SEARCH

SEARCH BY CITATION

Keywords:

  • apoptosis;
  • diabetic retinopathy;
  • antioxidant;
  • advanced glycation end-product

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Background

Pericyte ghosts and acellular capillaries are well known as early histological changes resulting from diabetic retinopathy. These histological changes mean that the cell death of retinal microvessels has accelerated. It was reported that apoptosis of retinal microvascular cells (RMCs) was increased in diabetic patients. Therefore, we investigated apoptosis of RMCs in Goto-Kakizaki (GK) rats, a type 2 diabetic model, and involvement with antioxidants (a combination of vitamins C and E) or a novel inhibitor of advanced glycation, OPB-9195.

Methods

GK rats were treated with the antioxidants combination or OPB-9195 for 36 weeks. We obtained isolated preparations of the vascular network from their retinas by trypsin digestion. Apoptosis of retinal vascular cells was detected with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay.

Results

We found that apoptosis of RMCs was increased in the diabetic GK rats. Furthermore, a combination of vitamins C and E and an advanced glycation end-products inhibitor mostly inhibited this increased apoptosis.

Conclusions

We concluded that apoptosis of RMCs was a good marker that indicates the progression of diabetic retinopathy in GK rats. Both oxidative stress and the accumulation of advanced glycation end-products appears to promote the apoptosis of retinal microvascular cells, and antioxidants or advanced glycation end-products inhibitors might ameliorate diabetic retinopathy. Copyright © 2005 John Wiley & Sons, Ltd.

Diabetic retinopathy remains one of the main causes of vision loss and blindness in many countries. There have been recent advancements in treatments for preproliferative or proliferative diabetic retinopathy, laser photocoagulation and intra-ocular surgery, such as vitrectomy; however, the prognosis of difficult cases with maculopathy, rubeotic glaucoma, retinal detachment, and so on, is still poor. Therefore, it is important to prevent the progression of retinopathy at an early stage, that is, when it is background retinopathy. In background retinopathy, before dot hemorrhages and microaneurysms are seen with funduscopy, microscopic changes, such as thickening of basement membranes or pericyte ghosts, have already occurred in microvascular walls. Pericyte ghosts, which are the pockets in basement membranes marking the space from which pericytes have disappeared, are well-known features in the histology of background retinopathy 1, 2 and indicate the demise of pericytes. Recently, we reported that the death of RMCs, that is, pericytes and endothelial cells, by apoptosis was accelerated in diabetic patients and in two animal models that are known to develop the early stages of retinopathy (the alloxan-diabetic rat and the galactose-fed rat) 3. We concluded that the increased apoptosis in these cells indicated the progression of diabetic retinopathy. Furthermore, we speculated that the progression of early diabetic retinopathy could be quantitatively evaluated by determining the number of apoptotic cells in diabetic animal models.

Goto-Kakizaki (GK) rats are considered to be a spontaneous, non-obese model of type 2 diabetes mellitus with glucose intolerance due to impaired insulin secretion 4–6. Mild hyperglycemia has been demonstrated already in 8-day-old GK rats 5, 7. Regarding diabetic complications, thickening of the glomerular capillary basement membrane 8, slowing of the conduction velocity of impulse in the tail motor nerve 9, and increased endothelial/pericyte ratio 10 were confirmed. However, there has been no study of apoptosis of RMCs.

Oxidative stress, accumulation of advanced glycation end-products (AGE), elevation of protein kinase C (PKC) activity, activation of a polyol pathway 11, and so on, were previously reported as pathogenesis of diabetic retinopathy, but the detailed mechanisms are unknown and prevention strategies have not been established except improving blood glucose levels and blood pressure. We were interested in oxidative stress and AGE, in particular, and speculated that an antioxidant or an inhibitor of advanced glycation might reduce apoptosis of RMCs. There is no report about the effect of antioxidant on apoptosis of RMCs in diabetic animal models.

Currently, many types of antioxidants are known and available. Vitamins C (ascorbic acid) and E (alpha-tocopherol) are well-known dietary antioxidants. Vitamin E is lipophilic and inhibits lipid peroxidation, scavenging lipid peroxyl radicals to yield lipid hydroperoxides and the tocopheroxyl radical 12. Vitamin C, a water-soluble vitamin, functions cooperatively with vitamin E by regenerating tocopherol from tocopheroxl radical. We used a combination of ascorbic acid and tocopherol as antioxidants.

