Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat

The transgenic (mRen2)27 (Ren2) rat overexpresses mouse renin in extrarenal tissues, causing increased local synthesis of ANG II, oxidative stress, and hypertension. However, little is known about the role of oxidative stress induced by the tissue renin-angiotensin system (RAS) as a contributing factor in pulmonary hypertension (PH). Using male Ren2 rats, we test the hypothesis that lung tissue RAS overexpression and resultant oxidative stress contribute to PH and pulmonary vascular remodeling. Mean arterial pressure (MAP), right ventricular systolic pressure (RVSP), and wall thickness of small pulmonary arteries (PA), as well as intrapulmonary NADPH oxidase activity and subunit protein expression and reactive oxygen species (ROS), were compared in age-matched Ren2 and Sprague-Dawley (SD) rats pretreated with the SOD/catalase mimetic tempol for 21 days. In placebo-treated Ren2 rats, MAP and RVSP, as well as intrapulmonary NADPH oxidase activity and subunits (Nox2, p22phox, and Rac-1) and ROS, were elevated compared with placebo-treated SD rats (P < 0.05). Tempol decreased RVSP (P < 0.05), but not MAP, in Ren2 rats. Tempol also reduced intrapulmonary NADPH oxidase activity, Nox2, p22phox, and Rac-1 protein expression, and ROS in Ren2 rats (P < 0.05). Compared with SD rats, the cross-sectional surface area of small PA was 38% greater (P < 0.001) and luminal surface area was 54% less (P < 0.001) in Ren2 rats. Wall surface area was reduced and luminal area was increased in tempol-treated SD and Ren2 rats compared with untreated controls (P < 0.05). Collectively, the results of this investigation support a seminal role for enhanced tissue RAS/oxidative stress as factors in development of PH and pulmonary vascular remodeling.     

Tempol improves renal hemodynamics and pressure natriuresis in hyperthyroid rats

Hyperthyroidism in rats is associated with increased oxidative stress. These animals also show abnormal renal hemodynamics and an attenuated pressure-diuresis-natriuresis (PDN) response. We analyzed the role of oxidative stress as a mediator of these alterations by examining acute effects of tempol, a superoxide dismutase mimetic. The effects of increasing bolus doses of tempol (25-150 micromol/kg) on mean arterial pressure (MAP), renal vascular resistance (RVR), and cortical (CBF) and medullary (MBF) blood flow were studied in control and thyroxine (T4)-treated rats. In another experiment, tempol was infused at 150 micromol.kg(-1).h(-1) to analyze its effects on the glomerular filtration rate (GFR) and on PDN response in these animals. Tempol dose dependently decreased MAP and RVR and increased CBF and MBF in control and T4-treated rats, but the T4 group showed a greater responsiveness to tempol in all of these variables. The highest dose of tempol decreased RVR by 13.5 +/- 2.1 and 5.5 +/- 1.2 mmHg.ml(-1).min(-1) in hyperthyroid (P < 0.01) and control rats, respectively. GFR was not changed by tempol in controls but was significantly increased in the hyperthyroid group. Tempol did not change the absolute or fractional PDN responses of controls but significantly improved those of hyperthyroid rats, although without attaining normal values. Tempol increased the slopes of the relationship between renal perfusion pressure and natriuresis (T4+tempol: 0.17 +/- 0.05; T4: 0.09 +/- 0.03 microeq.min(-1).g(-1).mmHg(-1); P < 0.05) and reduced 8-isoprostane excretion in hyperthyroid rats. These results show that antioxidant treatment with tempol improves renal hemodynamic variables and PDN response in hyperthyroid rats, indicating the participation of an increased oxidative stress in these mechanisms.     

Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress

Tempol catalyzes the formation of H(2)O(2) from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H(2)O(2) by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H(2)O(2). Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-46619, or norepinephrine. During U-46619, tempol caused a transient dilation (22 +/- 2%), whereas NBT was ineffective (2 +/- 1%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K(+) channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 +/- 7%. Tempol, but not NBT, increased H(2)O(2) in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (184 +/- 20%) of mouse cremaster arterioles exposed to angiotensin II for 30 min, which was not seen with NBT (9 +/- 4%). The vasodilation to tempol was reduced by 68 +/- 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-46619 or angiotensin II is mediated in part via production of H(2)O(2) and is largely independent of the endothelium and potassium channels.     

