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(Investigative Ophthalmology and Visual Science. 2003;44:5327-5334.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.03-0353
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Diabetes-Induced Mitochondrial Dysfunction in the Retina

Renu A. Kowluru and Saiyeda Noor Abbas

From the Kresge Eye Institute, Wayne State University, Detroit, Michigan.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-3 and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells.

METHODS. Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-3 activity and ELISA assay.

RESULTS. Diabetes of 8 months’ duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis.

CONCLUSIONS. Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.

Diabetes increases oxidative stress, which plays an important role in the development of diabetic complications.1 2 3 Oxidative stress is increased in retina in diabetes and in isolated retinal capillary cells (both endothelial cells and pericytes) incubated in high-glucose medium.4 The antioxidant defense system is impaired in the retina in diabetes, GSH levels are decreased, superoxide production is increased, and mRNA levels of superoxide dismutase (SOD) and glutathione reductase are downregulated.4 5 6 7 8 9 We have reported that the long-term administration of antioxidants inhibits the development of retinopathy in diabetic rats and in galactose-fed rats (another model of diabetic retinopathy),3 suggesting an important role for oxidative stress in the development of retinopathy in diabetes. Oxidative stress is involved directly in the upregulation of vascular endothelial growth factor in the retina during early diabetes.10 Recent studies from our laboratory have shown that oxidative stress plays an important role, not only in the development of retinopathy in diabetes, but also in the resistance of retinopathy to arrest after good glycemic control is initiated.11

Capillary cells and neurons are lost in the retina before other histopathology is detectable, and apoptosis has been implicated as one of the mechanism(s).12 13 14 15 Apoptosis execution enzyme, caspase-3, and nuclear transcriptional factor (NF-{kappa}B) are activated in the retina when the duration of diabetes in rats is such that the capillary cell death and histopathology are detectable, and antioxidants inhibit such activations.4 16 17 Oxidative stress is shown to be closely linked to apoptosis in a variety of cell types18 19 ; however, the signaling steps involved in oxidative-stress–induced retinal capillary cell apoptosis are not clear.

Mitochondria are the major endogenous source of superoxides and hydroxyl radicals.20 Reactive oxidant intermediates can trigger mitochondria to release cytochrome c, resulting in activation of caspase-3.21 22 23 Overproduction of superoxides by mitochondria is considered as a causal link between elevated glucose and the major biochemical pathways postulated to be involved in the development of vascular complications in diabetes.24 25 Increasing evidence indicates that mitochondria are intimately associated with the initiation of apoptosis. Mitochondrial changes are associated with the activation of apoptotic pathways resulting in diabetic neuropathy,26 27 impaired kidney function,28 and myocardial abnormalities.29 However, the involvement of mitochondria in the development of retinopathy in diabetes is not clear.

In the present study the effect of diabetes on mitochondrial dysfunction in the retina of rats and in the isolated retinal capillary cells was investigated by measuring the release of cytochrome c into the cytosol and translocation of Bax into the mitochondria. The effect of inhibition of mitochondrial oxidative stress on capillary cell death is also determined.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Rats
Wistar rats (male, 200–220 g) were randomly assigned to normal or diabetic groups. Diabetes was induced with streptozotocin injection (55 mg/kg body weight, intraperitoneal), and insulin was given as needed to allow slow weight gain while maintaining hyperglycemia (blood glucose levels of 20–25 mM). The rats were weighed two times a week, and their food consumption was measured once every week. Glycated hemoglobin (GHb) was measured at 2 months of diabetes, and every 3 months thereafter, using affinity columns (kit 442-B; Sigma-Aldrich). Diabetic rats and age-matched normal rats were killed at 2 and 8 months of diabetes, and the retina was immediately removed. These experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Capillary Cells
Endothelial cells and pericytes were prepared from bovine eyes by a method described by Kennedy et al.30 and routinely used by us.4 31 32 Endothelial cells were grown to 80% confluence in Petri dishes coated with 0.1% gelatin in Dulbecco’s modified Eagle’s medium (DMEM) containing heparin, 10% fetal calf serum (heat inactivated), 10% serum replacement (Nu-serum; BD Biosciences, Lincoln Park, NJ) endothelial growth supplement (25 µg/mL), and antibiotic-antimycotic in an environment of 95% O2 and 5% CO2. Confluent cells from passages 4 to 8 were split and incubated under normoglycemic (5 mM glucose) or hyperglycemic (20 mM glucose) conditions for 1 to 10 days in the presence or absence of 20 mU/mL SOD,33 34 200 µM MnTBAP (Mn(III)tetrakis(4-benzoic acid)porphyrin chloride; a cell-permeable SOD mimetic; Biomol, Plymouth Meeting, PA35 36 ), 250 µM N-acetyl cysteine, or 250 µM lipoic acid.4