OPB-9195, (±)-2-Isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide, is a novel inhibitor of advanced glycation that was developed by Otsuka Pharmaceutical Co., Japan. In diabetic rats, this drug inhibits the formation of AGE, but has no effect in lowering blood glucose, which prevents the development of diabetic nephropathy 13, 14. Recently, it was reported that OPB-9195 inhibited formation of both AGE and advanced lipoxidation end-products (ALE), such as malondialdehyde (MDA)-lysine and 4-hydroxynonenal (HNE)-protein adduct, through its ability to trap reactive carbonyl compounds (RCO) 15, 16.

We studied the effects of antioxidants (using a combination of ascorbic acid and tocopherol) or a novel inhibitor of advanced glycation, OPB-9195, on apoptosis of RMCs in diabetic GK rats.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

Fifteen 8-week-old male GK rats and five male Wistar rats (the control) were obtained from Charles River Japan, Inc., Kanagawa, Japan. The rats were maintained in an environmentally controlled room with a 12-h light/dark cycle and had free access to chow (CE-2; Clea Japan, Inc., Tokyo, Japan) and drinking water. When GK rats were 14 weeks old, they were randomly separated into three groups. In the first group (Vit. C + E), rats had drinking water with 0.1% ascorbic acid (Sigma Chemical Co., St. Louis, MO) and chow with 0.1% tocopherol acetate (Eisai Co., Ltd., Tokyo, Japan). In the second group (AGE-I), rats had chow with 0.1% OPB-9195 (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). In the third group (Diabetic), rats had normal water and chow. Treatment of animals conformed to the Association for Research in Vision and Ophthalmology resolution on the treatment of research animals.

Measurement

Blood samples were collected from tail veins. Fasting plasma glucose levels (FPG) were measured by a glucose oxidase method with heparinized blood after 20 h of fasting. Glycohemoglobin (HbA1c) levels were measured by latex photometric immunoassay (Unimate HBA1c, Roche Diagnostics K.K., Tokyo, Japan). Ascorbic acid (high performance liquid chromatography, HPLC), alpha-tocopherol (HPLC), total cholesterol, and triglyceride (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were measured with blood samples collected when the rats were sacrificed at 50 weeks of age.

Trypsin digestion of retinas

At 50 weeks of age, all rats were killed with a lethal dose of sodium pentobarbital. Their eyes were removed and immediately placed in 4% buffered paraformaldehyde. In each rat, one eye was used for apoptosis study and microvascular pathology, and the other was for immunohistochemical study. Trypsin digestion of the retinas was performed according to the method of Cogan et al.1 with some modifications 17. Briefly, retinas were removed from eyeballs in distilled water and placed in phosphate-buffered saline (PBS) overnight for rehydration. Then, the retinas were incubated at 37 °C for 120–150 min in trypsin solution (3% trypsin, 0.1M trishydroxymethylaminomethane (Tris), 0.2 M sodium fluoride (Sigma Chemical Co.), pH 7.8). After vitreous was removed, neural retina was swept away with rat whiskers. The vascular network was cleared by tapping tissue, except vascular tissue, away gently. Finally, preparations of retinal vascular networks were set onto Silane-coated slides (Muto Pure Chemicals Co., Ltd., Japan) in distilled water and dried. The preparations were stored at −20 °C until use.

Determination of apoptosis

Apoptosis of cells in retinal trypsin digests was detected with the TUNEL method 18 using the in situ Cell Death Detection Kit by Roche Diagnostics K.K., Tokyo, Japan. The kit is based on the principle that terminal deoxynucleotidyl transferase catalyzes a template-independent addition of deoxynucleotides to free 3-OH ends present in DNA breaks. This tailing reaction is especially sensitive to the type of DNA fragmentation occurring in apoptotic, rather than necrotic, cell death 19. The preparations of retinal vascular networks were rehydrated in PBS and permeabilized with 0.5% Triton X-100 in PBS for 1 h at room temperature (RT). Then, they were incubated with TUNEL reagents for 1 h at 37 °C under parafilm protection and mounted in Slow-Fade (Molecular Probes, Inc., Eugene, OR). The negative control received only the label solution without the terminal transferase. Before the TUNEL reaction, the positive control was exposed to DNase I (Takara Shuzo Co., Ltd., Shiga, Japan; 500 U/mL in 40 mM tris-HCl, 6 mM MgCl2 buffer, pH 7.5) for 10 min at RT.

