1 Departments of Medicine and Ophthalmology, and Center for Diabetes Research, Case Western Reserve University, Cleveland, Ohio
2 Department of Ophthalmology and Vision Research, University of Wisconsin, Madison, Wisconsin
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ABSTRACT |
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INTRODUCTION |
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Several biochemical sequelae of hyperglycemia have attracted particular attention as possible causes of the retinopathy, and methods to inhibit these biochemical defects continue to be identified. Chronic aspirin consumption was reported many years ago to be associated with protection from diabetic retinopathy, raising a possibility that alteration of prostaglandin production might influence the development of retinopathy. The effectiveness of aspirin in clinical trials has been controversial, however, showing a statistically significant (although modest) inhibitory effect of the drug on retinopathy in one clinical trial (9), but having no significant beneficial effect in another larger clinical trial (10). Another drug, aminoguanidine, was shown to inhibit many sequelae of advanced glycation end product (AGE) formation (11,12,13) and was observed to have beneficial effects on a number of diabetes-induced alterations of tissue function and structure (14,15,16,17,18,19,20). Moreover, aminoguanidine was found by Hammes et al. (21,22) and later also by Kern and Kowluru (23) to inhibit the development of some retinal lesions in diabetic rats. In our experience, diabetic rats develop the early stages of diabetic retinopathy, but do not reproducibly develop microaneurysms and advanced lesions of the retinopathy (7).
Dogs that develop diabetes either spontaneously or experimentally develop a retinopathy that is morphologically indistinguishable from that which is characteristic of diabetic patients (24,25). In the present study, we examined the effects of aminoguanidine and aspirin on the development of retinopathy in diabetic dogs over a 5-year interval. Unlike in clinical trials, where many patients have some retinopathy at the onset of the study, drug administration in our dog studies was initiated at the time of diabetes onset.
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RESEARCH DESIGN AND METHODS |
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To ensure that drug-treated and control groups would be comparably diabetic, blood and urine were monitored carefully throughout the 5-year study. Values reported in Table 1 are the mean over the entire 5 years of study. Animals were housed in metabolism cages, and 24-h urinary excretion of reducing sugar was measured every day (7 days/week). Fasting blood glucose was measured once per month using glucose oxidasebased methods. Every 2 months, HbA1 (Isolab minicolumns; Isolab, Akron, OH) was assayed in all animals after incubation of blood to remove labile adducts. Cholesterol and triglycerides were measured yearly. Blood was collected after an overnight fast and at different times after drug administration to assess plasma drug levels. Plasma levels of aspirin and aminoguanidine were measured by chemical assay (26) and high-performance liquid chromatography (HPLC), respectively.
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Biochemical effects of aminoguanidine.
Immunoreactive AGE adducts on hemoglobin and aortic proteins, and pentosidine levels in aorta and tail collagen, were measured at autopsy to obtain information on aminoguanidine efficacy and action. The parameters of AGE that were measured represent both intracellular (Hb-AGE, aortic AGE-adducts, and pentosidine) and extracellular (collagen pentosidine) environments. Hb-AGE was measured by immunoassay (28). Aortic AGE was measured by competitive enzyme-linked immunosorbent assay after digestion overnight in collagenase. Pentosidine was measured by HPLC in acid hydrolyzates of tail tendon. Results are expressed relative to collagen concentration as estimated by assay of hydroxy-proline. Measurements of collagen thermal breaking time (29) were attempted, but were abandoned because the fibrils did not break notwithstanding increasing test weight (up to 10 g), temperature of the urea solution, or incubation duration.
