Pharmacological inhibition of diabetic retinopathy: aminoguanidine and aspirin.

Effects of aminoguanidine and aspirin on the development of retinopathy have been examined in 5-year studies of diabetic dogs. Either agent was administered daily in doses of 20-25 mg. kg(-1). day(-1). Because severity of hyperglycemia greatly influences development of the retinopathy, special effort was devoted to maintaining comparable glycemia in experimental and control groups. The retinal vasculature was isolated by the trypsin digest method, and retinopathy was assessed by light microscopy. Diabetes for 5 years resulted, as expected, in saccular capillary aneurysms, pericyte ghosts, acellular capillaries, retinal hemorrhages, and other lesions. Administration of aminoguanidine essentially prevented the retinopathy, significantly inhibiting the development of retinal microaneurysms, acellular capillaries, and pericyte ghosts compared with diabetic controls. Aspirin significantly inhibited the development of retinal hemorrhages and acellular capillaries over the 5 years of study, but had less effect on other lesions. Although diabetes resulted in significantly increased levels of advanced glycation end products (AGEs) (namely, pentosidine in tail collagen and aorta, and Hb-AGE), aminoguanidine had no significant influence on these parameters of glycation. Nitration of a retinal protein was significantly increased in diabetes and inhibited by aminoguanidine. The biochemical mechanism by which aminoguanidine has inhibited retinopathy thus is not clear. Aminoguanidine (but not aspirin) inhibited a diabetes-induced defect in ulnar nerve conduction velocity, but neither agent was found to influence kidney structure or albumen excretion.

Retinal glutamate in diabetes and effect of antioxidants.

Diabetes results in various biochemical abnormalities in the retina, but which of these abnormalities are critical in the development of retinopathy is not known. The aim of this study is to examine the effect of antioxidant supplementation on diabetes-induced alterations of retinal glutamate, and to explore the inter-relationship between alterations of retinal glutamate, oxidative stress, and nitric oxide (NO) in diabetes. Glutamate was measured in the retina at 2 months of diabetes in rats receiving diets supplemented with or without a mixture of antioxidants containing ascorbic acid, Trolox, DL alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene and selenium. The relationship between glutamate, oxidative stress and NO was evaluated using both bovine retinal endothelial cells and normal rat retina. In diabetes, retinal glutamate was elevated by 40, thiobarbituric acid-reactive substances (TBARS) by 100, and NO by 70%, respectively. Administration of antioxidants inhibited the diabetes-induced increases in glutamate, TBARS and NO. Incubation of bovine retinal endothelial cells or normal rat retina with glutamate significantly increased TBARS and NO, and addition of either antioxidant (N-acetyl cysteine) or a NO synthase inhibitor prevented the glutamate-induced elevation in oxidative stress and NO. Incubation of retina with a glutamate agonist, likewise elevated oxidative stress and NO, and memantine inhibited such elevations. Thus, the alterations of retinal glutamate, oxidative stress and NO appear to be inter-related in diabetes, and antioxidant therapy may be a suitable approach to determine the roles of these abnormalities in the development of diabetic retinopathy.

Abnormalities of retinal metabolism in diabetes or experimental galactosemia VIII. Prevention by aminoguanidine.

PURPOSE: Aminoguanidine has been found to inhibit the development of some retinal lesions in diabetic rats and diabetic dogs, thereby raising a possibility that the formation of glycation end products (AGEs) may be an essential step in the pathogenesis of the retinopathy. The purpose of this study is to investigate the effect of aminoguanidine administration on other metabolic abnormalities which might be involved in the development of retinopathy in two models of the retinopathy, alloxan diabetes and experimental galactosemia. METHODS: Oxidative stress, nitric oxide (NO) and the activity of protein kinase C (PKC, total activity) were measured in the retina of the rats experimentally diabetic or galactosemic for 2 months. Effect of aminoguanidine administration on the inhibition of hyperglycemia-induced retinal dysmetabolism was investigated. RESULTS: Two months of diabetes or experimental galactosemia in rats resulted in elevation of retinal oxidative stress (increase in thiobarbituric acid reactive substances, TBARS, and decrease in glutathione, GSH), NO, and PKC activity. Aminoguanidine supplementation (2.5 g aminoguanidine/kg rat diet) significantly inhibited each of these abnormalities in retinas of diabetic rats and galactosemic rats, and did so without lowering the blood hexose levels of these animals. CONCLUSIONS: The ability of aminoguanidine to normalize the hyperglycemia-induced increases in retinal oxidative stress, NO and PKC in diabetic rats and galactose-fed rats suggests that these abnormalities may be inter-related in the retina, and that the biochemical mechanism by which aminoguanidine inhibits retinal microvascular disease in diabetes may be complex.