Pericytes were grown in DMEM supplemented with 10% fetal calf serum, antibiotics, and antimycotics, as described by us previously.4 31 32 Pericytes (passages 4–6) were incubated in DMEM containing 2.5% fetal bovine serum in 5 or 20 mM glucose in the presence and absence of antioxidants.

Control incubations containing 20 mM mannitol always were run simultaneously to rule out the effect of increased osmolarity. Each experiment was repeated with at least three separate cell preparations.

Isolation of Mitochondria and Cytosol
Mitochondria were isolated from the freshly removed retina or from cells by centrifugation.37 38 The retina was suspended in the mitochondria buffer containing 20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL aprotinin, and 250 mM sucrose, and gently homogenized with a glass homogenizer. The cells were removed from the incubation Petri dishes by trypsin digestion, washed with ice-cold PBS, and homogenized in the mitochondria buffer. The homogenate was centrifuged at 750g for 10 minutes at 4°C to remove nuclei and unbroken cells, and the supernatant was centrifuged at 10,000g for 15 minutes. The resultant mitochondrial pellet was lysed in 50 µL of 20 mM Tris (pH 7.4), 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 10 µg/mL aprotinin, and the supernatant was centrifuged at 100,000g for 60 minutes to obtain the cytosolic fraction. Protein was determined in both mitochondrial and cytosolic fractions by the bicinchoninic acid assay (Sigma-Aldrich).

Cytochrome c Release and Bax Translocation
Release of cytochrome c was quantitated by Western blot techniques by measuring the expression of cytochrome c in mitochondrial and cytosolic fractions. Mitochondrial (20 µg) and cytosolic (40 µg) proteins were separated on 15% reducing polyacrylamide gel and then transferred to nitrocellulose membranes. The membranes were blocked in 5% milk, followed by incubation with a polyclonal antibody against cytochrome c (Santa Cruz Biotechnology, Santa Cruz, CA). After washing, the membranes were incubated with anti-rabbit IgG horseradish peroxidase–conjugated antibody in blocking buffer for 1 hour, washed, and developed using a Western blot chemiluminescence detection kit (ECL-Plus; Amersham Biosciences, Arlington Heights, IL).

To ensure that the same content of mitochondrial or cytosolic protein was loaded in each lane, after they were blotted for cytochrome c, the membrane were incubated in stripping buffer (62.5 mM Tris-HCl [pH 6.8], 100 mM mercaptoethanol, and 2% sodium dodecyl sulfate) at 50°C for 30 minutes and washed (three times for 10 minutes each). The membranes were then incubated with anti-cytochrome c oxidase subunit IV (Cox IV; Molecular Probes, Eugene, OR) or ß-actin (Santa Cruz Biotechnology) and developed with the Western blot analysis detection kit.

Translocation of Bax into mitochondria was measured by performing Western blots for Bax in both mitochondrial and cytosolic fractions using rabbit polyclonal antibodies (Santa Cruz Biotechnology).