Identification of TUNEL-positive cells

Each preparation of retinal vascular network was surveyed systematically under a BX60 (Olympus Optical Co., Ltd., Tokyo, Japan) fluorescence microscope by scanning it with downward and upward motion beginning at the upper left margin. Specificity of any fluorescent signal was determined by switching to the rhodamine excitation wavelength. Pictures of TUNEL-positive cells were saved in a Macintosh computer through a cooled charge-coupled device (CCD) camera (CoolSNAP; Nippon Roper, Chiba, Japan). Recording the coordinates on the microscope platform identified their location in the preparation. After observing the TUNEL-positive cells, we stained the preparation with periodic acid-Schiff (PAS) and hematoxylin, and the morphological counterparts of TUNEL-positive images were identified and saved as pictures in the computer. These steps permitted the attribution of the majority of TUNEL-positive images in the preparations to pericyte or endothelial cell nuclei; for rare TUNEL-positive chromatin, generally of small size and/or with uninformative shape or topography, the cellular attribution remained undetermined. The results are thus reported as TUNEL-positive pericytes, endothelial cells, or undetermined. Positive images found at intersections of vessels and not confidently attributable to a given cell were not counted to prevent inclusion of artifacts. The preparations stained with PAS hematoxylin were also examined for the characteristic histological lesions of retinopathy (microaneurysms, pericyte ghosts, and acellular capillaries). The frequency of pericyte ghosts and acellular capillaries was determined in a masked fashion, as reported previously 20. In other words, an investigator counted the preparations with no knowledge of the identity of the samples. Saccular microaneurysms were not observed in the rat specimens.

Immunohistochemistry

The remaining eyes that were not used for trypsin digestion were embedded in paraffin. Sections of size 3 µm were used for immunohistochemistry with the DAKO LSAB2 kit, Peroxidase for use on RAT Specimens (DAKO A/S, Denmark). Briefly, the sections were deparaffinized and hydrated through xylenes and graded alcohol series. After being rinsed in tap water, they were incubated in DAKO Peroxidase Blocking Reagent (DAKO) for 10 min at RT. After being washed in PBS, the sections were incubated in DAKO protein block serum-free reagents (DAKO) for 30 min at RT. They were then incubated in a 5 µg/mL mouse anti-AGE monoclonal antibody 21–23 (Kumamoto Immunochemical Laboratory Co., Ltd., Kumamoto, Japan) overnight at 4 °C and, after being washed, they were incubated in the biotinylated secondary antibody for 30 min at RT. After another washing, sections were incubated in the streptavidin-peroxidase reagent for 30 min at RT, then washed again and incubated in the diaminobenzidine (DAB) substrate solution for 5 min. They were counterstained lightly with hematoxylin. In negative control sections, 5 µg/mL mouse IgG1 for negative control (DAKO) was used as the primary antibody.

Statistical analysis

The data are summarized with the mean ± SE. Statistical analysis was performed with ANOVA, followed by Scheffe's F-test to isolate differences between groups. P-values less than 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Measurements

One rat in the Vit. C + E group died at 46 weeks of age accidentally. One rat in the Diabetic group was excluded from the following analysis of data because its HbA1c was much lower than that of the other GK rats. With regard to data collected, the Control, Diabetic, Vit. C + E, and AGE-I groups ultimately included 5, 4, 4, and 5 rats respectively.

Table 1 shows the results of body weight (BW) measurement and blood analysis. At 13 weeks of age, the BW in the Diabetic, the Vit. C + E, and the AGE-I group was significantly lighter than in the Control group. Levels of FPG and HbA1c in the Diabetic, the Vit. C + E, and the AGE-I group were significantly higher than in the Control group. In BW and levels of FPG and HbA1c, there was no significant difference among the Diabetic, the Vit. C + E, and the AGE-I groups. At 50 weeks of age, the BW in the Diabetic, the Vit. C + E, and the AGE-I groups was significantly heavier than in the Control group. Levels of FPG and HbA1c in the Diabetic, the Vit. C + E, and the AGE-I groups were significantly higher than in the Control group. In BW and levels of FPG and HbA1c, there was no significant difference among the Diabetic, the Vit. C + E, and the AGE-I groups. Levels of plasma ascorbic acid and alpha-tocopherol in the Vit. C + E group were significantly higher than in the Diabetic group. Levels of T-cho in the Diabetic and the AGE-I group were significantly higher than in the Control group. In levels of TG, there was no significant difference among the groups.

Table 1. Results of body weight measurement and blood analysis
 ControlDiabeticVit. C + EAGE-I
  • *

    Shows there is a significant difference from the control group (p < 0.05).