Diabetes-induced metabolic abnormalities in myocardium: effect of antioxidant therapy.

Effects of hyperglycemia (both diabetes and experimental galactosemia) on cardiac metabolism have been determined. In addition, the effect of supplemental antioxidants on these hyperglycemia-induced abnormalities of cardiac metabolism has been investigated. Diabetes or experimental galactosemia of 2 months duration in rats significantly increased oxidative stress in myocardium, as demonstrated by elevation of thiobarbituric acid reactive substances (TBARS) and lipid fluorescent products in left ventricle. Activity of protein kinase C (PKC) was elevated in the myocardium, and the activities of (Na,K)-ATPase and calcium ATPases were subnormal. Administration of supplemental antioxidants containing a mixture of ascorbic acid, Trolox; alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene, and selenium prevented both the diabetes-induced and galactosemia-induced elevation of oxidative stress and PKC activity, and inhibited the decreases of myocardial (Na,K)-ATPase and calcium ATPases. The results show that these metabolic abnormalities are not unique to diabetes per se, but are secondary to elevated blood hexose levels, and supplemental antioxidants inhibit these metabolic abnormalities. Our findings suggest that antioxidants inhibit abnormal metabolic processes that may contribute to the development of cardiac disease in diabetes, and offer a potential clinical means to inhibit cardiac abnormalities in diabetes.

Aldose reductase and the development of renal disease in diabetic dogs.

Effects of 5 years administration of an aldose reductase inhibitor (Sorbinil) on renal structure and albumin excretion were evaluated in diabetic dogs. Glycemia, estimated by frequent measurements of HbA1, glycated plasma proteins and glucosuria, was kept comparable between the placebo- and Sorbinil-treated diabetic groups. Kidney structure was evaluated using morphometric techniques by light and electron microscopy, and excretion of immunoreactive albumin was measured yearly. Placebo-treated diabetic dogs developed nephromegaly, glomerular enlargement, increased mesangial volume, and basement membrane thickening during the 5 years of study, and by the fifth year, excreted greater than normal quantities of albumin. Sorbinil treatment prevented sorbitol accumulation in erythrocytes and tended to have a similar effect in renal cortex, but had no beneficial effect on renal structure or albuminuria. Experimental galactosemia, another model of polyol over-production, failed to produce nephromegaly, glomerular enlargement, or mesangial expansion in dogs even after 5 years of galactose-feeding. The results suggest that polyol over-production and/or accumulation per se are not sufficient to account for the nephromegaly, glomerular enlargement, or increased mesangial volume observed in diabetic dogs.

Abnormalities of retinal metabolism in diabetes or experimental galactosemia. VI. Comparison of retinal and cerebral cortex metabolism, and effects of antioxidant therapy.

Metabolic abnormalities observed in retina and in cerebral cortex were compared in diabetic rats and experimentally galactosemic rats. Diabetes or experimental galactosemia of 2 months duration significantly increased oxidative stress in retina, as shown by elevation of retinal thiobarbituric acid reactive substances (TBARS) and subnormal activities of antioxidant defense enzymes, but had no such effect in the cerebral cortex. Activities of sodium potassium adenosine triphosphatase [(Na,K)-ATPase] and calcium ATPase became subnormal in retina as well as in cerebral cortex. In contrast, protein kinase C (PKC) activity was elevated in retina but not in cerebral cortex in the same hyperglycemic rats. Dietary supplementation with an antioxidant mixture (containing ascorbic acid, Trolox, alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene, and selenium) prevented the diabetes-induced and galactosemia-induced elevation of retinal oxidative stress, the elevation of retinal PKC activity and the decrease of retinal ATPases. In cerebral cortex, administration of the antioxidant diet also prevented the diabetes-induced decreases in (Na,K)-ATPase and calcium ATPases, but had no effect on TBARS and activities of PKC and antioxidant-defense enzymes. The results indicate that retina and cerebral cortex differ distinctly in their response to elevation of tissue hexose, and that cerebral cortex is more resistant than retina to diabetes-induced oxidative stress. The greater resistance to oxidative stress in cerebral cortex, as compared to retina, is consistent with the resistance of cerebral cortex to microvascular disease in diabetes, and with a hypothesis that oxidative stress contributes to microvascular disease in diabetes. Dietary supplementation with these antioxidants offers a means to inhibit multiple hyperglycemia-induced retinal metabolic abnormalities.