Tempol improves vascular function in the mesenteric vascular bed of senescent rats

Ageing is associated with structural and functional alterations of the vasculature. The nature of age-related vascular disorders is not completely understood. Oxidative stress is hypothesized to play a crucial role in the pathophysiology of vascular complications. We investigated the effects of chronic treatment with the superoxide dismutase mimetic tempol (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl) on vascular function in the mesenteric vasculature of aged rats. Young (3 weeks) and old (40 weeks) Sprague-Dawley rats were treated with tempol (1 mM in drinking water) or vehicle for 3 weeks. Arterial blood pressure was slightly, but significantly, higher in old than in young rats. Tempol had no effect on arterial blood pressure. The vasoconstrictor responses to norepinephrine (NE) and serotonin (5-HT) were exaggerated in the mesenteric vascular bed (MVB) removed from old rats. Vasodilator responses to acetylcholine (ACh), papaverine (PPV), and isoprenaline (ISO) were reduced in the MVB of old rats in comparison with young rats. Chronic treatment of old rats with tempol normalized their responses to NE and 5-HT. The dilator responses to ACh, PPV, and ISO were similar between old rats receiving tempol and young rats. The present findings suggest that oxidative stress contributes to vascular dysfunction in the mesentery of old rats. The vasculoprotective effects of tempol remain to be elucidated.     

Acute antioxidant supplementation and skeletal muscle vascular conductance in aged rats: role of exercise and fiber type

Age-related increases in oxidative stress contribute to impaired skeletal muscle vascular control. However, recent evidence indicates that antioxidant treatment with tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) attenuates flow-mediated vasodilation in isolated arterioles from the highly oxidative soleus muscle of aged rats. Whether antioxidant treatment with tempol evokes similar responses in vivo at rest and during exercise in senescent individuals and whether this effect varies based on muscle fiber type composition are unknown. We tested the hypothesis that redox modulation via acute systemic tempol administration decreases vascular conductance (VC) primarily in oxidative hindlimb locomotor muscles at rest and during submaximal whole body exercise (treadmill running at 20 m/min, 5% grade) in aged rats. Eighteen old (25-26 mo) male Fischer 344 x Brown Norway rats were assigned to either rest (n = 8) or exercise (n = 10) groups. Regional VC was determined via radiolabeled microspheres before and after intra-arterial administration of tempol (302 μmol/kg). Tempol decreased mean arterial pressure significantly by 9% at rest and 16% during exercise. At rest, similar VC in 26 out of 28 individual hindlimb muscles or muscle parts following tempol administration compared with control resulted in unchanged total hindlimb muscle VC (control: 0.18 ± 0.02; tempol: 0.17 ± 0.05 ml·min(-1)·100 g(-1)·mmHg(-1); P > 0.05). During exercise, all individual hindlimb muscles or muscle parts irrespective of fiber type composition exhibited either an increase or no change in VC with tempol (i.e., ↑11 and ?17 muscles or muscle parts), such that total hindlimb VC increased by 25% (control: 0.93 ± 0.04; tempol: 1.15 ± 0.09 ml·min(-1)·100 g(-1)·mmHg(-1); P ≤ 0.05). These results demonstrate that acute systemic administration of the antioxidant tempol significantly impacts the control of regional vascular tone in vivo presumably via redox modulation and improves skeletal muscle vasodilation independently of fiber type composition during submaximal whole body exercise in aged rats.     