  • **

    Shows there is a significant difference from the diabetic group (p < 0.05).

13 weeks of age 
BW (g)456 ± 5344 ± 8*341 ± 6*353 ± 13*
FPG (mmol/L)4.9 ± 0.110.2 ± 0.7*10.2 ± 0.5*9.5 ± 0.6*
HbA1c (%)3.65 ± 0.075.41 ± 0.04*5.11 ± 0.43*5.01 ± 0.29*
50 weeks of age
BW (g)691 ± 15385 ± 17*406 ± 7*438 ± 7*
FPG (mmol/L)5.1 ± 0.310.0 ± 0.8*8.9 ± 0.7*10.3 ± 0.3*
HbA1c (%)3.53 ± 0.117.14 ± 0.25*6.88 ± 0.15*7.12 ± 0.13*
Ascorbic acid (mg/dL)0.25 ± 0.290.85 ± 0.87**  
Alpha-tocopherol (µg/mL)5.98 ± 0.5918.23 ± 1.01**  
T-cho (mg/dL)73 ± 12133 ± 19*119 ± 5132 ± 6*
TG (mg/dL)95 ± 1666 ± 587 ± 872 ± 6

TUNEL

Figure 1a shows a TUNEL-positive retinal endothelial cell, and Figure 1b shows the same cell stained by PAS-hematoxylin. Figure 1c shows a TUNEL-positive retinal pericyte, and Figure 1d shows the same cell stained by PAS.

thumbnail image

Figure 1. Apoptosis of retinal microvascular cells. (a) Shows a TUNEL-positive retinal endothelial cell (original magnification, ×400). (b) Shows the same cell with PAS staining (magnification, ×400). (c) Shows a TUNEL-positive retinal pericyte (original magnification, ×400). (d) Shows the same cell with PAS staining (magnification, ×400)

Download figure to PowerPoint

TUNEL-positive cells

The number of TUNEL-positive retinal capillary cells (both endothelial cells and pericytes) in the Diabetic group (8.8 ± 1.8) was significantly larger than in the Control group (0.6 ± 0.4) (Figure 2). The number in the Vit. C + E (2.3 ± 0.6) and in the AGE-I (1.8 ± 0.7) groups was significantly smaller than in the Diabetic group.

thumbnail image

Figure 2. Number of TUNEL-positive retinal capillary cells. The number of TUNEL-positive retinal capillary cells in the Diabetic group was significantly larger than in the control group. The number in the Vit. C + E and in the AGE-I groups was significantly smaller than in the Diabetic group. Error bars show standard errors

Download figure to PowerPoint

Figure 3 shows the number of TUNEL-positive endothelial cells and pericytes. The number of TUNEL-positive endothelial cells in the Control, the Diabetic, the Vit. C + E, and the AGE-I group was 0.4 ± 0.2, 4.5 ± 1.7, 1.0 ± 0.7, and 1.4 ± 0.7 respectively. The number of TUNEL-positive endothelial cells in the Diabetic group was significantly larger than in the Control group (p = 0.0481). The number of TUNEL-positive pericytes in the Diabetic, the Vit. C + E, and the AGE-I group was 3.5 ± 0.6, 1.0 ± 0.4, or 0.4 ± 0.4 respectively. No TUNEL-positive pericytes were found in the Control group. The number of TUNEL-positive pericytes in the Diabetic group was significantly larger than in the Control group (p = 0.0003). The number of TUNEL-positive pericytes in the Vit. C + E (p = 0.009) and in the AGE-I group (p = 0.001) was significantly smaller than in the Diabetic group. The number of TUNEL-positive retinal capillary cells that were not determined to be endothelial cells or pericytes in the Control, the Diabetic, and the Vit. C + E groups was 0.2 ± 0.2, 0.8 ± 0.3, and 0.3 ± 0.3 respectively. No TUNEL-positive undetermined retinal capillary cells were found in the AGE-I group.

thumbnail image

Figure 3. Number of TUNEL-positive endothelial cells and pericytes. The number of TUNEL-positive endothelial cells in the diabetic group was significantly larger than in the control group. The number of TUNEL-positive pericytes in the diabetic group was significantly larger than in the Control group. The number of TUNEL-positive pericytes in the Vit. C + E and in the AGE-I groups was significantly smaller than in the diabetic group. Error bars show standard errors

