AbstractŽ y . Objective: Previous studies suggest a role of superoxide anion radicals PO in impaired endothelium-dependent relaxation of 2 diabetic blood vessels; however, the role of secondary reactive oxygen species remains unclear. In the present study, we investigated a role of various potential reactive oxygen species in diabetic endothelial dysfunction. Methods: Thoracic aortic rings from 8-week streptozotocin-induced diabetic and age-matched control rats were mounted in isolated tissue baths. Endothelium-dependent relaxation to Ž . Ž . acetylcholine ACH and endothelium-independent relaxation to nitroglycerin NTG were assessed in precontracted rings. Results: ACH-induced relaxation was impaired in diabetic compared to control rings and was not improved with either indomethacin or daltroban. ACH-induced relaxation in both control and diabetic rings was completely blocked with the nitric oxide synthase inhibitors, Ž . L -nitroarginine methyl ester or L-nitroarginine L-NA . NTG-induced relaxation was insensitive to L-NA and was unaltered by diabetes.Ž . Pretreatment with superoxide dismutase SOD at activities which did not alter contractile tone failed to alter responses to ACH in diabetic rings. Similar results were obtained using either catalase or mannitol. In contrast, the combination of SOD plus catalase or Ž . DETAPAC, an inhibitor of metal-facilitated hydroxyl radical POH formation, markedly enhanced relaxation to ACH in diabetic but not in control rings. Neither the combination of SOD plus catalase nor DETAPAC altered the sensitivity or relaxation to NTG in control rings with or without endothelium. In diabetic rings with endothelium, both DETAPAC or SOD plus catalase increased sensitivity but not maximum relaxation to NTG. In diabetic rings without endothelium, relaxation and sensitivity to NTG were unaltered by either treatment. In L-NA-treated diabetic rings with endothelium, sensitivity and relaxation to NTG was unaltered by either DETAPAC or SOD plus catalase. Conclusion: Diabetic endothelium produces increases in both PO y and H O leading to enhanced intracellular production of 2 2 2 P OH. Thus, POH are implicated in diabetes-induced endothelial dysfunction.

Oxidative stress is believed to play an important role in the development of vascular complications associated with diabetes mellitus. In this study, we examined the efficacy of long-term treatment with the antioxidant, N-acetylcysteine, in preventing the development of defective endothelium-dependent relaxation in streptozotocin-induced, Sprague-Dawley diabetic rats. At 48 h after injection of streptozotocin, a portion of diabetic rats received 250 mg/L N-acetylcysteine in drinking water for a total duration of 8 weeks. Oral administration did not alter the increase in blood glucose or the reduction in serum insulin but did modestly reduce total glycosylated hemoglobin. In precontracted thoracic aortic rings suspended in isolated tissue baths, endothelium-dependent relaxation to acetylcholine was impaired in diabetic rings compared with control rings. Endothelium-independent relaxation to nitroglycerin was unaltered. Long-term oral administration of N-acetylcysteine did not alter responses to nitroglycerin but completely prevented the defective relaxation to acetylcholine. These studies indicate a dissociation between glycemic control and correction of endothelial dysfunction and suggest that long-term exposure to reactive oxygen subsequent to diabetes rather than hyperglycemia per se is responsible for the development of endothelial dysfunction in diabetes mellitus.

Recent studies have indicated impaired endotheliumdependent relaxation in arteries of insulin-dependent [1] diabetic (IDDM) patients. The mechanisms for this defect in human blood vessels is not yet understood. Several studies have observed defects in endothelium-dependent relaxation in both conduit and resistance arteries of streptozotocin (STZ)-or alloxaninduced diabetic rats or rabbits (see reviews [2,3]).Another experimental diabetic model which is not as frequently investigated is the spontaneously diabetic BB (Bio-Breeding) rat despite the fact that this model more closely approximates IDDM in man. Currently, only a limited number of studies regarding endothelial function have been conducted in this important model [4][5][6][7]. More importantly, there is no available information regarding specific defects in nitric oxide (NO) production contributing to defective endothelium-dependent relaxation in this model. In this study, we evaluated the efficacy of supplementation with l -arginine (l-ARG) to improve NO synthase-dependent, endothelium-dependent relaxation in the diabetic BB rat. Materials and methodsExperiments were performed in compliance with the National Institutes of Health "Principles of Laboratory Animal Care" (NIH publication No. 85-23, revised 1985). Progeny of the original BB rat colony from the University of Pennsylvania were used in breeding pairs to develop a local colony of diabetic and non-diabetic BB rats. Onset of diabetes was regularly monitored using test strips and glucometer and defined by 2 successive days of elevated glucose concentration less than 11 mmol/l. Diabetic animals received s. c. injections of NPH insulin which was varied to maintain a low level of hyperglycaemia (i. e. approximately 12-14 mmol/l). Female diabetic BB rats and age-matched, diabetic-resistant littermates were Diabetologia (1997) 40: 910-915 Reversal by L-arginine of a dysfunctional arginine/nitric oxide pathway in the endothelium of the genetic diabetic BB rat Summary We examined the effects of acute supplementation with arginine in vitro on endothelium-dependent relaxation in aortic rings taken from female genetic, diabetes-prone BB rats. Sensitivity to norepinephrine-induced contraction was unaltered in rings of diabetic BB rats compared to rings from non-diabetic littermates. In precontracted rings, acetylcholine produced a concentration-dependent relaxation which was impaired by diabetes. This relaxation was blocked by l -arginine had no effect on acetylcholine-induced relaxation in control rings. In contrast, relaxation-induced by nitroglycerin in diabetic rings without endothelium was not altered by l -arginine treatment. Thus, a defect in the utilization of arginine by nitric oxide synthase exists in the endothelium of the diabetic BB rat. [Diabetologia (1997) 40: 910-915]