Cytosolic, but not mitochondrial, oxidative stress is a likely contributor to cardiac hypertrophy resulting from cardiac specific GLUT4 deletion in mice

We hypothesized that oxidative stress may contribute to the development of hypertrophy observed in mice with cardiac specific ablation of the insulin sensitive glucose transporter 4 gene (GLUT4, G4H(-/-) ). Measurements of oxidized glutathione (GSSG) in isolated mitochondria and whole heart homogenates were increased resulting in a lower ratio of reduced glutathione (GSH) to GSSG. Membrane translocation of the p67(phox) subunit of cardiac NADPH oxidase 2 (NOX2) was markedly increased in G4H(-/-) mice, suggesting elevated activity. To determine if oxidative stress was contributing to cardiac hypertrophy, 4-week-old control (Con) and G4H(-/-) mice were treated with either tempol (T, 1 mm, drinking water), a whole cell antioxidant, or Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP, 10 mg·kg(-1) , intraperitoneally), a mitochondrial targeted antioxidant, for 28 days. Tempol attenuated cardiac hypertrophy in G4H(-/-) mice (heart : tibia, Con 6.82 ± 0.35, G4H(-/-) 8.83 ± 0.34, Con + T 6.82 ± 0.46, G4H(-/-) + T 7.57 ± 0.3), without changing GSH : GSSG, glutathione peroxidase 4 or membrane translocation of the p67(phox) . Tempol did not modify phosphorylation of glycogen synthase kinase 3β or thioredoxin-2. In contrast, MnTBAP lowered mitochondrial GSSG and improved GSH : GSSG, but did not prevent hypertrophy, indicating that mitochondrial oxidative stress may not be critical for hypertrophy in this model. The ability of tempol to attenuate cardiac hypertrophy suggests that a cytosolic source of reactive oxygen species, probably NOX2, may contribute to the hypertrophic phenotype in G4H(-/-) mice.     

Angiotensin II-mediated oxidative stress promotes myocardial tissue remodeling in the transgenic (mRen2) 27 Ren2 rat

Angiotensin II (ANG II) contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. ANG II stimulation of the ANG type 1 receptor (AT(1)R) generates reactive oxygen species via NADPH oxidase, which facilitates this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo AT(1)R blockade (AT(1)B) (valsartan) or superoxide dismutase/catalase mimetic (tempol) treatment in a rodent model of chronically elevated tissue levels of ANG II, the transgenic (mRen2) 27 rat (Ren2). Ren2 rats overexpress the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6-7 wk old) male Ren2 and age-matched Sprague-Dawley rats were treated with valsartan (30 mg/kg), tempol (1 mmol/l), or placebo for 3 wk. Heart tissue NADPH oxidase (NOX) activity and immunohistochemical analysis of subunits NOX2, Rac1, and p22(phox), heart tissue malondialdehyde, and insulin-stimulated protein kinase B (Akt) activation were measured. Structural changes were assessed with cine MRI, transmission electron microscopy, and light microscopy. Increases in septal wall thickness and altered systolic function (cine MRI) were associated with perivascular fibrosis and increased mitochondria in Ren2 on light and transmission electron microscopy (P < 0.05). AT(1)B, but not tempol, reduced blood pressure (P < 0.05); significant improvements were seen with both AT(1)B and tempol on NOX activity, subunit expression, malondialdehyde, and insulin-mediated activation/phosphorylation of Akt (each P < 0.05). Collectively, these data suggest cardiac oxidative stress-induced structural and functional changes are driven, in part, by AT(1)R-mediated increases in NADPH oxidase activity.     

Effects of Dipeptidyl-Peptidase 4 Inhibitor about Vascular Inflammation in a Metabolic Syndrome Model

Background

In this study, we used vidagliptin(V) to examine the role of the DDP-IV, incretin system component, in the activation of different molecular inflammatory cytokines, NF-kB and VCAM-1 to generate a microenvironment that supports cardiovascular remodeling.

Methods

Male WKY and SHR were separated into five groups: Control, FFR: WKY rats receiving a 10% (w/v) fructose solution during all 12 weeks, SHR, FFHR: SHR receiving a 10% (w/v) fructose solution during all 12 weeks and FFHR+V: (5 mg/kg per day for 6 weeks) (n = 8 each group). Metabolic variables and systolic blood pressure were measured. The TBRAS, eNOS activity, and NAD(P)H oxidase activity were estimated to evaluate oxidative stress. Cardiac and vascular remodeling were evaluated. To assess the cytokine, NF-kB and VCAM-1 immunostaining techniques were used.

