Fish Oil Blunted Nicotine-Induced Vascular Endothelial Abnormalities Possibly via Activation of PPARγ-eNOS-NO Signals

Nicotine exposure is associated with an induction of vascular endothelial dysfunction (VED), a hallmark of various cardiovascular disorders. The present study investigated the effect of fish oil in nicotine-induced experimental VED. VED was assessed by employing isolated aortic ring preparation, estimating aortic and serum nitrite/nitrate, aortic superoxide anion generation, and serum TBARS, and carrying out electron microscopic and histological studies of thoracic aorta. Nicotine (2 mg/kg/day, i.p., 4 weeks) administration produced VED in rats by attenuating acetylcholine-induced endothelium-dependent relaxation in the isolated aortic ring preparation, decreasing aortic and serum nitrite/nitrate concentration, impairing endothelial integrity, and inducing vascular oxidative stress. Treatment with fish oil (2 mL/kg/day p.o., 4 weeks) markedly prevented nicotine-induced endothelial functional and structural abnormalities and oxidative stress. However, administration of GW9662, a selective inhibitor of PPARγ, to a significant degree attenuated fish oil-associated anti-oxidant action and vascular endothelial functional and structural improvements. Intriguingly, in vitro incubation of L-NAME (100 μM), an inhibitor of nitric oxide synthase (NOS), markedly attenuated fish oil-induced improvement in endothelium-dependent relaxation in the aorta of nicotine-administered rats. Nicotine administration altered the lipid profile by increasing serum total cholesterol, which was significantly prevented by fish oil treatment. The vascular protective potential of fish oil in preventing nicotine-induced VED may pertain to its additional properties (besides its lipid-lowering effect) such as activation of PPARγ and subsequent possible activation of endothelial NOS and generation of nitric oxide, and consequent reduction in oxidative stress.     

The Novel Role of Fenofibrate in Preventing Nicotine- and Sodium Arsenite-Induced Vascular Endothelial Dysfunction in the Rat

The present study investigated the effect of fenofibrate, an agonist of PPAR-α, in nicotine- and sodium arsenite-induced vascular endothelial dysfunction (VED) in rats. Nicotine (2 mg/kg/day, i.p., 4 weeks) and sodium arsenite (1.5 mg/kg/day, i.p., 2 weeks) were administered to produce VED in rats. The scanning electron microscopy study in thoracic aorta revealed that administration of nicotine or sodium arsenite impaired the integrity of vascular endothelium. Further, administration of nicotine or sodium arsenite significantly decreased serum and aortic concentrations of nitrite/nitrate and subsequently reduced acetylcholine-induced endothelium-dependent relaxation. Moreover, nicotine or sodium arsenite produced oxidative stress by increasing serum thiobarbituric acid reactive substances (TBARS) and aortic superoxide generation. However, treatment with fenofibrate (30 mg/kg/day, p.o.) or atorvastatin (30 mg/kg/day p.o., a standard agent) significantly prevented nicotine- and sodium arsenite-induced VED and oxidative stress by improving the integrity of vascular endothelium, increasing the concentrations of serum and aortic nitrite/nitrate, enhancing the acetylcholine-induced endothelium-dependent relaxation and decreasing serum TBARS and aortic superoxide anion generation. Conversely, co-administration of L-NAME (25 mg/kg/day, i.p.), an inhibitor of nitric oxide synthase, markedly attenuated these vascular protective effects of fenofibrate. The administration of nicotine or sodium arsenite altered the lipid profile by increasing serum cholesterol and triglycerides and consequently decreasing high-density lipoprotein levels, which were significantly prevented by treatment with fenofibrate or atorvastatin. It may be concluded that fenofibrate improves the integrity and function of vascular endothelium, and the vascular protecting potential of fenofibrate in preventing the development of nicotine- and sodium arsenite-induced VED may be attributed to its additional properties (other than lipid lowering effect) such as activation of eNOS and generation of NO and consequent reduction in oxidative stress.     