Cell Death
Cell death was determined by performing ELISA and by apoptotic DNA laddering with cell death detection kits (Cell Death Detection ELISA and Apoptotic DNA ladder kits; Roche Diagnostics, Indianapolis, IN). Cell death was further confirmed by measuring the activity of the apoptosis executor enzyme caspase-3.4

Relative amounts of mono- and oligonucleosomes generated from apoptotic cells were quantitated with the ELISA kit, using monoclonal antibodies directed against DNA and histones, respectively. The cytoplasmic fraction of the cells was transferred to a streptavidin-coated microtiter plate and incubated for 2 hours at room temperature with a mixture of peroxidase-conjugated anti-DNA and biotin-labeled anti-histone. The plate was then thoroughly washed, incubated with ABTS (2,2'-Azino-di[3-ethylbenzithiazolinesulfonate(6)] disodium salt; Roche Diagnostics), and absorbance was measured at 405 nm against ABTS solution as a blank. After separation of the cytoplasmic fraction, the nuclear pellet was suspended in 50 mM sodium phosphate buffer (pH 7.5) containing 2 mM NaCl and 0.05 mM Na2HPO4 (pH 7.5) and sonicated. DNA was measured in this fraction, and apoptosis was normalized to micrograms of DNA.

DNA fragmentation was detected with a kit (Apoptotic DNA Ladder Kit; Roche Diagnostics). Briefly, the cells were washed twice with PBS, resuspended in PBS, and incubated for 10 minutes with an equal volume of lysis buffer containing 10 mM Tris-HCl (pH 7.4), 6 M guanidine-HCl, 10 mM urea and EDTA, and 0.2% Triton X-100. The samples were passed through glass fiber fleece by centrifugation and the nucleic acid bound to the glass fibers was eluted. The DNA was applied to a 1.5% agarose gel, and the bands were then visualized by ethidium bromide staining and photographed.

Statistical Analysis
Data are reported as the mean ± SD, and experimental groups were compared using the nonparametric Kruskal-Wallis test followed by the Mann-Whitney test for multiple-group comparison. Similar conclusions were reached also by using ANOVA with the Fisher or Tukey test.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Diabetic Rats
Glycated hemoglobin (an index of severity of hyperglycemia) and urine volumes were elevated by two- to threefold in the diabetic rats compared with their age-matched normal rats. The body weights were significantly lower in diabetic rats at 2 and 8 months of diabetes compared with the age-matched normal control (Table 1) .


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  		TABLE 1. Severity of Hyperglycemia in Diabetic Rats

 
Diabetes of 2 months’ duration in rats had no effect on the release of cytochrome into the cytosol. The cytochrome c content of the cytosolic fraction of the retina was similar in diabetic rats and age-matched normal rats, but 8 months of diabetes resulted in an approximate twofold increase in the cytosolic content of cytochrome c, compared with the age-matched normal rats (Fig. 1A) . However, the expression of ß-actin among the lanes did not vary.



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  		FIGURE 1. Effect of diabetes on the (A) release of cytochrome c in the cytosol and (B) translocation of Bax into mitochondria. The mitochondrial and cytosolic fractions were prepared from retina freshly harvested from rats diabetic for 2 or 8 months and age-matched normal control rats. Cytochrome c and Bax contents were determined by Western blot analyses, and the band intensities were adjusted to the expression of the ß-actin or Cox IV in cytosolic and mitochondrial fractions, respectively. The Western blots are representative of five rats in each group, and the retina from each rat was analyzed in duplicate in two separate experiments. *P < 0.05 and #P > 0.05 compared with the age-matched normal control.

 
Similarly, the expression of Bax was significantly increased in the mitochondria obtained from the retina of rats diabetic for 8 months compared with the age-matched normal control rats (P < 0.02). However, there was no significant difference in the Bax expression in the mitochondria obtained from the retina of rats diabetic for 2 months compared with that from age-matched normal rats (Fig. 1B) .