Results

The FFHR experimental model presents metabolic syndrome criteria, vascular and cardiac remodeling, vascular inflammation due to increased expression of NF-kB, VCAM-1, and pro-atherogenic cytokines. Chronic treatment with V was able to reverse total or partiality of variables studied.

Conclusions

Data demonstrated an important effect of DDP-IV in reducing vascular inflammation, accompanied by a favorable reduction in metabolic and structural parameters.     

Inhibition of in vivo leishmanicidal mechanisms by tempol: nitric oxide down-regulation and oxidant scavenging

Tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) has long been known to protect experimental animals from the injury associated with oxidative and inflammatory conditions. In the latter case, a parallel decrease in tissue protein nitration levels has been observed. Protein nitration represents a shift in nitric oxide actions from physiological to pathophysiological and potentially damaging pathways involving its derived oxidants such as nitrogen dioxide and peroxynitrite. In infectious diseases, protein tyrosine nitration of tissues and cells has been taken as evidence for the involvement of nitric oxide-derived oxidants in microbicidal mechanisms. To examine whether tempol inhibits the microbicidal action of macrophages, we investigated its effects on Leishmania amazonensis infection in vitro (RAW 264.7 murine macrophages) and in vivo (C57Bl/6 mice). Tempol was administered in the drinking water at 2 mM throughout the experiments and shown to reach infected footpads as the nitroxide plus the hydroxylamine derivative by EPR analysis. At the time of maximum infection (6 weeks), tempol increased footpad lesion size (120%) and parasite burden (150%). In lesion extracts, tempol decreased overall nitric oxide products and expression of inducible nitric oxide synthase to about 80% of the levels in control animals. Nitric oxide-derived products produced by radical mechanisms, such as 3-nitrotyrosine and nitrosothiol, decreased to about 40% of the levels in control mice. The results indicate that tempol worsened L. amazonensis infection by a dual mechanism involving down-regulation of iNOS expression and scavenging of nitric oxide-derived oxidants. Thus, the development of therapeutic strategies based on nitroxides should take into account the potential risk of altering host resistance to parasite infection.     

Oxidative stress alters renal D1 and AT1 receptor functions and increases blood pressure in old rats

Aging is associated with an increase in oxidative stress and blood pressure (BP). Renal dopamine D1 (D1R) and angiotensin II AT1 (AT1R) receptors maintain sodium homeostasis and BP. We hypothesized that age-associated increase in oxidative stress causes altered D1R and AT1R functions and high BP in aging. To test this, adult (3 mo) and old (21 mo) Fischer 344 × Brown Norway F1 rats were supplemented without/with antioxidant tempol followed by determining oxidative stress markers (urinary antioxidant capacity, proximal tubular NADPH-gp91phox, and plasma 8-isoprostane), D1R and AT1R functions, and BP. The D1R and AT1R functions were determined by measuring diuretic and natriuretic responses to D1R agonist (SKF-38393; 1 μg·kg(-1)·min(-1) iv) and AT1R antagonist (candesartan; 10 μg/kg iv), respectively. We found that the total urinary antioxidant capacity was lower in old rats, which increased with tempol treatment. In addition, tempol decreased the elevated NADPH-gp91phox and 8-isoprostane levels in old rats. Systolic, diastolic, and mean arterial BPs were higher in old rats and were reduced by tempol. Although SKF-38393 produced diuresis in both adult and old rats, urinary sodium excretion (UNaV) increased only in adult rats. While candesartan increased diuresis and UNaV in adult and old rats, the magnitude of response was greater in old rats. Tempol treatment in old rats reduced candesartan-induced increase in diuresis and UNaV. Our results demonstrate that diminished renal D1R and exaggerated AT1R functions are associated with high BP in old rats. Furthermore, oxidative stress may cause altered renal D1R and AT1R functions and high BP in old rats.     