Download figure to PowerPoint

Microvascular pathology

Table 2 shows retinal microvascular pathology. The number of acellular capillaries per 1 mm2 retina in the Control, the Diabetic, the Vit. C + E, and the AGE-I group was 2.7 ± 0.3, 6.4 ± 0.5, 3.3 ± 0.4, and 2.6 ± 0.3 respectively. There were significantly more acellular capillaries in the Diabetic group than in the Control group, whereas those in the Vit. C + E (p < 0.0001) and the AGE-I group (p < 0.0001) were significantly less than in the Diabetic group. The number of pericyte ghosts per 1000 microvascular cells in the Diabetic and the Vit. C + E group was 0.2 ± 0.1 and 0.1 ± 0.1 respectively. No pericyte ghost was found in the Control or the AGE-I group.

Table 2. Retinal microvascular pathology
 Acellular capillaries (/mm2 retina)Pericyte ghosts (/1000 capillary cells)
  • *

    Shows there is a significant difference from the control group (p < 0.05).

  • **

    Shows there is a significant difference from the diabetic group (p < 0.05).

Control2.7 ± 0.3Not found
Diabetic6.4 ± 0.5*0.2 ± 0.1
Vit. C + E3.3 ± 0.4**0.1 ± 0.1
AGE-I2.6 ± 0.3**Not found

Immunohistochemistry

Figures 4a,b,c, and d represent the retinas in the Control, the Diabetic, the Vit. C + E, and the AGE-I group respectively. AGE-like immunoreactivity (brown) in the inner segment of the photoreceptor layer (PRL) was not found in the Control group. It was found more in the Diabetic group and less in the Vit. C + E and in the AGE-I group than in the Diabetic group. The intensity of AGE-like immunoreactivity was almost equal among all groups in the inner limiting membrane (strong; not shown in Figure 4a, d), the ganglion cell layer (GCL, moderate), the inner plexiform layer (IPL, mild), and the outer plexiform layer (OPL, moderate). DAB staining was not found in the negative control section.

thumbnail image

Figure 4. Immunohistochemical staining of AGE in retinas. Figures (a), (b), (c), and (d) represent the retinas in the Control, the Diabetic, the Vit. C + E, and the AGE-I groups respectively. AGE-like immunoreactivity (brown) in the inner segment of the photoreceptor layer (PRL) was not found in the Control group. It was found much in the Diabetic group and less in the Vit. C + E and in the AGE-I group than in the Diabetic group (original magnification, ×400)

Download figure to PowerPoint

Figures 5a,b,c, and d represent retinal blood vessels of the GCL in the Control, the Diabetic, the Vit. C + E, and the AGE-I groups respectively. AGE-like immunoreactivity of retinal vascular walls in the Diabetic group was found more than that in the Control, whereas that in the Vit. C + E and the AGE-I groups was less than that in the Diabetic group (original magnification, ×1000).

thumbnail image

Figure 5. Immunohistochemical staining of AGE in retinal vascular walls. Figures (a), (b), (c), and (d) represent retinal blood vessels of the GCL in the Control, the Diabetic, the Vit. C + E, and the AGE-I groups respectively. AGE-like immunoreactivity of retinal vascular walls in the Diabetic group was found more than that in the Control, whereas that in the Vit. C + E and the AGE-I groups was less than that in the Diabetic group (original magnification, ×1000)

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

One of the novel findings of our study is that apoptosis of RMCs actually occurred in moderately diabetic GK rats. Apoptosis of RMCs has only been confirmed in diabetic patients, alloxan-diabetic rats, and galactose-fed rats 3, 24. It is interesting that apoptosis of RMCs was found in 1-year-old GK rats with much milder hyperglycemia than streptozotocin-induced or alloxan-induced diabetic rats. This fact disproves the assertion that only severe hyperglycemia or hypergalactosemia might lead to apoptosis of RMCs and shows that it was not caused by streptozotocin or alloxan.

Our data shows that the evaluation of apoptosis of RMCs is a good marker of diabetic retinopathy in animal models because the amount of apoptosis, as well as the number of acellular capillaries, increased in the diabetic rats more than in the control, and the difference in the amount of apoptosis between the diabetic GK rats and the controls was large. A cellular capillaries and pericyte ghosts are well known as early histological changes of diabetic retinopathy, and these changes mean that the nuclei of RMCs have disappeared from capillaries. An acellular capillary is a capillary from which all endothelial cells and all pericytes have disappeared, and a pericyte ghost is an empty pocket in the basement membrane from which a pericyte nucleus has disappeared. Because cells in which apoptosis has started will soon die and disappear, we presume that these two changes appear as a result of apoptosis of RMCs. The evaluation of apoptosis shows how many RMCs are dying now, and the evaluation of the two morphological changes show how many RMCs have died so far. Because the evaluation of apoptosis predicts prospective histological changes, it is useful as well as the evaluation of morphological changes.

