Maria Helena C. Carvalho

Vascular free radical release. Ex vivo and in vivo evidence for a flow-dependent endothelial mechanism.

Mechanisms underlying production of vascular free radicals are unclear. We hypothesized that changes in blood flow might serve as a physiological stimulus for endothelial free radical release. Intact isolated aortas from 45 rabbits were perfused with the spin trap alpha-phenyl-N-tert-butylnitrone (PBN, 20 mmol/L) and formed radical adducts detected by electron paramagnetic resonance spectroscopy (EPR). Sequential perfusion at 2, 7.5, and 12 mL/min changed cumulative vascular PBN radical adduct yields, respectively, from 3.2 +/- 0.9 to 4.1 +/- 0.7 (P < .05) and 7.0 +/- 1.5 (P < .005) pmol/mg with endothelium and from 3.6 +/- 1.6 to 3.8 +/- 1.4 and 2.2 +/- 0.8 pmol/mg without endothelium (P = NS). In endothelialized aortas, superoxide dismutase (SOD) completely blocked flow-induced free radical production, whereas inactivated SOD, indomethacin, and the nitric oxide synthetase antagonist nitro-L-arginine methyl ester (L-NAME) had no effect; relaxations to acetylcholine remained unchanged with higher flows. To assess the role of flow on in vivo radical production, femoral arterial plasma levels of the ascorbyl radical, a stable ascorbate oxidation product, were measured by direct EPR in 56 other rabbits. Ascorbyl levels were assessed at baseline (30.2 +/- 0.7 nmol/L) and at peak-induced iliac flow changes. Flow increases from 25% to 100% due to saline injections through an extracorporeal aortic loop induced significant dose-dependent increases in ascorbyl levels (n = 5). In addition, after papaverine bolus injections, flow increased by 114 +/- 8% versus baseline, and ascorbyl levels increased by 5.4 +/- 0.7 nmol/L (n = 31, P < .001); similar results occurred with adenosine, isoproterenol, or hyperemia after 30-second occlusions (P < .05, n = 4 or 5 in each group). Active SOD completely blocked papaverine-induced ascorbyl radical increase, despite preserved flow response (delta ascorbyl = 0.02 +/- 1.6 nmol/L, P = NS); inactivated SOD, catalase, indomethacin, and L-NAME had no effect. Blood flow decreases of 65% to 100% due to phenylephrine or 60-second balloon occlusions were accompanied by an average decrease of 4.4 nmol/L (P < .05) in ascorbyl levels. No change in ascorbyl signal was observed when rabbit blood alone was submitted to in vitro flow increases through a tubing circuit. Thus, increases in blood flow trigger vascular free radical generation; such a response seems to involve endothelium-derived superoxide radicals unrelated to cyclooxygenase or nitric oxide synthetase activities. This mechanism may contribute to explain vascular free radical generation in physiological or pathological circumstances.

Intrauterine undernutrition: expression and activity of the endothelial nitric oxide synthase in male and female adult offspring

OBJECTIVE Epidemiological studies suggest that intrauterine undernutrition plays an important role in the development of arterial hypertension in adulthood. In an attempt to define the mechanisms whereby blood pressure may be raised, we have hypothesized that arteries from offspring of nutritionally restricted dams exhibit abnormalities in the endothelial function and in nitric oxide synthesis. In order to investigate the existence of potential gender differences on the effects of intrauterine undernutrition, both male and female offspring of pregnant Wistar rats on normal and restricted diets were studied in adulthood. METHODS Female pregnant Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. At 14 weeks of age, the rats were used for the study of vascular reactivity, eNOS and iNOS gene expression, eNOS activity and, in the case of females, estrogen levels. RESULTS Intrauterine undernutrition induced hypertension in both male and female offspring, but hypertension was more severe in male rats. Endothelium-intact aortic rings from male and female rats in the restricted diet group exhibited increased responses to norepinephrine, decreased vasodilation to acetylcholine and unaltered responses to sodium nitroprusside in comparison to aortic rings from control rats. No gender-related differences were observed in the vascular reactivity studies. Intrauterine undernutrition promoted decreased gene expression for eNOS in aorta isolated from male, but not female, offspring, reduction in eNOS activity in both male and female offspring and impairment in synthesis of estrogen in female offspring. CONCLUSION Our data show that intrauterine undernutrition: (1) induces hypertension both in the male and female offspring, hypertension being more severe in male than in female rats; (2) alters endothelium-dependent responses in aortas from the resulting offspring. The endothelial dysfunction is associated with a decrease in activity/expression of eNOS in aortas from male offspring. The mechanism involved in altered response to ACh in female offspring might be a consequence of reduction in estrogen levels leading to reduced eNOS activity.