Abnormalities of retinal metabolism in diabetes or experimental galactosemia: V. Relationship between protein kinase C and ATPases.

In the retinas of diabetic animals, protein kinase C (PKC) activity is elevated, and Na+-K+-ATPase and calcium ATPase activities are subnormal. These abnormalities are also present in another model of diabetic retinopathy, experimental galactosemia. We have investigated the relationship between hyperglycemia-induced abnormalities of PKC and ATPases using a selective inhibitor of beta isoform of PKC (LY333531). Diabetes or experimental galactosemia of 2 months' duration resulted in > 50% elevation of PKC activity in the retina, and administration of LY333531 prevented the elevation. In retinas of the same rats, the LY333531 prevented hyperglycemia-induced decreases of both Na+-K+-ATPase and calcium ATPase activities. Retinal microvessels, the main site of lesions in diabetic retinopathy, likewise showed elevated activity of PKC and inhibition of ATPases in diabetes and in experimental galactosemia, and administration of LY333531 to diabetic animals prevented these abnormalities. PKC activity in sciatic nerves, in contrast, became subnormal in diabetes and experimental galactosemia, and LY333531 had no effect on PKC activity in the sciatic nerve. PKC activity in the cerebral cortex was not affected by diabetes or experimental galactosemia. The results suggest that diabetes-induced reductions in Na+-K+-ATPase and calcium ATPase in the retina are mediated in large part by PKC-beta. The availability of an agent that can normalize the hyperglycemia-induced increase in PKC activity in the retina should facilitate investigation of the role of PKC in the development of diabetic retinopathy.

Abnormalities of retinal metabolism in diabetes or experimental galactosemia. IV. Antioxidant defense system.

Activities of enzymes that protect the retina from reactive oxygen species were investigated in experimentally diabetic rats and experimentally galactosemic rats, two animal models known to develop vascular lesions consistent with diabetic retinopathy. Diabetes or experimental galactosemia of 2 months duration significantly decreased the activities of glutathione reductase and glutathione peroxidase in the retina while having no effect on the glutathione synthesizing enzymes glutathione synthetase and gamma-glutamyl cysteine synthetase. Activities of two other important antioxidant defense enzymes-superoxide dismutase (SOD) and catalase-also were decreased (by more than 25%) in retinas of diabetic rats and galactosemic rats. Administration of supplemental antioxidants, vitamins C and E, for the 2 months prevented the diabetes-induced impairment of antioxidant defense system in the retina. In experimentally galactosemic rats, the supplemental antioxidants were not as effective: SOD activity was normalized, but the enzymes of the glutathione redox cycle were only partly restored, and the subnormal catalase activity was unaffected. Diabetes or experimental galactosemia results in significant impairment of the antioxidant defense system in the retina, and exogenous antioxidant supplementation can help alleviate the subnormal activities of antioxidant defense enzymes.

Abnormalities of retinal metabolism in diabetes or experimental galactosemia. III. Effects of antioxidants.