Chronic Treatment with the 21-Aminosteroid U74389F, an Inhibitor of Lipid Peroxidation, Does Not Prevent Diabetic Endothelial Dysfunction

Oxygen-derived free radicals are believed to be involved in diabetes-induced vascular complications. The role of oxygen radicals in endothelial dysfunction in diabetes is not known with certainty. In this study we tested whether inhibition of lipid peroxidation using the potent inhibitor U74389F, a 21-aminosteroid also known as lazaroid, could prevent endothelial dysfunction in diabetes. Lewis strain rats were made diabetic by intravenous injection of streptozotocin. A subgroup of diabetic animals received daily oral doses of 10 mg/kg U74389F at 72 hours post streptozotocin and throughout the 8-week duration of diabetes. Thoracic aortas were isolated and suspended in isolated tissue baths and contracted with norepinephrine. Relaxation due to the endothelium-dependent vasodilator, acetylcholine, was impaired in diabetic aorta while relaxation due to A23187 and nitroglycerin was unaltered. Chronic treatment of diabetic animals with U74389F normalized the increase in plasma lipid peroxides as assessed by thiobarbituric acid–reactive substances but did not alter serum insulin levels, blood glucose concentration, nor total glycosylated hemoglobin. Increases in aortic catalase activity resulting from diabetes was not altered by U74389F. Despite reductions in lipid peroxides, U74389F did not prevent the diabetes-induced impairment in endothelium-dependent relaxation caused by acetylcholine. These data suggest that other pathways that are antecedent to lipid peroxidation may be responsible for endothelial dysfunction in diabetes.     

Irisin improves endothelial function in type 2 diabetes through reducing oxidative/nitrative stresses

Vascular complications are the major causes of death in patients with diabetes, and endothelial dysfunction is the earliest event in vascular complications of diabetes. It has been reported that plasma irisin level is significantly reduced in patients with type 2 diabetic patients. The present study aimed to investigate whether irisin improved endothelial function in type 2 diabetes as well as the underlying mechanisms. The type 2 diabetes model was established by feeding C57BL/6 mice with high-fat diet. The type 2 diabetic mice exhibited reduced serum irisin level and impaired endothelial function. Irisin treatment (0.5 mg/kg/d) for two weeks improved vascular function based on the evaluation of endothelium-dependent vasorelaxation and p-VASP levels. To investigate the direct endothelial protective effects of irisin, diabetic aortic segments were incubated with irisin (1 μg/ml) ex vivo. Exposure to irisin improved endothelium-dependent vasorelaxation of diabetic aortas. Mechanically, the diabetic aortic segments exhibited increased oxidative/nitrative stresses. Irisin reduced the diabetes-induced oxidative/nitrative stresses evidenced by reducing overproduction of superoxide and peroxynitrite, and down-regulation of iNOS and gp91^phox. To further investigate the protective effects of irisin on endothelial cells and the underlying mechanisms, human umbilical vein endothelial cells (HUVECs) cultured in high-glucose/high-fat (HG/HF) medium were pre-incubated with irisin. Irisin (1 μg/ml) reduced the oxidative/nitrative stresses and apoptosis induced by HG/HF in HUVECs probably via inhibiting activation of PKC-β/NADPH oxidase and NF-κB/iNOS pathways. Taken together, irisin alleviates endothelial dysfunction in type 2 diabetes partially via reducing oxidative/nitrative stresses through inhibiting signaling pathways implicating PKC-β/NADPH oxidase and NF-κB/iNOS, suggesting that irisin may be a promising molecule for the treatment of vascular complications of diabetes.     

Long-term treatment in vivo with NOX-101, a scavenger of nitric oxide, prevents diabetes-induced endothelial dysfunction

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) 41: 1220–1226] Received: 12 January 1998 and in revised form: 9 May 1998     

Effects of erythritol on endothelial function in patients with type 2 diabetes mellitus: a pilot study

Sugar substitutes are important in the dietary management of diabetes mellitus. Erythritol is a non-caloric dietary bulk sweetener that reverses endothelial dysfunction in diabetic rats. We completed a pilot study to examine the effects of erythritol on vascular function in patients with type 2 diabetes mellitus. Participants (n = 24) consumed erythritol 36 g/day as an orange-flavored beverage for 4 weeks and a single dose of 24 g during the baseline and final visits. We assessed vascular function before and after acute (2 h) and chronic (4 weeks) erythritol consumption. Acute erythritol improved endothelial function measured by fingertip peripheral arterial tonometry (0.52 ± 0.48 to 0.87 ± 0.29 au, P = 0.005). Chronic erythritol decreased central pulse pressure (47 ± 13 to 41 ± 9 mmHg, P = 0.02) and tended to decrease carotid-femoral pulse wave velocity (P = 0.06). Thus, erythritol consumption acutely improved small vessel endothelial function, and chronic treatment reduced central aortic stiffness. Erythritol may be a preferred sugar substitute for patients with diabetes mellitus.     