Effects of aminoguanidine on glycation and formation of nitrotyrosine in the retina were assessed also in other alloxan-diabetic dogs treated with (n = 3; 2 years) or without (n = 3; 2 years) aminoguanidine or comparably aged normal controls (n = 4). In all respects, these animals were treated like the animals described previously. Retina was homogenized in 10 mmol Tris containing 0.5 mmol EGTA and 0.5 mol sucrose (pH 7.4). Homogenates were centrifuged at low speed for 5 min, the protein content of the resulting supernatants measured using bicinchoninic acid reagent (Pierce), proteins in the supernatant solubilized in SDS, and 25100 µg protein per lane subjected to SDS-PAGE (12% acrylamide gels). After SDS-PAGE and transfer to a nitrocellulose membrane (BAS-85; Schleicher and Schuell, Keene, NH), blots were incubated in Tris-buffered saline (20 mmol Tris and 137 mmol NaCI, pH 7.6) containing 0.1% Tween 20 and 5% nonfat milk. To show comparable loading of lanes, membranes were stained with Ponceau S staining solution (Sigma, St. Louis, MO), and the intensity of bands was compared visually across lanes. Membranes then were incubated for 1 h with antibodies against 3-deoxyglucosonederived imidazolone (30) (monoclonal antibody; 1:5,000 dilution) or nitrotyrosine (rabbit polyclonal, 1:1,000 dilution; Upstate Biotechnology), and incubated for another hour in peroxidase-labeled goat antimouse IgG (1:3,000 dilution). Blots were developed using an enhanced chemiluminescence kit (Amersham Life Sciences, Amersham, England). Films were digitized, and intensity of bands was quantitated using the OS-Scan Image Analysis software (USB State Biochemical Corp., Cleveland, OH). The diabetes-induced increase in intensity of the aforementioned imidazolone-positive band was slight, so to further confirm comparable loading among lanes, the membrane was stripped and then restained with antibody against the intrinsic protein, GAPDH, and a ratio of intensity of the imidazolone-modified band to the intensity of the GAPDH band was calculated.
Biochemical effects of aspirin.
Platelet aggregation was measured three times each year using a collagen stimulus as reported previously (31). Plasma and urinary prostaglandins (6-keto F1 and thromboxane B2) were measured in representative dogs (three per group) using commercially available kits (Advanced Magnetics, Cambridge, MA). Formic acidtreated urine was first extracted using C18 columns. Prostaglandin release by retina (32) was measured using fresh retinas from nondiabetic controls, diabetic dogs, and diabetic dogs treated with aspirin for 2 months (n = 2 per experimental group; aspirin given as described above, 20 mg/kg twice a day). The retinas were incubated for 30 min in Krebs-Ringer bicarbonate buffer. Prostaglandins released into the incubation buffer were quantitated as above.
Kidney and nerve.
Conduction of the ulnar nerve was measured three times in the 5-year study; at baseline, at 2.5 years, and at the 5-year anniversary just before sacrifice. Conduction velocity was measured as reported previously (33,34). Data are presented herein only for year 5. Samples of renal cortex from mid-kidney were fixed for light microscopy in 10% neutral formalin, and 4-µm sections of paraffin-embedded kidney were cut. Sections were stained with methenamine silver for basement membrane and matrix. The area of 50 consecutive glomeruli was measured in each dog by tracing at 200x, and the fractional area of the silver-stained area per glomerulus was determined using computer-assisted image analysis. Total volume per glomerulus of silver-stained matrix was calculated from the average glomerular volume for each animal (35) and fractional area (determined using Image-Pro Plus Imaging system; Media Cybernetics, Silver Spring, MD). Urinary albumin was quantitated by immunoassay three times each year, using an antibody directed against dog albumin (Cappel) (36) and 24-h samples of urine. Table 2 includes the average of the three values used to calculate a yearly mean and also the mean excretion rate during the fifth year.
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RESULTS |
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Retinopathy.
Histological study of the retina in dogs of the control group diabetic for 5 years revealed the expected saccular capillary aneurysms, ghosts of intramural pericytes, acellular capillaries, varicose vessels, vessel sudanophilia, intraretinal neovascularization, and hemorrhages described previously (Table 2 and Fig. 1).
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Aspirin significantly inhibited the development of acellular capillaries, retinal hemorrhages, and capillary sudanophilia over the 5 years of study (P < 0.001, P < 0.002, and P < 0.05, respectively). The effect of aspirin on the number of microaneurysms and pericyte ghosts was equivocal, achieving statistical significance by Fishers test only for the worst eye, and not when using other statistical tests.