In the same retina, as reported previously,4 caspase-3 activity was increased by 40% at 8 months of diabetes compared with age-matched normal control rats, but 2 months of diabetes had no effect on retinal caspase-3 activity.4

Isolated Capillary Cells: Studies with Endothelial Cells
The cytosolic content of cytochrome c was similar in the cells incubated in 5 or 20 mM glucose for 3 days, but was increased by more than fourfold when the incubation in 20 mM glucose medium was extended to 5 days. The release of cytochrome c was not further increased when the duration of incubation with 20 mM glucose was further extended to 10 days (Fig. 2A) . Despite the significant differences in the content of cytochrome c among various cytosol preparations, the content of ß-actin did not vary.



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  		FIGURE 2. Time course of (A) glucose-induced release of cytochrome c and (B) translocation of Bax in retinal endothelial cells. Bovine retinal endothelial cells were incubated in 5 or 20 mM glucose for up to 10 days. At the end of the desired time of incubation, the cells were trypsinized and mitochondrial and cytosolic fractions were isolated by differential centrifugation. The purity of mitochondrial and cytosolic fractions was determined by measuring Cox IV expression. Each measurement was performed in duplicate with three to four separate cell preparations. The results obtained in 5 mM glucose medium did not change with the duration of incubation and are considered as 100%. *P < 0.05 compared with 5 mM glucose.

 
As shown in Figure 2B , the expression of Bax in the mitochondria obtained from the same preparations was increased by 40% in the cells incubated with 20 mM glucose for 3 days compared with the cells incubated in 5 mM glucose, but was increased by fourfold when the incubation was extended to 5 days. There was no additional increase in Bax content in the mitochondria when the incubation with glucose was allowed to continue for 10 days. The expression of Cox IV in various lanes was identical.

Cell Death
Apoptosis levels, as determined by measuring the cytoplasmic nucleosomal DNA, were not changed in the endothelial cells incubated in 20 mM glucose for 3 days compared with the cells incubated in 5 mM, but were increased by 50% when the incubation with 20 mM glucose was increased to 5 days (Fig. 3) . Similarly, increased DNA laddering was observed in these cells at 5 days of incubation with 20 mM glucose (data not shown).



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  		FIGURE 3. Glucose-induced apoptosis of retinal endothelial cells. Apoptosis was measured by performing ELISA for cytoplasmic histone-associated DNA fragments. Data were obtained from cells incubated with glucose for 5 days, and the values were adjusted to the total DNA. No significant apoptosis was observed in these cells when the glucose exposure was 3 days or less. *P < 0.05 compared with 5 mM glucose; P < 0.05 compared with 20 mM glucose.

 
Inclusion of SOD or MnTBAP in the medium for 5 days inhibited glucose-induced release of cytochrome c from the mitochondria into the cytosol, and in the same mitochondrial fractions, inhibited increased Bax expression. As stated earlier, despite the significant differences in the content of cytochrome c and Bax among various preparations, the content of ß-actin and Cox IV did not vary in their respective fractions (Fig. 3) . Other antioxidants, including N-acetyl cysteine and lipoic acid had beneficial effects similar to those of SOD or its mimetic, MnTBAP (data not shown).

Similarly, the addition of SOD or MnTBAP inhibited glucose-induced increased apoptosis of endothelial cells, and this was confirmed by ELISA assays (Fig. 4) . Both SOD and MnTBAP also inhibited activation of caspase-3 induced by glucose.



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  		FIGURE 4. Effect of SOD on glucose-induced mitochondrial dysfunction. Retinal endothelial cells were incubated with 5 or 20 mM glucose for 5 days in the presence or absence of 20 mU/mL SOD or 200 µM MnTBAP. The cytochrome c and Bax contents were determined in the mitochondrial and cytosolic fractions by Western blot analysis. The experiments were repeated with at least three separate cell preparations, and each measurement was made in duplicate. Similar beneficial effects were observed when the cells were incubated in the presence of 250 µM N-acetyl cysteine or 250 µM {alpha}-lipoic acid.