Antioxidants attenuate early up regulation of retinal vascular endothelial growth factor in streptozotocin-diabetic rats

Aims/hypothesis: A strong positive correlation has been found between lipid peroxidation product and vascular endothelial growth factor concentrations in the vitreous of patients with proliferative diabetic retinopathy. To establish a causal relation between diabetes-associated enhanced oxidative stress and vascular endothelial growth factor production, we evaluated two antioxidants, dl-α-lipoic acid and taurine, on retinal vascular endothelial growth factor protein and mRNA expression and on parameters of oxidative stress in streptozotocin-diabetic rats. Methods: Our experiments were on control rats and streptozotocin-diabetic rats with a 6-week duration of diabetes, treated with or without dl-α-lipoic acid (100 mg · kg^–1· d^–1, i. p.) or taurine (1 % in the diet) starting from induction of diabetes. Vascular endothelial growth factor protein in retinal homogenates was assessed by sandwich ELISA with an affinity-purified polyclonal antibody and vascular endothelial growth factor mRNA by ribonuclease protection assay. Retinal lipid peroxidation products i. e. malondialdehyde plus 4-hydroxyalkenals were quantified with n-methyl-2-phenylindole. Retinal reduced and oxidized glutathione, ascorbate, dehydroascorbate, and sorbitol pathway intermediates were measured spectrofluorometrically, and taurine by reverse-phase HPLC. Results: Vascular endothelial growth factor protein concentration (means ± SD) was increased in diabetic rats compared with control rats (33 ± 7 vs 19 ± 5 pg/mg total protein, p < 0.01) This increase was attenuated by taurine (26 ± 8, p < 0.05) and prevented by dl-α-lipoic acid (21 ± 4, p < 0.01). Vascular endothelial growth factor mRNA abundance was reduced by 1.4-fold in diabetic rats compared with control rats and this decrease was attenuated but not completely prevented by both antioxidants. Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats compared with control rats, and both antioxidants arrested accumulation of lipid peroxidation products. Taurine, reduced glutathione, oxidized glutathione, ascorbate, dehydroascorbate and sorbitol pathway intermediate concentrations as well as oxidized glutathione/reduced glutathione and dehydroascorbate/ascorbate ratios were similar in control and diabetic rats treated with or without taurine. Conclusion/interpretation: Oxidative stress is directly involved in up regulation of vascular endothelial growth factor protein in the retina during early diabetes. [Diabetologia (2001) 44: 1102–1110] Received: 29 December 2001 and in revised form: 21 May 2001     

Doxycycline Ameliorates Vascular Endothelial and Contractile Dysfunction in the Thoracic Aorta of Diabetic Rats

Studies have shown that tetracycline class antibiotics exhibit an ameliorating action with its antioxidant property on increased oxidative stress in tissues, including heart. Since endothelial vascular dysfunction in diabetes is associated with increased oxidative stress and prevented with antioxidants, herein, we aimed to test a hypothesis whether a low-dose doxycycline treatment of diabetic rats for 4 weeks can ameliorate endothelial vascular dysfunction of thoracic aortas. Results of the present study shows that both direct and alpha receptor-mediated contractile responses as well as endothelium-dependent and endothelium-independent vasodilatory responses were preserved with low-dose doxycycline treatment (30 μmol/kg, daily; for 4 weeks) compared with untreated diabetic group. Furthermore, doxycycline treatment normalized increased lipid peroxidation and cellular glutathione level measured in plasma and prevented diabetes-induced impaired body weight gain without significant effect on high blood glucose level. Increased membrane protein level of caveolin-1, elevated ratio of PKC in particulate and cytosolic fraction, and increased protein level of cytosolic endothelin-1 in diabetic rats were also significantly prevented with doxycycline treatment. Moreover, diabetes-induced another type of oxidative stress markers in rats, matrix metalloproteinases, MMP-2, and MMP-9 were also normalized with doxycycline treatment in blood. Taken together, our data address that amelioration and/or prevention of vascular endothelial and contractile dysfunction by doxycycline is accompanied by a clear reduction in oxidative stress markers of diabetes, which provides evidence for doxycycline’s potential antioxidant action as a therapeutic agent for amelioration and/or prevention of vascular disorders in diabetic subjects.     