Biochemical effects of aminoguanidine.
Diabetes resulted in significantly increased levels of pentosidine in tail collagen and aorta (P < 0.005 and 0.005) and of protein-bound AGE on hemoglobin and aortic protein (P < 0.05 and < 0.01, respectively; Table 1). Administration of aminoguanidine had no significant influence on any of these parameters. Western blots of retinal protein (from dogs studied 2 years) stained with antibody against the AGE, imidazolone, revealed numerous bands, but in only one of those bands (50 kDa) did stain intensity tend to be increased in diabetes and seem also inhibited by aminoguanidine. The density of immunostain of this imidazolone-modified protein (expressed as a ratio to the density of GAPDH immunostaining) was 1.36 ± 0.43, 1.75 ± 0.18, and 1.23 ± 0.40, respectively, in normal, diabetic control, and aminoguanidine-treated diabetic animals (not statistically significant, P = 0.22). Staining of Western blots with antibody against nitrotyrosine likewise revealed numerous bands, but in only one of those bands (
80 kDa) was stain intensity increased in diabetes and was stain intensity inhibited by aminoguanidine. The increase in stain intensity of this band in diabetes (440 ± 35 arbitrary units in those with diabetes vs. 279 ± 42 arbitrary units in those without diabetes) and inhibition of staining by aminoguanidine (309 ± 25) both were statistically significant (P < 0.05). No other protein bands were observed to have greater than normal immunostain in samples from the diabetic animals or inhibition of immunostain intensity in samples from aminoguanidine-treated diabetic animals.
Biochemical effects of aspirin.
As we discovered in previous studies (31), diabetic dogs showed no abnormality of collagen-induced platelet aggregation. Nevertheless, aspirin treatment significantly inhibited collagen-induced platelet aggregation (P < 0.001), even 14 h after the previous dose of drug (the longest interval between administration of aspirin). Plasma levels of 6-keto F1 and thromboxane B2 tended to be elevated in diabetic animals, but the results did not achieve statistical significance. Aspirin significantly reduced the diabetes-induced rise in prostaglandins only for plasma 6-keto F1
(data not shown). In short-term studies, release of prostaglandins from the retina tended to be increased (6-keto F1
: 2.4 ± 0.8 ng · mg-1 · min-1 vs. 5.9 ± 4.0 for nondiabetic and diabetic, respectively, and thromboxane: 13 ± 5 ng · mg-1 · min-1 vs. 20 ± 4 for nondiabetic and diabetic, respectively), although the small sample sizes preclude statistical comparison. Aspirin consumption inhibited production of these prostaglandins by retinas of diabetic animals by >90% (0.4 ± 0.1 and 0.5 ± 0.1, respectively).
Drug side effects.
No evidence of gross anatomical pathology was observed at autopsy in either the aminoguanidine- or aspirin-treated group. No tumors were noted in any animals, and aspirin-treated animals had no evidence of stomach hemorrhages or ulcerations. One aminoguanidine-treated dog developed cyclical insulin resistance in association with going into heat, but no clear relationship with aminoguanidine was evident.
Kidney.
Moderate glycemic control in the diabetic animals resulted in statistically significant, although modest, changes in renal structure, including nephromegaly and glomerular enlargement with increased absolute volume of matrix/basement membrane. The fractional area of the silver-stained matrix/basement membrane in these diabetic animals in moderate glycemic control tended to be greater than normal (51 ± 12 and 60 ± 3% for normal and control diabetic animals, respectively), but the increase did not achieve statistical significance in this small sample. Albumin excretion progressed with duration of diabetes, becoming statistically greater than normal during the fifth year of moderate glycemic control (P < 0.01). Neither aspirin nor aminoguanidine had a significantly beneficial effect on albumin excretion in diabetic dogs (P = 0.68 and 0.11, respectively).
Nerve.