Effects of antioxidants on hyperglycemia-induced alterations of retinal metabolism were evaluated in rats diabetic or experimentally galactosemic for 2 months. Oxidative stress was estimated by measuring lipid peroxides (measured as thiobarbituric acid reactive substances [TBARS]) in retina and plasma. Erythrocyte osmotic fragility, another measure of oxidative stress, also was determined in the same groups of rats. In diabetic rats, TBARS were elevated by 74% in retina and 87% in plasma. In galactose-fed rats, TBARS were significantly elevated in retina (P < 0.05), but were normal in plasma. The administration of supplemental dietary ascorbic acid and alpha-tocopherol acetate for 2 months prevented the elevation of retinal TBARS and the decrease of Na(+)-K(+)-ATPase and calcium ATPase activities in retinas of diabetic animals without having any beneficial effect on plasma TBARS. In galactosemic rats, these antioxidants had a partial beneficial effect on the activity of retinal Na(+)-K(+)-ATPase, but failed to have any effect on calcium ATPase. The beneficial effects of antioxidants in diabetes and experimental galactosemia were not caused by the amelioration of hyperglycemia or retinal polyol accumulation. Erythrocyte osmotic fragility was increased by more than twofold in diabetes, but was normal in experimental galactosemia, and antioxidants prevented diabetes-induced increases in erythrocyte osmotic fragility-Diabetes-induced increased oxidative stress and subnormal ATPase activities in the retina can be inhibited by dietary supplementation with antioxidants.

A mouse model of diabetic retinopathy.

OBJECTIVE: To obtain a model of diabetic retinopathy to which modern methods of genetic engineering may be applied, by determining the response of 2 strains of mice to long-term galactose feeding. METHODS: Both C57BL/6 mice BALB/c mice were fed each of 2 galactose-rich diets (30% and 50% galactose), and trypsin digests of their retinas were compared with those of controls at durations of up to 26 months. RESULTS: The mortality rate in galactose-fed animals was lower in C57BL/6 mice than in BALB/c mice, and both strains tolerated the 30% galactose diet significantly better than the 50% galactose diet. In C57BL/6 mice fed 30% galactose for 21 to 26 months, saccular microaneurysms were observed in the retina, together with significant increases in the thickness of capillary basement membrane and the prevalence of acellular capillaries and pericyte ghosts. The 50% galactose diet caused significantly more acellular capillaries than normal by 15 months, but excessive mortality precluded study at longer durations. The frequency of acellular capillaries also was greater than normal in BALB/c mice fed 30% galactose for 21 months. Retinal polyol levels in galactose-fed mice were found to be lower than those in galactosemic rats. CONCLUSION: The mouse may provide an inexpensive model suitable for in vivo study of the pathogenesis of diabetic retinopathy using molecular biological techniques.

Evidence of a glycemic threshold for the formation of pentosidine in diabetic dog lens but not in collagen.

The relationship between long-term glycemic control and the advanced Maillard reaction was investigated in dura mater collagen and lens proteins from dogs that were diabetic for 5 years. Diabetic dogs were assigned prospectively to good, moderate, and poor glycemic control and maintained by insulin. Biochemical changes were determined at study exit. Mean levels of collagen digestibility by pepsin decreased (NS) whereas collagen glycation (P < 0.001), pentosidine cross-links (P < 0.001), and collagen fluorescence (P = 0.02) increased with increasing mean HbA1 values. Similarly, mean levels of lens crystallin glycation (P < 0.001), fluorescence (P < 0.001), and the specific advanced lens Maillard product 1 (LM-1) (P < 0.001) and pentosidine (P < 0.005) increased significantly with poorer glycemic control. Statistical analysis revealed very high Spearman correlation coefficients between collagen and lens changes. Whereas pentosidine cross-links were significantly elevated in collagen from diabetic dogs with moderate levels of HbA1 (i.e., 8.0 +/- 0.4%), lens pentosidine levels were normal in this group and were elevated (P < 0.001) only in the animals with poor glycemic control (HbA1 = 9.7 +/- 0.6%). Thus, whereas protein glycation and advanced glycation in the extracellular matrix and in the lens are generally related to the level of glycemic control, there is evidence for a tissue-specific glycemic threshold for pentosidine formation, i.e., glycoxidation, in the lens. This threshold may be in part linked to a dramatic acceleration in crystallin glycation with HbA1 values of > 8.0% and/or a loss of lens membrane permeability. This study provides support at the molecular level for the growing concept that glycemic thresholds may be involved in the development of some of the complications in diabetes.