 
Isolated Retinal Pericytes
Incubation of retinal pericytes with 20 mM glucose showed mitochondrial dysfunction similar to that observed with endothelial cells (Fig. 5) . Cytochrome c release into the cytosol and Bax translocation into the mitochondria were increased by three to fourfold in the cells incubated in 20 mM glucose for 5 days compared with that in 5 mM glucose. This was accompanied by a twofold increase in apoptosis. No significant translocation of cytochrome c or Bax and apoptosis was observed in these cells when the glucose exposure was of 3 days’ or less duration. Addition of MnTBAP inhibited glucose-induced mitochondrial dysfunction and apoptosis in these retinal pericytes (Fig. 5) .



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  		FIGURE 5. Glucose-induced mitochondrial dysfunction and apoptosis in retinal pericytes. Pericytes isolated from bovine retina were incubated in 5 or 20 mM glucose for up to 10 days in the presence or absence of SOD or MnTBAP. Top: release of cytochrome c into the cytosol and Bax into mitochondria after a 5-day incubation with 20 mM glucose. The results are representative of five separate experiments using three different preparations. Bottom: apoptosis measured by ELISA. Results are the mean ± SD obtained from three separate preparations, and each measurement was made in duplicate. *P < 0.05 compared with 5 mM glucose and #P < 0.05 compared with 20 mM glucose.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The results presented herein show for the first time that the retinal mitochondria become leaky and the cytochrome c starts to accumulate in the cytosolic fraction when the duration of diabetes in rats is such that capillary cell apoptosis can be detected in the retina. Further, both in retinal endothelial cells and pericytes, high glucose increases the release of cytochrome c into the cytosol and Bax into the mitochondria, which can be prevented by reducing superoxide levels, thus suggesting that retinal mitochondria experiences dysfunction in diabetes. Retina and its isolated capillary cells experience increased oxidative stress in high-glucose conditions, and increased oxidative stress is postulated to play an important role in the development of diabetic complications.1 2 3 High glucose can induce apoptosis and activate caspases, including caspase-3, in retinal capillary and Müller cells,4 15 16 17 39 and we provide data showing that glucose-induced apoptosis can be inhibited by lowering superoxides levels.

Oxidative stress is closely linked to apoptosis in a variety of cell types. It can alter both signal transduction and genomic processes.18 19 The mechanism by which oxidative stress can increase apoptosis may involve increased membrane lipid peroxidation, increased oxidative injury to other macromolecules, alterations in signal transduction, change in cellular redox potentials or depletion of glutathione (GSH).40 41 An altered gene profile of scavenging enzymes is reported in the retinal pericytes obtained from patients with diabetes, and this correlates with the overexpression of the cell death protease gene, suggesting an important role for oxidative stress in the pericyte dropout that occurs in diabetic retinopathy.10 Increased oxidative stress in diabetes is shown to play a critical role in advanced glycation end-product (AGE)–induced and palmitate-induced apoptosis of retinal capillary cells that can be inhibited by antioxidants.42 Our previous studies have shown that increased oxidative stress plays an important role in the activation of retinal caspase-3 and in the development of retinopathy in diabetes.3 4 The results of the present study show that the inhibition of glucose-induced release of cytochrome c into the cytosol and translocation of Bax into the mitochondria in retinal endothelial cells and pericytes by SOD is accompanied by the inhibition of their apoptosis.