Conduction velocity of ulnar nerve decreased with duration of diabetes, and the decrease was statistically significant during the fifth year of moderate glycemic control (P < 0.0001). Aspirin had no apparent effect on the conduction velocity. Aminoguanidine had a modest beneficial effect (P = 0.04), the diabetes-induced decrease in conduction velocity in year five being reduced by about half.
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DISCUSSION |
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Both aminoguanidine and aspirin were found to significantly inhibit the development of acellular capillaries in diabetes. An acellular capillary consists of the remnant basement membrane skeleton of a once-functional capillary from which all capillary cells have disappeared. Acellular capillaries are of interest because they are seen to be not perfused (39). Thus, increased numbers of acellular capillaries in diabetes likely are causally related to the development of retinal ischemia and clinically significant retinal neovascularization. Inhibition of the development of acellular capillaries by agents such as aminoguanidine and aspirin can be expected to inhibit the development of retinal ischemia and neovascularization.
Aminoguanidine was viewed originally as an inhibitor of sequelae of AGE formation (11). Hammes et al. (21,22) observed that the drug inhibited retinal pathology in their diabetic rats, and concluded that the drug did so by inhibiting formation of AGEs because they found retinal arterioles of drug-treated animals had less in situ fluorescence at wavelengths characteristic of AGEs than did untreated diabetic animals. These wavelengths, however, are not specific for AGEs, and likely include also a variety of oxidation products. We have found that the inhibition of retinal lesions by aminoguanidine in diabetic rats occurs without systemic reductions in parameters of AGEs, such as Hb-AGE (mainly carboxymethyl lysine), retinal pentosidine, and tail collagen fluorescence and pentosidine (23). In the present study, aminoguanidine had no significantly beneficial effect on systemic AGEs (such as Hb-AGE and pentosidine). The therapy tended to inhibit accumulation of retinal imidazolone, which is derived from the AGE, 3-dexyglucosone (30), but the effect was not statistically significant. Whether or not the therapy had a more dramatic effect on other retinal AGEs remains to be investigated. Aminoguanidine has been reported to inhibit biochemical processes other than those merely related to AGE formation, including activities of semicarbizide-sensitive amine oxidase and the inducible isoform of nitric oxide synthase (iNOS), as well as diabetes-induced oxidative stress in the retina and other tissues (15,17,40,41,42,43,44,45). In the present study, diabetes increased the amount of nitration of especially one retinal protein (presumably secondary to formation of peroxynitrite from nitric oxide), and aminoguanidine inhibited this nitration (consistent with aminoguanidine-mediated inhibition of iNOS). Nevertheless, determination of the biochemical mechanism by which aminoguanidine has inhibited retinopathy will require additional study.
Kern et al. (46) have found aminoguanidine to inhibit a diabetes-induced increase in apoptosis of retinal capillary cells. Rapid advances in understanding of the biochemical basis for apoptosis offer a valuable bridge between diabetes-induced alterations in metabolism and the histopathology characteristic of diabetic retinopathy. It seems likely that the inhibition of apoptosis by aminoguanidine contributes to the drugs inhibition of acellular capillaries and pericyte loss.
In rats, aminoguanidine has been reported to have beneficial effects also on diabetes-induced complications in kidney and nerve (14,15,16,17,18,19,20). Aminoguanidine did have a modest beneficial effect on nerve conduction velocity in our diabetic dogs, although the results barely achieved statistical significance in this small sample. Aspirins effect on the development of diabetic complications, in contrast, has been studied little in laboratory animals and chiefly in humans. In the present study of diabetic dogs, neither aspirin nor aminoguanidine had a significant effect on any of the parameters of renal structure and function examined. Dogs in the present experiment intentionally were kept "moderately" insulin deficient, so the severity of renal disease is less than that reported previously by us for dogs in poor glycemic control (47,48) and consists largely of hypertrophic changes. The difference in conclusions reached between previous rat studies and the present dog study might be due to species differences, or to the great difference in duration of the experiments in the two species. Nevertheless, the ability of aminoguanidine to inhibit diabetic retinopathy in dogs while having little or no effect on renal disease in diabetes is consistent with our previous evidence that the pathogenesis of the retinopathy differs appreciably from that of nephropathy (49).