Effect of bis (maltolato) oxovanadium (BMOV) in uric acid and sodium arsenite-induced vascular endothelial dysfunction in rats

The study has been designed to investigate the effect of BMOV, a protein tyrosine phosphatase (PTPase) inhibitor in uric acid and sodium arsenite-induced vascular endothelial dysfunction (VED). Uric acid (150 mg kg(-)(1) day(-)(1), i.p., 3 weeks) and sodium arsenite (1.5 mg kg(-)(1) day(-)(1), i.p., 2 weeks) were administered to produce VED in rats. VED was assessed by employing isolated aortic ring preparation, electron microscopy of thoracic aorta and estimating serum concentration of nitrite/nitrate. Further, serum thiobarbituric acid reactive substances (TBARS) and aortic production of superoxide anion were estimated to assess oxidative stress. Uric acid and sodium arsenite were noted to produce VED by attenuating acetylcholine-induced endothelium dependent relaxation, impairing the integrity of vascular endothelial lining, decreasing serum nitrite/nitrate concentration and increasing serum TBARS and aortic superoxide anion generation which were significantly attenuated by BMOV (0.2 mg ml(-)(1) day(-)(1) in drinking water) or atorvastatin (30 mg kg(-)(1) day(-)(1)p.o., a standard agent). However, these ameliorative effects of BMOV have been prevented by N-omega-nitro-L-arginine methyl ester (L-NAME) (25 mg kg(-)(1) day(-)(1), i.p.), an inhibitor of NOS and glibenclamide (5 mg kg(-)(1) day(-)(1), i.p.), a blocker of ATP-sensitive K(+) channel. It may be concluded that BMOV-induced inhibition of PTPase may activate eNOS by opening of ATP-sensitive K(+) channels and consequently decrease oxidative stress to prevent uric acid and sodium arsenite-induced vascular endothelial dysfunction.     

不同HO-1表达水平对糖尿病模型大鼠血管舒张功能及NOS的影响

目的 探讨改变血红素加氧酶-1(HO-1)表达水平对糖尿病(DM)大鼠血管舒张功能的影响及与一氧化氮合酶(NOS)/一氧化氮(NO)的关系.方法 以链脲佐菌素(STZ)诱导DM大鼠模型.SD大鼠分成4组:对照组、DM组、正铁血红素(HO-1诱导剂)组、锌原卟啉(HO-1抑制剂)组.应用离体血管张力检测技术观察胸主动脉舒张功能变化;RT-PCR法及比色法分别检测血管组织和血清中诱生型NOS(iNOS)及内皮型NOS(eNOS)的表达和NO含量.结果 与DM组相比,正铁血红素组血管环对乙酰胆碱舒张百分率有所提高,而锌原卟啉组血管舒张反应继续下降.应用正铁血红素可在提高DM大鼠血管和血清eNOS表达的同时降低iNOS/NO表达;而锌原卟啉组血清中iNOS活性及其在血管组织表达均增高.结论 提高HO-1的表达水平有益于改善DM大鼠血管舒张反应失调,这种保护作用与抑制iNOS/NO的生成、上调eNOS表达水平有关.     

Effects of chronic exercise on endothelial dysfunction and insulin signaling of cutaneous microvascular in streptozotocin-induced diabetic rats.