Mitochondria play a key role in regulating apoptosis, reactive oxidant intermediates can trigger mitochondria to release cytochrome c and apoptosis-inducing factor, and increased lipid peroxidation itself can damage mitochondrial membrane potential and provoke apoptosis.21 22 Once cytochrome c is released from mitochondria, it activates caspase-9, which initiates a cascade of events that activates caspase-3 and results in DNA fragmentation. Cytochrome c can induce apoptosis if it is present in the cytoplasm in the oxidized state, and under normal conditions cytoplasmic GSH maintains cytochrome c in the reduced state.41 In diabetes, retinal GSH levels are decreased and glutathione redox cycle enzymes are impaired,5 6 7 which raises the possibility that the reduction of cytochrome c is also impaired, and here we provide data that clearly show that the release of cytochrome c into the cytosol is increased by approximately twofold. The increased release of cytochrome c is seen also when the endothelial cells and pericytes from the retina are incubated in high-glucose medium for 5 days, but not for 3 days. This time course of mitochondrial dysfunction is similar to the activation of caspase-3 that we have reported previously.6 Thus, the present study provided data to show that the inhibition of mitochondrial changes in retinal endothelial cells and pericytes are accompanied by inhibition of apoptosis in these cells.

Bax, a proapoptotic protein, enhances the release of cytochrome c by translocating to the mitochondria and by inducing a mitochondrial permeability transition.43 44 Others have reported that the expression of Bax is increased in the retina in diabetes and in retinal pericytes incubated in high-glucose medium, and this overexpression in retinal pericytes is associated with their apoptosis.16 In retinal sections, Bax immunostaining is shown to be present in ganglion and vascular cells, the cell types known to undergo accelerated cell death in diabetes.13 15 16 The results of the present study demonstrate that it is the mitochondrial fraction of the retina where Bax expression is increased in diabetes, and we have confirmed our in vivo results using isolated retinal endothelial cells and pericytes incubated in high-glucose medium for 5 days. This strengthens the possible involvement of mitochondria in the apoptosis of both pericytes and endothelial cells that occurs in diabetes.12 13 Romeo et al.39 have suggested a possible involvement of Bax in retinal capillary cell apoptosis in diabetes, but their study did not identify the effect of diabetes on subcellular distribution of Bax in the retina or its capillary cells. Our study is the first to provide data that demonstrate the possible involvement of mitochondrial dysfunction in the apoptosis of both retinal endothelial cells and pericytes in diabetes.

Release of cytochrome c is considered a key event in the activation of caspase-3, a downstream pivotal step in the initiation of apoptosis.29 Cells deficient in caspase-3 are resistant to apoptosis,45 and activation of caspase-3 alone is sufficient to cause cell death in cardiac muscle.46 The results presented herein demonstrate that caspase-3 activation in diabetes is associated with mitochondrial dysfunction.

Hyperglycemia-induced overproduction of superoxides by mitochondria is considered as a causal link between elevated glucose and the major biochemical pathways postulated to be involved in the development of vascular complications in diabetes,25 47 and overexpression of Mn-SOD is reported to suppress glucose-induced collagen accumulation in cultured mesangial cells.48 We have provided evidence that mitochondrial dysfunction, apoptosis, and caspase-3 activation induced by high glucose in both retinal endothelial cells and pericytes are inhibited by SOD and its mimetic, MnTBAP suggesting that a mitochondria-dependent pathway is operating in both the diabetic retina and its isolated capillary cells, and SOD production is causally involved in the hyperglycemia-induced apoptosis of retinal capillary cells.

Thus, our data strongly suggest that hyperglycemia-induced retinal capillary cell death most likely is initiated by the mitochondrial cytochrome c–mediated caspase-3 activation pathway. Understanding the signaling pathway(s) involved in the retinal capillary cell death will elucidate important molecular targets for future pharmacological interventions.


   Acknowledgements
 
The authors thank Prashant Koppolu for technical assistance.


   Footnotes
 
Supported in part by grants from the Juvenile Diabetes Research Foundation, The Thomas Foundation, and Research to Prevent Blindness.

Submitted for publication April 7, 2003; revised June 9, 2003; accepted June 19, 2003.

Disclosure: R.A. Kowluru, None; S.N. Abbas, 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
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 Discussion
 References
 

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