ETA Receptor Blockade Decreases Vascular Superoxide Generation in DOCA-Salt Hypertension

Abstract—Development and progression of end-organ damage in hypertension have been associated with increased oxidative stress. Superoxide anion accumulation has been reported in deoxycorticosterone acetate (DOCA)-salt hypertension, in which endothelin-1 plays an important role in cardiovascular damage. We hypothesized that blockade of ETA receptors in DOCA-salt rats would decrease oxidative stress. Both systolic blood pressure (SBP, 210±9 mm Hg; P <0.05) and vascular superoxide generation in vivo were increased in DOCA-salt (44.9±10.3% of ethidium bromide–positive nuclei; P <0.05) versus control uninephrectomized (UniNx) rats (118±3 mm Hg; 18.5±3%, respectively). In DOCA-salt rats, the ETA antagonist BMS 182874 (40 mg/kg per day PO) lowered SBP (170±4 versus UniNx, 120±3 mm Hg) and normalized superoxide production (21.7±6 versus UniNx, 11.9±7%). Vitamin E (200 mg/kg per day PO) decreased superoxide formation in DOCA-salt rats (18.8±7%) but did not alter SBP. Oxidative stress in nonstimulated circulating polymorphonuclear cells (PMNs) or in PMNs treated with zymosan, an inducer of superoxide release, was similar in DOCA-salt and UniNx groups. Superoxide formation by PMNs was unaffected by treatment with BMS 182874. Western blot analysis showed increased nitrotyrosine-containing proteins in mesenteric vessels from DOCA-salt compared with UniNX. Treatment with either BMS 182874 or vitamin E abolished the differences in vascular nitrotyrosine-containing proteins between DOCA-salt and UniNX. Maximal relaxation to acetylcholine was decreased in DOCA-salt aortas (75.8±4.2% versus UniNx, 95.4±1.9%, P <0.05). BMS 182874 treatment increased acetylcholine-induced relaxation in DOCA-salt aortas to 93.5±4.5%. These in vivo findings indicate that increased vascular superoxide production is associated with activation of the endothelin system through ETA receptors in DOCA-salt hypertension, in apparently blood pressure–independent fashion.

Enhanced oxidative stress as a potential mechanism underlying the programming of hypertension in utero.

Maternal undernutrition during critical periods of organ development is known to impair fetal growth and predispose to the development of adulthood diseases, such as hypertension, coronary heart disease and type II diabetes that are linked to low birth weight and are characterized by endothelial dysfunction. Increased oxidative stress, in rats submitted to intrauterine undernutrition, provides a potential explanation for the endothelial dysfunction development. The aim of this study was to determine the oxidative stress and its consequence on mesenteric arteriolar responses to vasoactive agents in offspring from diet-restricted dams. For this, female pregnant Wistar rats were fed either normal or 50% of normal intake diets, during the whole gestational period. In male offspring, arterial blood pressure was determined by the tail cuff method in anesthetized rats, mesenteric arteriolar reactivity and superoxide anion generation were studied using intravital microscopy and superoxide dismutase activity was determined in mesentery by spectrophotometric assay. Intrauterine undernutrition induced hypertension, decreased vasodilation to acetylcholine and bradykinin but did not alter the responses to sodium nitroprusside. Topical application of superoxide dismutase and superoxide dismutase mimetic manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin significantly improved the altered arteriolar responses to acetylcholine and bradykinin. A decreased superoxide dismutase activity and an increased superoxide anion concentration were observed in the offspring of diet-restricted dams. This study shows for the first time that intrauterine undernutrition enhances oxidative stress in vivo and relates this to the impaired endothelium-dependent vasodilation.