BACKGROUND: Abnormalities of the modulatory roles played by the endothelium and/or smooth muscle may be critical and initiating factors in the development of diabetic vascular disease. Decreased phosphatidylinositol 3-kinase (PI3-K)/Akt pathway activity and impaired nitric oxide production through this pathway may play pivotal roles in the diabetes-induced vascular dysfunction. Several findings have demonstrated that exercise training has therapeutic and protective effects in type 1 diabetes and could correct endothelial dysfunction. The molecular mechanisms, however, are only partially understood. METHOD: Male Wistar rats (220+/-10 g, N=60) were made diabetic by streptozotocin (60 mg/kg, subcutaneously). After 1 week of diabetes induction, animals were submitted to exercise training for 10 weeks on a treadmill. To characterize cutaneous microvascular responses by laser Doppler flowmetery, animals were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg) and placed on a heating pad. A rectal thermometer was inserted and body temperature was maintained at 37+/-0.5 degrees C. A tracheotomy was performed to minimize respiratory difficulties. Systemic arterial blood pressure and heart rate were measured by using a tail-cuff during assessment of cutaneous blood flow. RESULTS: (i) Acetylcholine-induced cutaneous perfusion were increased significantly by training in the diabetic groups; (ii) Cutaneous microvascular responses to sodium nitroprusside did not alter in control and diabetic animals by training; and (iii) Local microinjection of insulin increased cutaneous blood flow in trained diabetic and trained control rats compared with age-matched sedentary diabetic and sedentary control normal rats. The administration of wortmannin (PI3K inhibitor) and N-nitro-L-arginine ( nitric oxide synthase inhibitor) before insulin, however, attenuated the increase in cutaneous blood flow in trained diabetic and normal rats. CONCLUSIONS: Chronic exercise improved endothelium-dependent dilatation and potentiated insulin vascular function, possibly by PI3-kinase pathway in diabetic rats.     

Prevention of endothelial dysfunction in streptozotocin-induced diabetic rats by gliclazide treatment

The aim of the present work was to analyze whether the oral hypoglycemic drug gliclazide affects diabetic endothelial dysfunction in streptozotocin-induced diabetic rats. Gliclazide was compared with glibenclamide, ascorbic acid, and aminoguanidine. An insulin-dependent model of diabetes was selected to exclude insulin-releasing effects of the drugs. Both in isolated aortic segments and mesenteric microvessels, endothelium-dependent relaxation evoked by acetylcholine (ACh, 1 nM to 10 microM) was significantly reduced in vessels from diabetic animals. This impairment was reversed when the segments were previously incubated with 100 U/ml superoxide dismutase. When streptozotocin-induced diabetic rats were orally treated from the time of diabetes induction with gliclazide (10 mg/kg) or ascorbic acid (250 mg/kg), ACh-induced endothelium-dependent relaxation was well preserved both in aortic segments and mesenteric microvessels. In addition, the impaired vasodilatation to exogenous nitric oxide (NO) in aortic segments was also improved in gliclazide-treated diabetic rats. On the other hand, oral treatment with glibenclamide (1 and 10 mg/kg) or aminoguanidine (250 mg/kg) did not produce significant improvements in diabetic endothelial dysfunction. We conclude that gliclazide reverses the endothelial dysfunction associated with diabetes. This effect appears to be due not to the metabolic actions of the drug but rather to its antioxidant properties, as it can be mimicked by other antioxidants. We propose that the mechanism involved is the inactivation of reactive oxygen species, which are increased in diabetes probably as a result of increased early protein glycosylation products, such as glycosylated hemoglobin (HbA(1c)). These effects of gliclazide are not shared by other oral hypoglycemic agent such as glibenclamide, or by blockade of advanced glycosylation end product (AGE) generation with aminoguanidine.     

Genetic deletion of p66(Shc) adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress

Increased production of reactive oxygen species (ROS) and loss of endothelial NO bioavailability are key features of vascular disease in diabetes mellitus. The p66(Shc) adaptor protein controls cellular responses to oxidative stress. Mice lacking p66(Shc) (p66(Shc-/-)) have increased resistance to ROS and prolonged life span. The present work was designed to investigate hyperglycemia-associated changes in endothelial function in a model of insulin-dependent diabetes mellitus p66(Shc-/-) mouse. p66(Shc-/-) and wild-type (WT) mice were injected with citrate buffer (control) or made diabetic by an i.p. injection of 200 mg of streptozotocin per kg of body weight. Streptozotocin-treated p66(Shc-/-) and WT mice showed a similar increase in blood glucose. However, significant differences arose with respect to endothelial dysfunction and oxidative stress. WT diabetic mice displayed marked impairment of endothelium-dependent relaxations, increased peroxynitrite (ONOO(-)) generation, nitrotyrosine expression, and lipid peroxidation as measured in the aortic tissue. In contrast, p66(Shc-/-) diabetic mice did not develop these high-glucose-mediated abnormalities. Furthermore, protein expression of the antioxidant enzyme heme oxygenase 1 and endothelial NO synthase were up-regulated in p66(Shc-/-) but not in WT mice. We report that p66(Shc-/-) mice are resistant to hyperglycemia-induced, ROS-dependent endothelial dysfunction. These data suggest that p66(Shc) adaptor protein is part of a signal transduction pathway relevant to hyperglycemia vascular damage and, hence, may represent a novel therapeutic target against diabetic vascular complications.     