Systemic arterial pressure response to two weeks of Tempol therapy in SHR: involvement of NO, the RAS, and oxidative stress

The roles of nitric oxide (NO) and plasma renin activity (PRA) in the depressor response to chronic administration of Tempol in spontaneously hypertensive rats (SHR) are not clear. The present study was done to determine the effect of 2 wk of Tempol treatment on blood pressure [mean arterial pressure (MAP)], oxidative stress, and PRA in the presence or absence of chronic NO synthase inhibition. SHR were divided into four groups: control, Tempol (1 mmol/l) alone, nitro-L-arginine methyl ester (L-NAME, 4.5 mg x g(-1).day(-1)) alone, and Tempol + L-NAME or 2 wk. With Tempol, MAP decreased by 22%: 191 +/- 3 and 162 +/- 21 mmHg for control and Tempol, respectively (P < 0.05). L-NAME increased MAP by 16% (222 +/- 2 mmHg, P < 0.01), and L-NAME + Tempol abolished the depressor response to Tempol (215 +/- 3 mmHg, P < 0.01). PRA was not affected by Tempol but was increased slightly with L-NAME alone and 4.4-fold with L-NAME + Tempol. Urinary nitrate/nitrite increased with Tempol and decreased with L-NAME and L-NAME + Tempol. Tempol significantly reduced oxidative stress in the presence and absence of L-NAME. In conclusion, in SHR, Tempol administration for 2 wk reduces oxidative stress in the presence or absence of NO, but in the absence of NO, Tempol is unable to reduce MAP. Therefore, NO, but not changes in PRA, plays a major role in the blood pressure-lowering effects of Tempol. These data suggest that, in hypertensive individuals with endothelial damage and chronic NO deficiency, antioxidants may be able to reduce oxidative stress but not blood pressure.     

Rosiglitazone attenuates NF-κB-mediated Nox4 upregulation in hyperglycemia-activated endothelial cells

Vascular complications, a major cause of morbidity and mortality in diabetic patients, are related to hyperglycemia-induced oxidative stress. Previously, we reported that rosiglitazone (RSG) attenuated vascular expression and activity of NADPH oxidases in diabetic mice. The mechanisms underlying these effects remain to be elucidated. We hypothesized that RSG acts directly on endothelial cells to modulate vascular responses in diabetes. To test this hypothesis, human aortic endothelial cells (HAECs) were exposed to normal glucose (NG; 5.6 mmol/l) or high glucose (HG; 30 mmol/l) concentrations. Select HAEC monolayers were treated with RSG, caffeic acid phenethyl ester (CAPE), diphenyleneiodonium (DPI), small interfering (si)RNA (to NF-κB/p65 or Nox4), or Tempol. HG increased the expression and activity of the NADPH oxidase catalytic subunit Nox4 but not Nox1 or Nox2. RSG attenuated HG-induced NF-κB/p65 phosphorylation, nuclear translocation, and binding to the Nox4 promoter. Inhibiting NF-κB with CAPE or siNF-κB/p65 also reduced HG-induced Nox4 expression and activity. HG-induced H(2)O(2) production was attenuated by siRNA-mediated knockdown of Nox4, and HG-induced HAEC monocyte adhesion was attenuated by treatment with RSG, DPI, CAPE, or Tempol. These results indicate that HG exposure stimulates HAEC NF-κB activation, Nox4 expression, and H(2)O(2) production and that RSG attenuates HG-induced oxidative stress and subsequent monocyte-endothelial interactions by attenuating NF-κB/p65 activation and Nox4 expression. This study provides novel insights into mechanisms by which the thiazolidinedione peroxisome proliferator-activated receptor-γ ligand RSG favorably modulates endothelial responses in the diabetic vasculature.     

