Ethanol consumption increases blood pressure and alters the responsiveness of the mesenteric vasculature in rats

Chronic ethanol Consumption and hypertension are related. In the current study we investigated whether changes in reactivity of the mesenteric arterial bed could account for the increased blood pressure associated with chronic ethanol intake. Changes in reactivity to phenylephrine and acetylcholine were investigated in the perfused mesenteric bed from rats treated with ethanol for 2 or 6 weeks and their age-matched controls. Mild hypertension was observed in chronically ethanol-treated rats. Treatment of rats for 6 weeks induced an increase in the contractile response of endothelium-intact mesenteric bed to phenylephrine, but not denuded rat mesenteric bed. The phenylephrine-induced increase in perfusion pressure was not altered after 2 weeks` treatment with ethanol. Moreover, acetylcholine-induced endothelium-dependent relaxation was reduced by ethanol treatment for 6 weeks, but not 2 weeks. Pre-treatment with indometacin, a cyclooxygenase inhibitor, reduced the maximum effect induced by phenylephrine (E-max) in endothelium-intact mesenteric bed from both control and ethanol-treated rats. No differences in the E-max values for phenylephrine were observed between groups in the presence of indometacin. L-NNA, a nitric oxide (NO) synthase (NOS) inhibitor, increased the E-max for phenylephrine in endothelium-intact mesenteric bed from control rats but not from ethanol-treated rats. Levels of endothelial NOS (eNOS) mRNA were not altered by chronic ethanol consumption. However, chronic ethanol intake strongly reduced eNOS protein levels in the mesenteric bed. This study shows that chronic ethanol consumption increases blood pressure and alters the reactivity of the mesenteric bed. Moreover, the increased vascular response to phenylephrine observed in the mesenteric bed is maintained by two mechanisms: an increased release of endothelial-derived vasoconstrictor prostanoids and a reduced modulatory action of endothelial NO, which seems to be associated with reduced post-transcriptional expression of eNOS.

Combining two potential causes of metalloproteinase secretion causes abdominal aortic aneurysms in rats: a new experimental model

P>Progress in understanding the pathophysiology of abdominal aortic aneurysms (AAA) is dependent in part on the development and application of effective animal models that recapitulate key aspects of the disease. The objective was to produce an experimental model of AAA in rats by combining two potential causes of metalloproteinase (MMP) secretion: inflammation and turbulent blood flow. Male Wistar rats were randomly divided in four groups: Injury, Stenosis, Aneurysm and Control (40/group). The Injury group received a traumatic injury to the external aortic wall. The Stenosis group received an extrinsic stenosis at a corresponding location. The Aneurysm group received both the injury and stenosis simultaneously, and the Control group received a sham operation. Animals were euthanized at days 1, 3, 7 and 15. Aorta and/or aneurysms were collected and the fragments were fixed for morphologic, immunohistochemistry and morphometric analyses or frozen for MMP assays. AAAs had developed by day 3 in 60-70% of the animals, reaching an aortic dilatation ratio of more than 300%, exhibiting intense wall remodelling initiated at the adventitia and characterized by an obvious inflammatory infiltrate, mesenchymal proliferation, neoangiogenesis, elastin degradation and collagen deposition. Immunohistochemistry and zymography studies displayed significantly increased expressions of MMP-2 and MMP-9 in aneurysm walls compared to other groups. The haemo-dynamic alterations caused by the stenosis may have provided additional contribution to the MMPs liberation. This new model illustrated that AAA can be multifactorial and confirmed the key roles of MMP-2 and MMP-9 in this dynamic remodelling process.

P2X(7) receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1 beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1 beta into mature IL-1 beta, which is then secreted. Because both LPS (in vivo) and IL-1 beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1 beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco`s modified Eagle`s medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1 beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1 beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-L-arginine methyl ester or N-([3-(aminomethyl) phenyl] methyl) ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1 beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1 beta induced iNOS protein expression. Thus, activation of P2X 7 receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1 beta-mediated release of nitric oxide by iNOS.