Aspirin, like aminoguanidine, significantly inhibited retinal hemorrhage and the formation of acellular capillaries, but less effectively diminished the frequency of microaneurysms and pericyte ghosts. A lower than expected severity of retinopathy in diabetic subjects with arthritis led to a suggestion many years ago that aspirin might be a potentially effective therapy against diabetic retinopathy (50,51), but prospective clinical trials to assess this possibility have yielded contradictory results. Aspirin treatment resulted in a statistically significant (although weak) inhibition of the mean yearly increase in number of microaneurysms in the DAMAD trial (9), whereas no beneficial effect was observed on any aspect of retinopathy in the Early Treatment of Diabetic Retinopathy Study (ETDRS) trial (10). Differences in design of the two studies may have contributed to the divergent conclusions, but two differences in particular seem especially important. Patients in the DAMAD study had only little retinopathy at onset of the trial, whereas patients in the ETDRS had a more advanced stage of retinopathy (mild to severe nonproliferative or early proliferative retinopathy). Thus, the lack of effect of aspirin in the ETDRS might be attributable to the greater severity of retinopathy, especially since animal (27,52) and clinical (2) studies have shown that retinopathy, once initiated, tends to resist arrest. Moreover, patients in the DAMAD study received more aspirin than those in the ETDRS (990 vs. 660 mg/day). Aspirin did not make retinal hemorrhages worse in diabetic patients (10) or in diabetic dogs; in the present studies, it actually inhibited their development.
Our study of aspirin in dogs differed from the clinical studies in humans in at least two respects. Aspirin was administered to the diabetic dogs from the onset of diabetes; in the clinical trials with human subjects, however, diabetes had persisted for extended periods before aspirin therapy was initiated. In addition, the histological methods we used to detect retinal lesions are more sensitive than methods available clinically, allowing us to detect lesions on individual capillaries and at an earlier stage than is feasible clinically. The animal and human studies, taken together, are consistent with a hypothesis that aspirin therapy is more effective if initiated early in diabetes rather than later. Aspirin is capable of altering prostaglandin-mediated processes (via cyclooxygenase) and inflammatory processes independently of one another, but the dose of aspirin used in our dog studies was sufficiently high (when expressed relative to body weight) to inhibit presumably both of these processes. Whether aspirin at a different dose might result in a more complete inhibition of the retinopathy remains to be learned.
Aminoguanidine and aspirin, in addition to the independent effects, also share activities that might constititute a mechanism in common for their observed beneficial effects on retinopathy. Both aspirin and aminoguanidine reportedly can alter blood flow and vessel permeability (15,53,54), inhibit nonenzymatic glycation or its sequelae (55,56,57,58), and inhibit oxidative stress (44,45,59,60). The identification of multiple therapies by which retinopathy can be inhibited can be expected to reveal the various biochemical and physiological steps responsible for the retinopathy, and at which the retinopathy might be best inhibited. One mechanism for the inhibition of vaso-occlusion by aspirin might be via inhibiting formation of the microthrombi reported recently in diabetic retinopathy.
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ACKNOWLEDGMENTS |
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We sincerely appreciate the assistance of Dr. R. Nagaraj for measurements of collagen pentosidine, anti-imidazolone antibody from Dr. T. Niwa, and the skillful technical assistance of M. Larson, M. Garment, C. Miller, and L. Martin. Plasma aminoguanidine, Hb-AGE, and aorta pentosidine were measured by Alteon, Inc.
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FOOTNOTES |
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Received for publication 28 December 1999 and accepted in revised form 20 March 2001.
AGE, advanced glycation end product; ETDRS, Early Treatment of Diabetic Retinopathy Study; HPLC, high-performance liquid chromatography; iNOS, inducible isoform of nitric oxide synthase.
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REFERENCES |
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