Tempol modulates changes in xenobiotic permeability and occludin oligomeric assemblies at the blood-brain barrier during inflammatory pain

Our laboratory has shown that λ-carrageenan-induced peripheral inflammatory pain (CIP) can alter tight junction (TJ) protein expression and/or assembly leading to changes in blood-brain barrier xenobiotic permeability. However, the role of reactive oxygen species (ROS) and subsequent oxidative stress during CIP is unknown. ROS (i.e., superoxide) are known to cause cellular damage in response to pain/inflammation. Therefore, we examined oxidative stress-associated effects at the blood-brain barrier (BBB) in CIP rats. During CIP, increased staining of nitrosylated proteins was detected in hind paw tissue and enhanced presence of protein adducts containing 3-nitrotyrosine occurred at two molecular weights (i.e., 85 and 44 kDa) in brain microvessels. Tempol, a pharmacological ROS scavenger, attenuated formation of 3-nitrotyrosine-containing proteins in both the hind paw and in brain microvessels when administered 10 min before footpad injection of λ-carrageenan. Similarly, CIP increased 4-hydroxynoneal staining in brain microvessels and this effect was reduced by tempol. Brain permeability to [(14)C]sucrose and [(3)H]codeine was increased, and oligomeric assemblies of occludin, a critical TJ protein, were altered after 3 h CIP. Tempol attenuated both [(14)C]sucrose and [(3)H]codeine brain uptake as well as protected occludin oligomers from disruption in CIP animals, suggesting that ROS production/oxidative stress is involved in modulating BBB functional integrity during pain/inflammation. Interestingly, tempol administration reduced codeine analgesia in CIP animals, indicating that oxidative stress during pain/inflammation may affect opioid delivery to the brain and subsequent efficacy. Taken together, our data show for the first time that ROS pharmacological scavenging is a viable approach for maintaining BBB integrity and controlling central nervous system drug delivery during acute inflammatory pain.     

Endothelial and vascular smooth muscle cell dysfunction mediated by cyclophylin A and the atheroprotective effects of melatonin

AimsThis study evaluated the role of cyclophilin A (CyPA) in early phase of atherosclerosis and also examined the atheroprotective effects of melatonin due to its antioxidant properties.Main methodsAPOE null mice at 6 and 15 weeks of age were treated with melatonin at a dose of 0.1 mg/kg/day or 10 mg/kg/day. We evaluated both histopathological alterations in endothelial and vascular smooth muscle cells by CyPA and rolling mononuclear cell expression during the early phase of atherosclerosis development.Key findingsOur study showed that CyPA expression increases and may modulate inflammatory cell adhesion and interleukin-6 expression inducing vascular smooth muscle cell migration and inflammatory cell extravasation in a time-dependent manner. Moreover, we observed an indirect atheroprotective effect of melatonin on vascular injury; it inhibited CyPA mediated inflammatory cell extravasation and oxidative stress.SignificanceThe melatonin treatment may represent a new atheroprotective approach that contributes to reducing the early phase of atherosclerosis involving the rolling of monocytes, their passage to subendothelial space and inhibition of CyPA expression.     

Antioxidant treatment reduces matrix metalloproteinase-2-induced vascular changes in renovascular hypertension

Mounting evidence indicates that structural and functional vascular changes associated with two-kidney, one-clip (2K-1C) hypertension result, at least in part, from altered activity of matrix metalloproteinases (MMPs). Because MMPs are upregulated by increased formation of reactive oxygen species (ROS), we hypothesized that antioxidant approaches could attenuate the increases in MMP-2 expression/activity and the vascular dysfunction and remodeling associated with 2K-1C hypertension. Sham-operated or 2K-1C hypertensive rats were treated with tempol 18 mg/kg/day or apocyanin 25 mg/kg/day (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and -independent relaxation. Quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin sections. Aortic and systemic ROS levels were measured using dihydroethidine and thiobarbituric acid-reactive substances, respectively. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry, and immunohistochemistry. Tempol and apocyanin attenuated 2K-1C hypertension (181 ± 20.8 and 192 ± 17.6 mm Hg, respectively, versus 213 ± 18 mm Hg in hypertensive controls; both p < 0.05) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Tempol, but not apocyanin (p > 0.05), prevented the vascular remodeling found in 2K-1C rats (all p < 0.01). Tempol was more effective than apocyanin in attenuating hypertension-induced increases in oxidative stress (both p < 0.05), MMP-2 levels, and MMP-2 activity in hypertensive rats (all p < 0.05). Our results suggest that antioxidant approaches decrease MMP-2 upregulation and attenuate the vascular dysfunction and remodeling during 2K-1C hypertension.     