Because it has been known that TUNEL labeling is not an apoptosis-exclusive assay, our data cannot suggest that apoptosis is the only way in which vascular cells die during diabetes. However, it suggested that it was a useful evaluation method of diabetic retinopathy to count TUNEL-positive cells, which are dying mainly through apoptosis presumably.

Another of the novel findings in our study is that antioxidants (Vitamin C and E) and an AGE inhibitor (OPB-9195) ameliorated the increase of apoptosis of RMCs and acellular capillaries in diabetic GK rats. Vitamin C is water soluble and one of the most powerful natural antioxidants 12. It scavenges reactive oxygen species in aqueous phase (plasma, cytoplasm, and so on). Moreover, there is evidence from in vitro studies that it is capable of regenerating tocopherol from the tocopheroxyl radical that is formed upon the inhibition of lipid peroxidation by vitamin E. Vitamin E is lipophilic, operating in membranes or lipoprotein particles. It scavenges lipid peroxyl radicals and inhibits lipid peroxidation. It was reported that, in the lipid phase, it might be the most efficient of lipophilic antioxidants 25. Therefore, a combination of vitamins C and E is thought to work as a powerful antioxidant in aqueous and lipid phases.

Our data demonstrated that the combination of vitamins C and E reduced apoptosis of RMCs and acellular capillaries in diabetic rats and suggested that the combination of vitamins C and E might inhibit the progression of early retinopathy in diabetic patients.

Brownlee M et al. reported 26 that high concentrations of glucose increased the production of reactive oxygen species (ROS) in endothelial cells, and inhibition of mitochondrial ROS production prevented high glucose-induced activation of PKC, formation of AGE, sorbitol accumulation, and NFκB activation. This means oxidative stress though mitochondrial ROS production should be the most important cause of diabetic endothelial dysfunction and inhibition of mitochondrial ROS production or elimination of intracellular excess ROS will be a effective and reasonable therapeutic approach to prevent diabetic complications.

A couple of recent articles studied whether a combination of vitamins C and E can improve diabetic retinopathy in diabetic rats. One of them 27 reported that a combination of vitamins C (10 g/kg diet) and E (1 g/kg diet) inhibited 50% of acellular capillaries significantly and tended to inhibit pericyte ghosts in alloxan-induced diabetic Sprague-Dawley rats. Moreover, multiple-antioxidant diet including vitamins C and E improved both histological changes significantly. The other article 28 reported a combination of vitamins C (1 g/L drinking water) and E (10 IU/kg BW) inhibited retinal superoxide production significantly and tended to inhibit pericyte ghosts or acellular capillaries (but the differences were not significant) in streptozotocin-induced diabetic Lewis rats. Interestingly, green tea with antioxidant property inhibited 23% of acellular capillaries significantly.

It is difficult to compare our data with theirs because of the levels of hyperglycemia, amount of administered vitamins C and E, and strains of rats. Hyperglycemia was milder in our GK rats than in diabetic rats used in the above studies. Fortunately, because glucose levels of GK rats were adequate for a treatment of vitamins C and E to work well, the difference between the Diabetic and the Vit. C + E groups might have become significant even if the number of experimental animals was low.

It was previously reported that aminoguanidine ameliorated the increase of apoptosis of RMCs and acellular capillaries in alloxan-induced diabetic rats 24. We showed that OPB-9195 ameliorated it in diabetic GK rats. These results demonstrate that advanced glycation is also cross related to the pathogenesis of diabetic retinopathy and suggest that AGE inhibitors might inhibit the progression of early retinopathy in diabetic patients. However, the antioxidative effects of aminoguanidine 29–32 and OPB-9195 15, 16 have recently been reported. Therefore, these AGE inhibitors may improve diabetic retinopathy through not only the inhibition of advanced glycation but also through an antioxidative effect.

Immunohistochemical analysis of AGE demonstrated that the antioxidants combination and OPB-9195 reduced AGE-like immunoreactivity, which increased in retinal vascular walls and the inner segment of the photoreceptor layer in a subject with diabetes. The epitope of anti AGE antibody we used was N-(carboxymethyl) lysine (CML). Oxidative stress is known to be related to the formation of CML, and it has been reported that CML is a biomarker of not only advanced glycation but also oxidative stress 33–38. We speculate that the antioxidants combination might reduce the accumulation of AGE through the inhibition of oxidative stress.

