abstract
 Methods
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 Discussion
 References
 

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