Toll-like receptor 4 contributes to blood pressure regulation and vascular contraction in spontaneously hypertensive rats

Activation of Toll-like receptors (TLR) induces gene expression of proteins involved in the immune system response. TLR4 has been implicated in the development and progression of cardiovascular diseases. Innate and adaptive immunity contribute to hypertension-associated end-organ damage, although the mechanism by which this occurs remains unclear. In the present study we hypothesize that inhibition of TLR4 decreases blood pressure and improves vascular contractility in resistance arteries from spontaneously hypertensive rats (SHR). TLR4 protein expression in mesenteric resistance arteries was higher in 15 weeks-old SHR than in same age Wistar controls or in 5 weeks-old SHR. In order to decrease activation of TLR4, 15 weeks-old SHR and Wistar rats were treated with anti-TLR4 antibody or non-specific IgG control antibody for 15 days (1µg per day, i.p.). Treatment with anti-TLR4 decreased mean arterial pressure as well as TLR4 protein expression in mesenteric resistance arteries and interleukin-6 (IL-6) serum levels from SHR when compared to SHR treated with IgG. No changes in these parameters were found in Wistar treated rats. Mesenteric resistance arteries from anti-TLR4-treated SHR exhibited decreased maximal contractile response to noradrenaline compared to IgG-treated-SHR. Inhibition of cyclooxygenase-1 (Cox) and Cox-2, enzymes related to inflammatory pathways, decreased noradrenaline responses only in mesenteric resistance arteries of SHR treated with IgG. Cox-2 expression and thromboxane A2 release were decreased in SHR treated with anti-TLR4 compared with IgG-treated-SHR. Our results suggest that TLR4 activation contributes to increased blood pressure, low grade inflammation and plays a role in the augmented vascular contractility displayed by SHR.

Synergistic effect of angiotensin-(1-7) on bradykinin arteriolar dilation in vivo.

The interaction between angiotensin [Ang-(1-7)] and bradykinin (BK) was determined in the mesentery of anesthetized Wistar rats using intravital microscopy. Topical application of BK and Ang-(1-7) induced vasodilation that was abolished by the BK B2 receptor antagonist HOE-140 and the Ang-(1-7) antagonist A-779, respectively. BK (1 pmol)-induced vasodilation, but not SNP and ACh responses, was potentiated by Ang-(1-7) 10 pmol and 100 pmols. The effect of 100 pmol of Ang-(1-7) on BK-induced vasodilation was abolished by A-779, indomethacin, and L-nitroarginine methyl esther, whereas losartan was without effect. Enalaprilat treatment enhanced the BK- and Ang-(1-7)-induced vasodilation and the potentiating effect of Ang-(1-7) on BK vasodilation. The potentiation of BK-induced vasodilation by Ang-(1-7) is a receptor-mediated phenomenon dependent on cyclooxygenase-related products and NO release.

NADPH oxidase and enhanced superoxide generation in intrauterine undernourished rats: involvement of the renin–angiotensin system

OBJECTIVE We previously reported that intrauterine undernutrition increased the oxidative stress by decreasing superoxide dismutase activity. In the present study, we tested whether NADPH oxidase, xanthine oxidase, cyclooxygenase or nitric oxide synthase are responsible for the increased O(2)(-) generation observed in rats submitted to intrauterine undernutrition. In addition, we investigated the effect of angiotensin II (ANG II) on O(2)(-) production via activation of NADPH oxidase. METHODS Female pregnant Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. At 16 weeks of age, the rats were used for the study of intravital fluorescence microscopy; microvascular reactivity, local ANG II concentration and AT(1), p22(phox) and gp91(phox) gene expression. In this study only the male offspring was used. RESULTS Treatment of mesenteric arterioles with the xanthine oxidase inhibitor oxypurinol, the nitric oxide synthase inhibitor L-NAME or the cyclooxygenase inhibitor diclofenac did not significantly change superoxide production. Thus, these vascular sources of superoxide were not responsible for the increased superoxide concentration. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased superoxide generation and improved vascular function. On the other hand, intrauterine undernutrition did not alter the gene expression for p22(phox) and gp91(phox). The fact that the local ANG II concentration was increased and the attenuation of oxidative stress by blocking AT(1) receptor with losartan, led us to suggest that ANG II induces O(2)(-) generation in intrauterine undernourished rats. CONCLUSION Our study shows that NADPH oxidase inhibition attenuated superoxide anion generation and ameliorated vascular function in rats submitted to intrauterine undernutrition. Although it is not clear which mechanisms are responsible for the increase in NADPH oxidase activity, a role for ANG II-mediated superoxide production via activation of NADPH oxidase is suggested.