In conclusion, we showed that retinal pathogenic changes were suppressed by antioxidants and AGE inhibitors in diabetic GK rats and the combination of Vitamin C + E, and OPB-9195 had beneficial effects.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr Giulio Romeo and Dr Mara Lorenzi of Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, and Sayon Roy of the Department of Ophthalmology, School of Medicine, Boston University, for information about the trypsin digest method of rat retina.

This study was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) and by a Health Sciences Research Grant (Research on Human Genome and Gene Therapy) from the Ministry of Health and Welfare. This study was also supported by a grant from the Japanese Ministry of Education, Science, Sports and Culture and by a grant for Project Research from the University of Tsukuba.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Cogan DG, Troussaint D, Kuwabara T. Retinal vascular patterns IV. Diabetic retinopathy. Arch Ophthalmol 1961; 66: 366378.
  • 2
    Speiser P, Gittelsohn A, Patz A. Studies on diabetic retinopathy III. Influence of diabetes on intramural pericytes. Arch Ophthalmol 1968; 80: 322337.
  • 3
    Mizutani M, Kern T, Lorenzi M. Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy. J Clin Invest 1996; 97(12): 28832890.
  • 4
    Kimura K, Toyota T, Kakizaki M, et al. Impaired insulin secretion in the spontaneous diabetes rats. Tohoku J Exp Med 1982; 137(4): 453459.
  • 5
    Abdel Halim SM, Guenifi A, Luthman H, et al. Impact of diabetic inheritance on glucose tolerance and insulin secretion in spontaneously diabetic GK-Wistar rats. Diabetes 1994; 43(2): 281288.
  • 6
    Ostenson CG, Khan A, Abdel Halim SM, et al. Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat. Diabetologia 1993; 36(1): 38.
  • 7
    Abdel Halim SM, Ostenson CG, Andersson A, et al. A defective stimulus-secretion coupling rather than glucotoxicity mediates the impaired insulin secretion in the mildly diabetic F1 hybrids of GK-Wistar rats. Diabetes 1995; 44(11): 12801284.
  • 8
    Yagihashi S, Goto Y, Kakizaki M, et al. Thickening of glomerular basement membrane in spontaneously diabetic rats. Diabetologia 1978; 15(4): 309312.
  • 9
    Goto Y, Kakizaki M, Yagihashi S. Neurological findings in spontaneously diabetic rats. Excerpta Med Int Cong Ser 1982; 581: 2638.
  • 10
    Agardh CD, Agardh E, Zhang H, et al. Altered endothelial/pericyte ratio in Goto-Kakizaki rat retina. J Diabetes Complications 1997; 11(3): 158162. DOI: 10.1016/S1056-8727(96)00049-9.
  • 11
    Feener EP, King GL. Vascular dysfunction in diabetes mellitus. Lancet 1997; 350(Suppl. 1): SI9S13.
  • 12
    Stahl Wa, Sies H. Antioxidant defense: vitamins E and C and carotenoids. Diabetes 1997; 46(Suppl. 2): S14S18.
  • 13
    Nakamura S, Makita Z, Ishikawa S, et al. Progression of nephropathy in spontaneous diabetic rats is prevented by OPB-9195, a novel inhibitor of advanced glycation. Diabetes 1997; 46(5): 895899.
  • 14
    Tsuchida K, Makita Z, Yamagishi S, et al. Suppression of transforming growth factor beta and vascular endothelial growth factor in diabetic nephropathy in rats by a novel advanced glycation end product inhibitor, OPB-9195. Diabetologia 1999; 42(5): 579588. DOI: 10.1007/s001250051198.
  • 15
    Miyata T, Ueda Y, Asah K, et al. Mechanism of the inhibitory effect of OPB-9195 [(+/−)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide] on advanced glycation end product and advanced lipoxidation end product formation. J Am Soc Nephrol 2000; 11(9): 17191725.
  • 16
    Miyata T, Kurokawa K, De-Strihou CV. Advanced glycation and lipoxidation end products: role of reactive carbonyl compounds generated during carbohydrate and lipid metabolism. J Am Soc Nephrol 2000; 11(9): 17441752.
  • 17
    Boeri D, Cagliero E, Podesta F, et al. Vascular wall von Willebrand factor in human diabetic retinopathy. Invest Ophthalmol Vis Sci 1994; 35(2): 600607.
  • 18