Hypoadiponectinemia is associated with impaired endothelium-dependent vasodilation

Adiponectin may have an antiatherogenic effect by reducing endothelial activation. We hypothesized that plasma adiponectin levels were correlated with endothelial function. Plasma adiponectin level was determined by an in-house RIA assay using a rabbit polyclonal antibody in 73 type 2 diabetic patients and 73 controls. Endothelium-dependent and independent vasodilation of the brachial artery was measured by high-resolution vascular ultrasound. Plasma adiponectin level was lower in diabetic patients than in controls (4.73 +/- 1.96 vs. 7.69 +/- 2.80 microg/ml, respectively; P < 0.001), and they also had impaired endothelium-dependent (5.6 +/- 3.6 vs. 8.6 +/- 4.5%, respectively; P < 0.001) and -independent vasodilation (13.3 +/- 4.9 vs. 16.5 +/- 5.6%, respectively; P < 0.001). Plasma adiponectin correlated with endothelium-dependent vasodilation in controls (P = 0.02) and diabetic patients (P = 0.04). On general linear-model univariate analysis, brachial artery diameter, the presence of diabetes, plasma adiponectin, and high-density lipoprotein were significant independent determinants of endothelium-dependent vasodilation. In vitro experiments showed that endothelial cells expressed adiponectin receptors, and adiponectin increased nitric oxide production in human aortic endothelial cells. In conclusion, low plasma adiponectin level is associated with impaired endothelium-dependent vasodilation, and the association is independent of diabetes mellitus. Adiponectin may act as a link between adipose tissue and the vasculature.     

Endothelial dysfunction in type 2 diabetes: effect of antioxidants.

Individuals with insulin resistance and diabetes mellitus have increased cardiovascular morbidity and mortality, caused in part by vascular complications. Endothelial dysfunction has been implicated in the pathogenesis of vascular diabetic disease. This abnormal function of the vasculature precedes cardiovascular disease and is associated with impaired endothelium-dependent vasorelaxation. The main etiology of the increased mortality and morbidity of type 2 diabetic patients is atherosclerosis. Increased production of free radicals is associated with the pathophysiology of diabetes, resulting in oxidative damage to lipids and proteins. Reduction of oxidative stress in diabetic patients may delay the onset of atherogenesis and the appearance of micro- and macrovascular complications. Alpha-lipoic acid (LA) is a multifunctional antioxidant that has been shown to have beneficial effects on polyneuropathy and on markers of oxidative stress in various tissues. This study was conducted to investigate the effects of LA on endothelial function in diabetic and hyperlipidemic animal models. Carbohydrate and lipid metabolism, endothelial function, plasma malondialdehyde (MDA) and urinary 8-hydroxydeoxyguanosine (8-OHdG) were assessed in non-diabetic controls (Wistar rats), untreated diabetic Goto-Kakizaki (GK) rats and, atherogenic diet (AD)-fed GK rats (fed with atherogenic diet only, treated with alpha-lipoic acid and treated with vehicle, for 3 months). AD resulted in a 3-fold increase in both total and non-HDL serum cholesterol levels and in a 2-fold increase triglyceride levels while endothelial function was significantly reduce MDA and 8-OHdG levels were higher in the GK and GK hyperlipidemic groups and were completely reversed by the antioxidant. Hyperlipidemic GK diabetic rats showed significantly reduced endothelial function that was partially improved with LA. Furthermore, lipoic acid significantly reduced serum cholesterol levels, without lowering HDL cholesterol. Alpha-lipoic acid supplementation represents an achievable adjunct therapy to improve endothelial function and reduce oxidative stress, factors that are implicated in the pathogenesis of atherosclerosis in diabetes.     