Influence of Female Sex Hormones on Endothelium-Derived Vasoconstrictor Prostanoid Generation in Microvessels of Spontaneously Hypertensive Rats

Although female sex hormones may attenuate endothelial dysfunction in spontaneously hypertensive rats (SHR) by increasing endothelium-derived relaxing factors (EDRFs), the influence of ovarian hormones on the generation of endothelium-derived contracting factors (EDCFs) remains unknown. The aim of this study was to evaluate the effect of estrogen and progesterone on the generation of vasoconstrictor prostanoids and superoxide anion (O2(-)) by microvessels from SHR. Vascular reactivity to norepinephrine (NE), acetylcholine (ACh), and sodium nitroprusside (SNP) were evaluated in the mesenteric arteriolar bed from estrous (OE) and ovariectomized (OVX) SHR. OVX-SHR were treated for 24 hours or 15 days with estradiol and for 15 days with estradiol+progesterone. The vascular reactivity was evaluated in the absence or presence of indomethacin (INDO, 10 micromol/L) and sodium diclofenac (DIC, 10 micromol/L), ridogrel (RID, 50 micromol/L), dazoxiben (DAZ, 10 micromol/L), or superoxide dismutase (SOD, 100 U/mL). Prostanoid levels in the arteriolar perfusate of mesenteries with or without endothelium were measured by enzyme immunoassay. An increased reactivity to NE and reduced sensitivity to ACh were observed in microvessels from OVX-SHR compared with OE-SHR. There were no differences in the responses to SNP. Treatments with estradiol and estradiol+progesterone similarly restored these altered responses. INDO, DIC, RID, and SOD also restored the NE and ACh responses in OVX-SHR. DAZ had no effect on the vascular reactivities. The release of PGF(2alpha), but not of TXB(2) and 6-keto-PGF(1alpha), was greater in OVX-SHR than in OE-SHR microvessels with endothelium when stimulated by NE. This response was normalized by hormonal treatments. Neither NE nor ACh stimulated prostanoid production by microvessels without endothelium. These results suggest that estrogen may protect female SHR against severe hypertension partly by decreasing the synthesis of EDCFs such as PGH(2)/PGF(2alpha) and O2(-).

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

AIMS Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. METHODS AND RESULTS NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO2-) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 microM). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. CONCLUSION Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Influence of Hypoxia on Nitric Oxide Synthase Activity and Gene Expression in Children With Congenital Heart Disease A Novel Pathophysiological Adaptive Mechanism

BackgroundChronic hypoxia has been shown to modulate nitric oxide (NO) responses in different cell models, but the relationship between hypoxia and NO synthase (NOS) regulation in humans was not studied. We studied the relationship between endothelial and inducible NOS (eNOS and iNOS) activities and expression and chronic hypoxia in children with cyanotic and acyanotic congenital heart defects. Methods and ResultsRight atrial tissue was excised from 18 patients during cardiac surgery. eNOS and iNOS activities were measured by conversion of l-[H3]arginine to l-[H3]citrulline. Gene expression of eNOS and iNOS was quantified by competitive reverse transcription–polymerase chain reaction. The eNOS activity and expression were significantly reduced in cyanotic hearts compared with acyanotic hearts: 0.38±0.14 versus 1.06±0.11 pmol · mg−1 · min−1 (P <0.0001) and 0.54±0.08 versus 0.80±0.10 relative optical density (ROD) of cDNA (P <0.0001), respectively. In contrast, iNOS activity and expression were significantly higher in cyanotic than in acyanotic children: 7.04±1.20 versus 4.17±1.10 pmol · mg−1 · min−1 (P <0.0001) and 2.55±0.11 versus 1.91±0.18 ROD of cDNA (P <0.0001), respectively. ConclusionsHypoxia downregulates eNOS activity and gene expression in cardiac tissue from patients with cyanotic congenital heart defects. By contrast, iNOS activity and expression are increased in cyanotic children and may represent an alternative mechanism to counteract the effects of hypoxia in the cardiovascular system. Therefore, a novel adaptive mechanism during hypoxia is suggested.