    Gavrieli Y, Sherman Y, Ben Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119(3): 493501.
  • 19
    Gold R, Schmied M, Giegerich G, et al. Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab Invest 1994; 71(2): 219225.
  • 20
    Kern TS, Engerman RL. Comparison of retinal lesions in alloxan-diabetic rats and galactose-fed rats. Curr Eye Res 1994; 13(12): 863867.
  • 21
    Horiuchi S, Araki N, Morino Y. Immunochemical approach to characterize advanced glycation end products of the Maillard reaction. Evidence for the presence of a common structure. J Biol Chem 1991; 266(12): 73297332.
  • 22
    Araki N, Ueno N, Chakrabarti B, et al. Immunochemical evidence for the presence of advanced glycation end products in human lens proteins and its positive correlation with aging. J Biol Chem 1992; 267(15): 10 21110 214.
  • 23
    Miyata T, Oda O, Inagi R, et al. Beta 2-microglobulin modified with advanced glycation end products is a major component of hemodialysis-associated amyloidosis. J Clin Invest 1993; 92(3): 12431252.
  • 24
    Kern TS, Tang J, Mizutani M, et al. Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia. Invest Ophthalmol Vis Sci 2000; 41(12): 39723978.
  • 25
    Ingold K, Webb A, Witter D, et al. Vitamin E remains the major lipid-soluble, chain-breaking antioxidant in human plasma even in individuals suffering severe vitamin E deficiency. Arch Biochem Biophys 1987; 259: 224225.
  • 26
    Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000; 404: 787790. DOI: 1038/35008121.
  • 27
    Kowluru RA, Tang J, Kern TS. Abnormalities of retinal metabolism in diabetes and experimental galactosemia. VII. Effect of long-term administration of antioxidants on the development of retinopathy. Diabetes 2001; 50: 19381942.
  • 28
    Mustata GT, Rosca M, Monnier VM. Paradoxical effects of green tea (Camellia Sinensis) and antioxidant vitamins in diabetic rats. Improved retinopathy and renal mitochondrial defects but deterioration of collagen matrix glycoxidation and cross-linking. Diabetes 2005; 54: 517526.
  • 29
    Courderot Masuyer C, Dalloz F, Maupoil V. et al. Antioxidant properties of aminoguanidine. Fundam Clin Pharmacol 1999; 13(5): 535540.
  • 30
    Ihm SH, Yoo HJ, Park SW, et al. Effect of aminoguanidine on lipid peroxidation in streptozotocin-induced diabetic rats. Metabolism 1999; 48(9): 11411145.
  • 31
    Kedziora Kornatowska K, Luciak M. Effect of aminoguanidine on lipid peroxidation and activities of antioxidant enzymes in the diabetic kidney. Biochem Mol Biol Int 1998; 46(3): 577583.
  • 32
    Giardino I, Fard AK, Hatchell DL, et al. Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation, and oxidant-induced apoptosis. Diabetes 1998; 47(7): 11141120.
  • 33
    Ikeda K, Higashi T, Sano H, et al. N (epsilon)-(carboxymethyl)lysine protein adduct is a major immunological epitope in proteins modified with advanced glycation end products of the Maillard reaction. Biochemistry 1996; 35(24): 80758083. DOI: 10.1021/bi9530550.
  • 34
    Nakayama M, Izumi G, Nemoto Y, et al. Suppression of N(epsilon)-(carboxymethyl)lysine generation by the antioxidant N-acetylcysteine. Perit Dial Int 1999; 19(3): 207210.
  • 35
    Nerlich AG, Schleicher ED. N(epsilon)-(carboxymethyl)lysine in atherosclerotic vascular lesions as a marker for local oxidative stress. Atherosclerosis 1999; 144(1): 4147. DOI: 10.1016/S0021-9150(99)00038-6.
  • 36
    Horie K, Miyata T, Maeda K, et al. Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in diabetic renal glomerular lesions. Implication for glycoxidative stress in the pathogenesis of diabetic nephropathy. J Clin Invest 1997; 100(12): 29953004.
  • 37
    Elgawish A, Glomb M, Friedlander M, et al. Involvement of hydrogen peroxide in collagen cross-linking by high glucose in vitro and in vivo. J Biol Chem 1996; 271(22): 12 96412 971.
  • 38
    Fu MX, Requena JR, Jenkins AJ, et al. The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem 1996; 271(17): 99829986.