Mechanisms underlying the biphasic effect of vitamin K-1 (phylloquinone) on arterial blood pressure

Phylloquinone (vitamin K-1, VK1) is widely used therapeutically and intravenous administration of this quinone can induce hypotension. We aimed to investigate the mechanisms underlying the effects induced by VK1 on arterial blood pressure. With this purpose a catheter was inserted into the abdominal aorta of male Wistar rats for blood pressure and heart rate recording. Bolus intravenous injection of VK1 (0.5-20 mg kg(-1)) produced a transient increase in blood pressure followed by a fall. Both the pressor and depressor response induced by VK1 were dose-dependent. On the other hand, intravenous injection of VK1 did not alter heart rate. The nitric oxide synthase (NOS) inhibitor N-G-nitro-L-arginine methyl ester (L-NAME, 10 and 20 mg kg(-1)) reduced both the increase and decrease in blood pressure induced by VK1 (5 mgkg(-1)). On the other hand, indometacin (10 mg kg(-1)), a non-selective cyclooxygenase inhibitor, did not alter the increase in mean arterial pressure (MAP) induced by VK1. However, VK1-induced fall in MAP was significantly attenuated by indometacin. We concluded that VK1 induces a dose-dependent effect on blood pressure that consists of an acute increase followed by a more sustained decrease in MAP. The hypotension induced by VK1 involves the activation of the nitric oxide (NO) pathway and the release of vasodilator prostanoid(s).

Can eccentric arterial plaques alone cause flow stagnation points and favour thrombus incorporation?

We have used an experimental model of aorta stenosis, with a Plexiglas plug, simulating a stable atheromatous plaque that promotes local turbulence and thrombosis. With animal survival of more than 24 h, we followed the partial fibrinolysis of the thrombus as well as its posterior organization and incorporation to the arterial wall as a neointima for up to 30 days. The mushroom plug form permitted the development of recirculation and stasis areas around it, favouring this evolution. Despite noted limitations, this study demonstrates that thrombus incorporation can contribute to plaque extension, as it can promote recirculation and stasis areas.

Lower Extremities Magnetic Resonance Angiography With Blood Pressure Cuff Compression: Quantitative Dynamic Analysis

Purpose: To quantitatively evaluate changes induced by the application of a femoral blood-pressure cuff (BPC) on run-off magnetic resonance angiography (MRA). which is a method generally previously proposed to reduce venous contamination in the leg. Materials and Methods: This study was Health Insurance Portability and Accountability Act (HIPAA)- and Institutional Review Board (IRB)-compliant, We used time-resolved gradient-echo gadolinium (Gd)-enhanced MRA to measure BPC effects on arterial, venous, and soft-tissue enhancement. Seven healthy volunteers (six men) were studied with the BPC applied at the mid-femoral level unilaterally using a 1.5T MR system after intravenous injection of Gd-BOPTA. Different statistical tools were used such as the Wilcoxon signed rank test and a cubic smoothing spline fit. Results: We found that BPC application induces delayed venous filling (as previously described), but also induces significant decreases in arterial inflow, arterial enhancement, vascular-soft tissue contrast, and delayed peak enhancement (which have not been previously measured). Conclusion: The potential benefits from using a BPC for run-off MRA must be balanced against the potential pitfalls, elucidated by our findings.