Capillary lesions develop in retina rather than cerebral cortex in diabetes and experimental galactosemia.

OBJECTIVE: To isolate microvessels from cerebral cortex of dogs with alloxan-induced diabetes and dogs with experimental galactosemia to compare the prevalence of microvascular lesions in cerebral cortex with that in retina. METHODS: Microvessels were isolated from cerebral cortex of experimental animals using a sieving method, and compared with the retinal vasculature isolated from the same animals using the trypsin digestion method. RESULTS: Dogs with diabetes or experimental galactosemia of 5 years' duration had retinopathy that was morphologically indistinguishable from that of humans with diabetes, including microaneurysms, acellular capillaries, and pericyte ghosts. These lesions never were seen in cerebral cortical vessels of the same animals. The only morphologic abnormality observed in cerebral capillaries of dogs with diabetes and dogs fed galactose was thickening of basement membrane. CONCLUSIONS: Local influences in the eye apparently play an important role in the development of diabetic retinopathy. Current hypotheses about the pathogenesis of the retinopathy do not account adequately for such differences in the tissue distribution of vascular lesions.

Vascular lesions in diabetes are distributed non-uniformly within the retina.

Microaneurysms, acellular capillaries and pericyte ghosts are characteristic of diabetic retinopathy, but it is not clear what causes these lesions or whether they are causally related to each other. The distribution of microaneurysms, acellular capillaries and pericyte ghosts has been evaluated in two animal models of diabetic retinopathy, diabetic dogs (n = 25) and galactose-fed dogs (n = 12). After 5 years of diabetes or galactosemia, retinas were divided into four quadrants at the optic disk, prepared by the trypsin-digest method, and the frequency of the lesions compared among the quadrants. Numbers of lesions were expressed relative to area of trypsin-digested retina examined or to total number of capillary cells examined. Microaneurysms and acellular capillaries were not uniformly distributed across the retina in diabetes or in galactosemia, both lesions being significantly (more than two-fold) more prevalent in the superior temporal retina than in the inferior nasal quadrant of retina. In contrast, the distribution of pericyte ghosts in these same eyes was not significantly different between the quadrants. These findings suggest that pericyte loss may not be sufficient to account for the development of microaneurysms and acellular (occluded) capillaries in diabetes, and raise a possibility that the lesions occur by different mechanisms. Currently available hypotheses regarding the pathogenesis of diabetic retinopathy fail to account for regional differences in the distribution of the vascular lesions within the same retina. Local factors within the eye apparently play an important role in the response of the retinal microvasculature to hyperglycemia.

Retinopathy in galactosemic dogs continues to progress after cessation of galactosemia.

BACKGROUND: Progression of diabetic retinopathy in human subjects and animal models is difficult to halt promptly by intensified insulin therapy and strict glycemic control. OBJECTIVE: To learn whether this resistance to arrest is peculiar to diabetes and insulin therapy or is a characteristic of hyperglycemia itself, we have determined the effect of intervention on diabetic-like retinopathy in a non-diabetic animal model, the galactose-fed dog. METHODS: Dogs were given a 30% galactose diet. At the end of 24 months, the dogs were divided into two groups, one of which continued to receive the galactose diet, while the second immediately began receiving the diet minus galactose. All animals were killed after 60 months of study. RESULTS: Consumption of the galactose-rich diet resulted, as expected, in galactosemia evident by elevated hemoglobin A1, plasma nonenzymatically glycated protein, and erythrocyte polyol concentrations, each of which decreased to normal levels following withdrawal of dietary galactose. Retinopathy was found to be equivocal at the end of 24 months of the galactose diet and subsequently progressed significantly despite cessation of the galactose diet. CONCLUSION: The fact that retinopathy did not halt promptly at intervention is consistent with previous data from diabetic dogs and suggests that the vascular lesions may be due to sequelae of excessive tissue aldohexose that are not promptly corrected by correction of the aldohexose level.

Galactose-induced retinal microangiopathy in rats.