Oxygen radicals are believed to play a role in vascular complications of diabetes mellitus. In this study, we evaluated whether long-term treatment with an iron chelator and inhibitor of metal-catalyzed hydroxyl radicals (.OH) could prevent diabetes-induced defects in endothelium-dependent relaxation. Diabetes was induced in Sprague-Dawley rats by injection of streptozotocin. At 48 h after streptozotocin, a subgroup of diabetic rats received daily injections of 50 mg/kg hydroxyethyl starch conjugated-deferoxamine (HES-DFO) for a total of 8 weeks. Long-term treatment with HES-DFO did not modify serum insulin or blood glucose taken at the end of the study; however, a modest reduction in glycosylated hemoglobin was present. In precontracted aortic rings suspended in tissue baths, endothelium-dependent relaxation to acetylcholine was impaired in diabetic rings compared with control rings in the presence or absence of indomethacin. Endothelium-independent relaxation to nitroglycerin was unaltered. Long-term treatment with HES-DFO had no effect on relaxation to nitroglycerin but completely prevented the impaired relaxation to acetylcholine in diabetic rings in either the presence or absence of indomethacin. These data suggest that iron-catalyzed .OH formation contributes to the development of diabetes-associated endothelial dysfunction.

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Considerable evidence using experimental models [1±5] have shown that diabetes mellitus is an independent risk factor for the development of impaired endothelium-dependent relaxation ± that is, endothelial dysfunction. This defect has been confirmed in Type I (insulin-dependent) [6,7] and Type II (non-insulindependent) [8] diabetic patients. The factor(s) which contribute to endothelial dysfunction in diabetic patients is not known but data derived in experimental models have suggested several possibilities including: (a) concurrent release of an endothelium-derived constricting factor arising from the cyclooxygenase pathway [9,10] Summary Substantial evidence exists that diabetes results in impaired endothelial dysfunction suggesting diminished nitric oxide production from diabetic endothelium. It is not known what factors contribute to the development of this defect. In this study, we tested whether chronic treatment in vivo with NOX-101, a water-soluble nitric oxide scavenger, prevents endothelial dysfunction in diabetes. Sprague-Dawley rats were made diabetic by an intravenous injection of streptozotocin. A subgroup of control or diabetic animals received twice daily subcutaneous injections of 80 mg/kg NOX-101 beginning at 48 h after streptozotocin was injected and throughout 8 weeks of diabetes. Body weights and glucose concentrations were monitored weekly. At the end of 8 weeks, blood glucose and glycosylated haemoglobin was raised in diabetic rats but serum insulin concentrations were reduced. Treatment with NOX-101 did not alter glucose or insulin concentrations in control or diabetic rats; however, total glycosylated haemoglobin was partially reduced compared with untreated rats. In a subgroup of 2-week diabetic and age-matched rats fasted for 24 h, NOX-101 abolished total urinary nitrate plus nitrite (an index of nitric oxide production in vivo). In isolated tissue baths, relaxation to the endothelium-dependent vasodilator, acetylcholine, was impaired in diabetic aortic rings and relaxation to nitroglycerin was unaltered. Treatment of control rats with NOX-101 did not alter maximum relaxation to acetylcholine but shifted the response curve slightly to the right. In contrast in diabetic rats, NOX-101 prevented the impairment in endothelium-dependent relaxation but had no effect on relaxation induced by nitroglycerin. These data suggest the possibility that diabetes-induced endothelial dysfunction in diabetes results, in part, from a paradoxical increase in nitric oxide production during the course of the disease. This suggests a novel pathway of vascular complications. [Diabetologia (1998

Oxidative stress is believed to play a role in diabetes-induced vascular complications. In this study, we tested whether chronic treatment with a known hydroxyl radical scavenger, dimethylthiourea (DMTU), could prevent endothelial dysfunction in diabetes. Lewis strain rats were made diabetic by an intravenous injection of streptozotocin. A subgroup of diabetic animals received daily intraperitoneal injections of 50 mg/kg DMTU beginning at 72 h after streptozotocin and throughout 8 weeks of diabetes. Diabetes caused an increase in aortic catalase activity (an index of compensatory in vivo oxidative stress) that was not prevented by long-term DMTU treatment. Long-term treatment of diabetic animals with DMTU did not alter serum insulin levels, blood glucose concentrations, or total glycosylated hemoglobin. Descending thoracic aortas were isolated, sectioned into rings and suspended in isolated tissue baths, and contracted with a submaximal concentration of norepinephrine. Relaxation to the endothelium-dependent vasodilator, acetylcholine, was impaired in diabetic aortas, whereas relaxation to A23187 and nitroglycerin was unaltered. DMTU treatment prevented the diabetes-induced impairment in endothelium-dependent relaxation to acetylcholine but had no effect on relaxations induced by either A23187 or nitroglycerin. These data suggest that chronic exposure to increased levels of hydroxyl radicals in vivo likely play a significant role in the origin of diabetes-associated endothelial dysfunction.

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