Endothelial dysfunction: Possible mechanisms and ways of correction

BACKGROUND: Endothelial function is impaired in atherosclerosis, hypertension, diabetes and aging, and this may be associated with an attenuated ability of endothelial cells to generate nitric oxide (NO). OBJECTIVES: To evaluate possible alterations in endothelium-dependent relaxation in rat aortic rings, the activity of constitutive NO synthase and endothelial electrical responses to acetylcholine (Ach) in rat aorta, and the effect of one month of treatment with the angiotensin-converting enzyme inhibitor enalapril on endothelial function. METHODS: Endothelial membrane potential was measured in excised rat aorta using the perforated patch-clamp technique. Enzyme activity was determined by measuring the rate of formation of L-citrulline from L-arginine. RESULTS: In old rats and rats with experimental diabetes, the relaxation response to Ach and the activity of constitutive NO synthase were significantly depressed compared with control rats, and the endothelial resting membrane potential was significantly depolarized (-32.7+/-0.8 mV and -28.4+/-3.1 mV, respectively) compared with the control rats (-42.9+/-0.6 mV). The membrane potential attained during peak hyper-polarization to Ach in the arteries of diabetic and old rats (-57.6+/-1.1 mV and -55.7+/-2.1 mV, respectively) did not reach the level attained in the arteries of control rats (-63.1+/-0.6 mV). Enalapril treatment restored the relaxation response to Ach and increased the activity of constitutive NO synthase in aortic rings from diabetic and old rats. CONCLUSIONS: Altered electrical properties of endothelial cells and attenuated NO synthase activity underpin the suppressed relaxation to Ach in aging and experimental diabetes. Enalapril treatment improves endothelium-dependent relaxation and the activity of constitutive NO synthase.     

Ramipril prevents hypersensitivity to phenylephrine in aorta from streptozotocin-induced diabetic rats

Summary This study investigated the protective effect of the angiotensin converting enzyme inhibitor, ramipril, on endothelium-dependent responses in arteries from control (CON) and streptozotocin-induced (STZ) diabetic rats. Three hypotheses were tested: 1) there is an endothelium-dependent component to the increased alpha-adrenergic responsiveness characteristic of diabetes; 2) endothelium-dependent, acetylcholine-induced relaxation is attenuated in aorta from diabetic rats; and 3) ramipril (3 mg/kg daily in the food, 12–15 weeks) will prevent functional vascular changes in diabetic rats. Vascular function was assessed in aortic rings using standard muscle bath procedures for measurement of isometric force. Sensitivity to phenylephrine was increased in aortic rings from diabetic compared to control values [pD₂ values (-log ED₅₀): CON=6.22±0.12, STZ=7.54±0.11), and removal of the endothelium (-Endo) increased phenylephrine sensitivity (CON-Endo=7.40±0.11, STZ-Endo=8.32±0.18). The magnitude of the shift in responsiveness following endothelium removal was greatest in control rats. Ramipril treatment (Ram) partially normalized phenylephrine responsiveness in intact (STZ + Ram=6.55±0.11) and denuded (STZ-Endo + Ram=7.75±0.10) vessels. Vasodilatation to acetylcholine and nitroglycerin was not altered in diabetic rats nor was it affected by ramipril treatment. Diabetes increases contractile sensitivity to phenylephrine but not to vasodilators and chronic ramipril treatment prevents this increase in contractile sensitivity. Ramipril treatment did not alter the hyperglycaemic condition induced by streptozotocin. The changes in phenylephrine sensitivity appear to involve an endothelial and a smooth muscle component.Abbreviations STZ streptozotocin - ACE angiotensin converting enzyme - EC₅₀ agonist concentration resulting in a half maximal contraction - NO nitric oxide     

Effect of quercetin on altered vascular reactivity in aortas isolated from streptozotocin-induced diabetic rats

The present work examined ex vivo the acute effect of quercetin on diabetic rat aortic ring reactivity in response to endothelium-dependent (acetylcholine, ACh) and endothelium-independent (sodium nitroprusside, SNP) relaxants, and to the alpha(1)-adrenergic agonist phenylephrine (PE). Responses were compared to those of aortic rings from age- and sex-matched euglycemic rats. Compared to euglycemic rat aortic rings, diabetic rings showed less relaxation in response to ACh and SNP, and greater contraction in response to PE. Pretreatment with quercetin (10microM, 20min) increased ACh-induced relaxation and decreased PE-induced contraction in diabetic, but did not affect euglycemic rat aortic ring responses. Following pretreatment with the nitric oxide synthase inhibitor Nomega-nitro-l-arginine methyl ester (l-NAME, 10microM), quercetin reduced PE-induced contractions in both aortic ring types, although l-NAME attenuated the reduction in the diabetic rings. Quercetin did not alter SNP vasodilatory effects in either ring type compared to their respective controls. These findings indicate that quercetin acutely improved vascular responsiveness in blood vessels from diabetic rats, and that these effects were mediated, at least in part, by enhanced endothelial nitric oxide bioavailability. These effects of quercetin suggest the possible beneficial effects of quercetin in vivo in experimental diabetes and possibly in other cardiovascular diseases.     