Chronic alcoholism associated with diabetes impairs erectile function in rats

OBJECTIVE To investigate the effects of chronic ethanol consumption and diabetes on nitric oxide (NO)-mediated relaxation of cavernosal smooth muscle (CSM). MATERIAL AND METHODS Male Wistar rats were divided into four groups: control, isocaloric, diabetic and ethanol-diabetic. The CSMs were mounted in organ chambers for measurement of isometric tension. Contraction of the strips was induced by electrical field stimulation (EFS, 1-32 Hz) and phenylephrine. We also evaluated the effect of ethanol consumption on the relaxation induced by acetylcholine (ACh; 0.01-1000 mu mol/L), sodium nitroprusside (SNP, 0.01-1000 mu mol/L) or EFS (1-32 Hz) in strips pre-contracted with phenylephrine (10 mu mol/L). Immunoexpression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) was also accessed. RESULTS The endothelium-dependent relaxation induced by ACh was decreased in CSM from ethanol-diabetic rats when compared with the controls, with a mean (sem) of 21 (4) vs 37 (2)%. Similarly, the potency and maximal responses induced by SNP were reduced in the ethanol-diabetic [3.97 (0.38) and 85 (1)%, respectively] and diabetic groups [3.78 (0.56) and 81 (2)%, respectively] when compared with the controls [5.3 (0.22) and 90 (3)%, respectively] and isocaloric [5.3 (0.19) and 92 (1)%, respectively] groups. Noradrenergic nerve-mediated contractions of CSM in response to EFS were increased in rats from ethanol-diabetic and diabetic groups when compared with the control and isocaloric groups. Conversely, there were no differences in EFS-induced relaxation among the groups. The immunostaining assays showed overexpression of eNOS and iNOS in the CSM from diabetic and ethanol-diabetic rats when compared with the control and isocaloric rats. CONCLUSION There was an impairment of relaxation of CSM from ethanol-diabetic and diabetic rats that involved a decrease in the NO-cyclic guanosine monophosphate signalling pathway by endothelium-dependent mechanisms accompanied by a change in the CSM contractile sensitivity.

Tissue diffusion and retention of metalloproteinases in ascending aortic aneurysms and dissections

Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases. (c) 2009 Elsevier Inc. All rights reserved.

Holt-Oram syndrome: novel TBX5 mutation and associated anomalous right coronary artery

The Holt-Oram syndrome was confirmed in an asymptomatic 36-year-old man by a novel TBX5-gene mutation (exon 8 acceptor splicing site, c.663-1G greater than A). Computed tomography showed an atrial septal defect and an anomalous right coronary artery crossing between the aorta and pulmonary arteries. Surgery corrected the septal defect and the initial segment of the anomalous vessel was unroofed and enlarged. Anomalous coronary arteries were not previously described in the Holt-Oram syndrome patients and should be added to the list of possible associated cardiac defects.

Arterial supply of the penis in agoutis (Dasyprocta prymnolpha, Wagler, 1831)

The arterial vascularization of agoutis` penis (Dasyprocta prymnolopha) were analysed using ten male adults from `Nucleo de Estudos e Preservacao de Animais Silvestres da Universidade Federal do Piaui` (FUFPI/IBAMA n degrees 02/99). Among the total number of specimens, six animals had natural death and were members of the research collection of the Laboratory of Anatomy, and four were killed after anaesthesia. Stained bi-centrifugated-Cis-I-4 latex was injected in arterial vessels responsible for penis vascularization throughout the abdominal portion of aorta. The samples were fixed in 10% formaldehyde solution and arteries were dissected. The penile artery is originated as a branch of internal pudendal artery. At the level of ischiatic arch, the penile artery project two branches, the penile dorsal and the deep arteries; those arteries irrigates the penile dorsal surface and the corpus cavernosum penis. The penile dorsal arteries have an independent course up to the glans penis. Based on the conditions of this work a remarkable similarity regarding the distribution of vessels destined to the agouti penis when compared to other domestic, wild and lagomorph rodents as rabbits.

Spironolactone and hydrochlorothiazide exert antioxidant effects and reduce vascular matrix metalloproteinase-2 activity and expression in a model of renovascular hypertension