PURPOSE: The suitability of the galactose-fed rat as a model of diabetic retinopathy was examined in nondiabetic rats fed diets enriched with either 30% or 50% galactose for up to 2 years. METHODS: Retinal capillaries were examined by light and electron microscopy, and the prevalence or severity of diabetic-like lesions was quantitated. RESULTS: Histologic evaluation of trypsin digests of retina revealed significantly greater than normal frequencies of pericyte ghosts and acellular capillaries at both 15 and 23 months receiving a 50% galactose diet. Similar lesions were observed in rats receiving a 30% galactose diet for 23 months. Capillary basement membrane thickening, dilated hypercellular capillaries (or intra-retinal microvascular abnormalities), and foci of vascular cells appeared in rats fed 50% galactose, but saccular microaneurysms characteristic of retinopathy in diabetic patients, diabetic dogs, and experimentally galactosemic dogs were not observed. Administration of the aldose reductase inhibitor, Sorbinil, to rats fed 50% galactose resulted in a significant inhibition of cataract and of galactitol accumulation in nerve and blood (by more that 90%) and retina (by 62%), but did not inhibit development of the retinal microvascular lesions. CONCLUSIONS: Two years of galactosemia in rats seems to reproduce only a portion of the lesions characteristic of diabetic retinopathy in patients or dogs. Nevertheless, lesions characteristic of at least the early stages of retinopathy clearly do develop in this galactosemic rat model, and are not restrained by inhibition of retinal polyol accumulation by 62%.

Comparison of retinal lesions in alloxan-diabetic rats and galactose-fed rats.

Galactose-fed rats develop a retinal microvascular disease, but retinopathy has not been found to develop reproducibly in diabetic rats. We sought to determine which retinal lesions can be reproducibly produced by long-term diabetes in rats, the extent to which the capillary lesions in diabetic rats and galactosemic rats are similar, and whether the retinopathy induced by 50% galactose can be reproduced satisfactorily by a lower concentration of galactose. Alloxan-diabetic rats and rats fed either a 50% galactose diet or a 30% galactose diet were killed after comparable durations of study (18 to 22 months). Rats fed 50% galactose showed greater than normal frequency of retinal pericyte ghosts and acellular capillaries, and thickening of capillary basement membranes by 18 months of galactosemia. Rats eating 30% galactose developed similar retinal lesions, and tended to be healthier than rats fed 50% galactose. Diabetes of 1 1/4 years or more likewise resulted in retinal pericyte ghosts, acellular capillaries and thickened capillary basement membrane. IRMAs and other vascular abnormalities were not reproducibly demonstrated at this duration of study, and saccular microaneurysms were not seen in any groups. In a number of diabetic rats, the severity of diabetes diminished spontaneously (after 1 to 1 1/2 years of insulin deficiency), thus making it essential that glycemia be systematically monitored. Both diabetic rats and experimentally galactosemic rats develop microvascular lesions that are consistent with at least the early stages of diabetic retinopathy, and these models should be useful to screen potential therapies for their ability to inhibit the development of retinopathy.

Abnormalities of retinal metabolism in diabetes or galactosemia. II. Comparison of gamma-glutamyl transpeptidase in retina and cerebral cortex, and effects of antioxidant therapy.

Levels of the intracellular antioxidant, glutathione, become subnormal in retina in diabetes or experimental galactosemia. In order to investigate the cause and significance of this abnormality, activity of gamma-glutamyl transpeptidase (an enzyme important in the synthesis and degradation of glutathione) and levels of reduced glutathione have been measured in retinas of diabetic rats and dogs and of experimentally galactosemic rats and dogs. Retinal gamma-glutamyl transpeptidase activity and glutathione level were significantly less than normal after 2 months of diabetes or galactosemia. In contrast, cerebral cortex from the same diabetic rats and galactosemic rats showed no significant reduction in either gamma-glutamyl transpeptidase activity or glutathione level. These different responses of the two tissues to hyperglycemia might help account for the difference in microvascular disease in these two tissues in diabetes. Consumption of the antioxidants, ascorbic acid (1.0%) plus alpha-tocopherol (0.1%), by diabetic rats and galactosemic rats inhibited the decrease of gamma-glutamyl transpeptidase activity and glutathione levels in retina, suggesting that defects in glutathione regulation in the retina are secondary to hyperglycemia-induced 'oxidative stress'.