Influence of mulberry (Morus indica L.) leaves on antioxidants and antioxidant enzymes in STZ-diabetic rats

To assess the influence of mulberry (Morus indica L.) leaves on antioxidants and antioxidant enzymes in STZ- diabetic rats as the leaves of mulberry (Morus indica L.) of Moraceae, are reported to be rich in a no. of bioactive principles i.e. antioxidant vitamins, flavonoids and moracins that can fight against oxidative stress in diabetes. STZ-diabetic rats were treated with dried mulberry leaves incorporated in the feed at 25 % level (as per dose response) for 8 weeks in standard experimental conditions in comparison with diabetic glibenclamide–treated and diabetic control rats. At the end of experimental period, fasting blood glucose, serum non-enzymatic antioxidants, hepatic lipid peroxidation and antioxidant enzymes were assayed in all the experimental groups. Hyperglycemia, a 274 % (P?<?0.01) rise in fasting blood glucose and increased oxidative stress as shown by a two fold increase in lipid peroxidation in hepatic tissue, decreased serum non enzymatic antioxidants viz. vit.C (51 %) and E (47 %), significantly decreased activities of hepatic antioxidant enzymes such as glucose-6-phosphate dehydrogenase (43 %), glutathione peroxidase (41 %) and superoxide dismutase (44 %) and significantly increased activity of catalase (39 %) in STZ-diabetic rats were ameliorated by mulberry leaves which is evidenced by significant fall in fasting glucose, and lipid peroxidation, rise in antioxidants as well as the activities of defense enzymes and decrease in the activity of catalase while the antidiabetic drug, glibenclamide showed lesser effects than mulberry leaves. Mulberry leaves protected STZ-diabetic rats against oxidative stress by improving antioxidants and the activities of defense enzymes and controlling hyperglycemia and lipid peroxidation in a better way than the drug.     

Increased vascular endothelial growth factor serum concentrations may help to identify patients with onset of type 1 diabetes during childhood at risk for developing persistent microalbuminuria

This study was designed to evaluate whether vascular endothelial growth factor serum concentrations may identify adolescents with onset of type 1 diabetes during childhood at greater risk to develop persistent microalbuminuria and incipient diabetic nephropathy. In January 1989, vascular endothelial growth factor serum levels were measured in 101 normoalbuminuric diabetic children and adolescents (aged 7-14.9 yr; onset of diabetes before age 18 yr; duration of diabetes >7 yr). Participants were clinically examined at baseline and annually thereafter. Vascular endothelial growth factor serum concentrations were measured every year during the 8-yr follow-up period. Over 8 yr, 11 of 101 patients (10.9%) developed persistent microalbuminuria; no patient developed overt nephropathy. The risk of developing microalbuminuria was higher in children with increased vascular endothelial growth factor serum levels (using 160 pg/ml as the arbitrary cut-off point; group 1) compared with those with normal vascular endothelial growth factor serum levels at the beginning of the study (group 2; 19.2 vs. 2.0%; P < 0.01; sensitivity, 90.9%; specificity, 53.3%). The odds ratio for the occurrence of microalbuminuria after adjustment for confounding variables (albumin excretion rate, sex, hemoglobin A(1c), mean blood pressure, cholesterol, and triglycerides) in type 1 diabetic adolescents with elevated vascular endothelial growth factor serum levels was 4.1 (95% confidence interval, 2.0-10.9). These results suggest that vascular endothelial growth factor serum concentrations may be one of the predictors and risk factors for microalbuminuria and incipient diabetic nephropathy in adolescents and young adults with onset of diabetes during childhood. Persistently increased vascular endothelial growth factor serum levels may help to identify normotensive, normoalbuminuric patients with type 1 diabetes who are predisposed to develop persistent microalbuminuria later in life.