Red wine polyphenols improve an established aging-related endothelial dysfunction in the mesenteric artery of middle-aged rats: role of oxidative stress

Aging is associated with blunted endothelium-dependent relaxations and vascular oxidative stress. Our previous study has indicated that daily intake of red wine polyphenols (RWPs) by young rats retards aging-related endothelial dysfunction in middle-aged rats. The aim of the present study is to determine whether intake of RWPs also improves an established endothelial dysfunction in middle-aged rats and, if so, to determine the underlying mechanism. Middle-aged rats (51 weeks) received either solvent (3% ethanol), RWPs extract (100mg/kg/day) or the antioxidant and NADPH oxidase inhibitor apocynin (100mg/kg/day) in the drinking water for 4 weeks. Vascular reactivity of mesenteric artery rings from control young (12 weeks) and middle-aged rats was assessed in organ chambers. The expression level of endothelial NO synthase (eNOS), arginase I, angiotensin II receptors (AT1R and AT2R), NADPH oxidase subunits and nitrotyrosines was assessed by immunohistochemistry, and the vascular formation of reactive oxygen species (ROS) by dihydroethidine. Aging is associated with blunted endothelium-dependent relaxations, an excessive vascular formation of ROS and peroxynitrites, and an up-regulation of eNOS, arginase I, NADPH oxidase subunits (nox-1, p22phox), and AT1R and AT2R expression. RWPs and apocynin treatments improved endothelial dysfunction, normalized oxidative stress and the expression of the different proteins in the mesenteric artery of middle-aged rats. The present findings indicate that aging is associated with blunted endothelium-dependent relaxations involving an increased oxidative stress, and that these responses are improved by the intake of RWPs or apocynin for 4weeks most likely by normalizing the expression of eNOS, arginase I, NADPH oxidase and angiotensin receptors.     

Mechanisms for suppressing NADPH oxidase in the vascular wall

Oxidative stress underlies many forms of vascular disease as well as tissue injury following ischemia and reperfusion. The major source of oxidative stress in the artery wall is an NADPH oxidase. This enzyme complex as expressed in vascular cells differs from that in phagocytic leucocytes both in biochemical structure and functions. The crucial flavin-containing catalytic subunits, Nox1 and Nox4, are not found in leucocytes, but are highly expressed in vascular cells and upregulated with vascular remodeling, such as that found in hypertension and atherosclerosis. The difference in catalytic subunits offers the opportunity to develop "vascular specific" NADPH oxidase inhibitors that do not compromise the essential physiological signaling and phagocytic functions carried out by reactive oxygen and nitrogen species. Nitric oxide and targeted inhibitors of NADPH oxidase that block the source of oxidative stress in the vasculature are more likely to prevent the deterioration of vascular function that leads to stroke and heart attack, than are conventional antioxidants. The roles of Nox isoforms in other inflammatory conditions are yet to be explored.     

人SOD1基因在大鼠血管平滑肌细胞的表达及对抗氧自由基损伤的作用

本实验构建含人铜锌超氧化物歧化酶（ｈＳＯＤ１）基因的逆转录病毒载体,将其导入离体培养的鼠血管平滑肌细胞,观察ｈＳＯＤ１基因表达及其抗氧自由基损害作用,结果表明：（１）载体构建策略和方法正确,ｈＳＯＤ１基因可在靶细胞中高效稳定表达;（２）转化ｈＳＯＤ１的ＶＳＭＣｓ可对抗大剂量氧自由基对细胞的直接损伤作用;（３）小剂量氧自由基刺激ＶＳＭＣｓ增殖,而转化ｈＳＯＤ１的ＶＳＭＣｓ增殖反应受到抑制,本研究结果