Background and purpose: Increased oxidative stress and up-regulation of matrix metalloproteinases (MMPs) may cause structural and functional vascular changes in renovascular hypertension. We examined whether treatment with spironolactone (SPRL), hydrochlorothiazide (HCTZ) or both drugs together modified hypertension-induced changes in arterial blood pressure, aortic remodelling, vascular reactivity, oxidative stress and MMP levels and activity, in a model of renovascular hypertension. Experimental approach: We used the two-kidney,one-clip (2K1C) model of hypertension in Wistar rats. Sham-operated or hypertensive rats were treated with vehicle, SPRL (25 mg center dot kg-1 center dot day-1), HCTZ (20 mg center dot kg-1 center dot day-1) or a combination for 8 weeks. Systolic blood pressure was monitored weekly. Aortic rings were isolated to assess endothelium-dependent and -independent relaxations. Morphometry of the vascular wall was carried out in sections of aorta. Aortic NADPH oxidase activity and superoxide production were evaluated. Formation of reactive oxygen species was measured in plasma as thiobarbituric acid-reactive substances. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry and immunohistochemistry. Key results: Treatment with SPRL, HCTZ or the combination attenuated 2K1C-induced hypertension, and reversed the endothelial dysfunction in 2K1C rats. Both drugs or the combination reversed vascular aortic remodelling induced by hypertension, attenuated hypertension-induced increases in oxidative stress and reduced MMP-2 levels and activity. Conclusions and implications: SPRL or HCTZ, alone or combined, exerted antioxidant effects, and decreased renovascular hypertension-induced MMP-2 up-regulation, thus improving the vascular dysfunction and remodelling found in this model of hypertension.

Melatonin inhibits LPS-induced NO production in rat endothelial cells

Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF-kappa B) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein-coupled melatonin receptors. The nuclear NF-kappa B translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium-intact aortic rings, and melatonin (10 mu m) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra-pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS-induced vasodilation by inhibiting the NF-kappa B pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.

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 (NO(2)) 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 mu M). 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.

Chronic ouabain treatment exacerbates blood pressure elevation in spontaneously hypertensive rats: the role of vascular mechanisms

Objective Hypertensive rats are more sensitive to the pressor effects of acute ouabain than normotensive rats. We analyzed the effect of chronic ouabain (similar to 8.0 mu g/day, 5 weeks) treatment on the blood pressure of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats and the contribution of vascular mechanisms. Methods Responses to acetylcholine and phenylephrine were analyzed in isolated tail arteries. Protein expression of endothelial nitric oxide synthase and cyclooxygenase-2 (COX-2) were also investigated. Results Ouabain treatment enhanced blood pressure only in SHRs. The pD(2) for acetylcholine was decreased in arteries from SHRs compared with Wistar-Kyoto rats, and ouabain did not change this parameter. However, ouabain was able to increase the pD(2) to phenylephrine in SHRs. Nitric oxide synthase inhibition with N(G)-nitro-L-arginine methyl ester or potassium channel blockade by tetraetylamonium increased the response to phenylephrine in SHRs, with a smaller increase in response observed in ouabain-treated SHRs. In addition, indomethacin (a COX inhibitor) and ridogrel (a thromboxane A(2) synthase inhibitor and prostaglandin H(2)/thromboxane A(2) receptor antagonist) decreased contraction to phenylephrine in tail rings from ouabain-treated SHRs. Protein expression of endothelial nitric oxide synthase was unaltered following ouabain treatment in SHRs, whereas COX-2 expression was increased. Conclusion Chronic ouabain treatment further increases the raised blood pressure of SHRs. This appears to involve a vascular mechanism, related to a reduced vasodilator influence of nitric oxide and endothelium-derived hyperpolarizing factor and increased production of vasoconstrictor prostanoids by COX-2. These data suggest that the increased plasma levels of ouabain could play an important role in the maintenance of hypertension and the impairment of endothelial function. J Hypertens 27:1233-1242 (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Dehydroepiandrosterone protects against oxidative stress-induced endothelial dysfunction in ovariectomized rats

Cardiovascular disease is less frequent in premenopausal women than in age-matched men or postmenopausal women. Moreover, the marked age-related decline in serum dehydroepiandrosterone (DHEA) level has been associated to cardiovascular disease. The aim of this study was to evaluate the effects of DHEA treatment on vascular function in ovariectomized rats. At 8 weeks of age, female Wistar rats were ovariectomized (OVX) or sham (SHAM) operated and 8 weeks after surgery both groups were treated with vehicle or DHEA (10 mg kg-1 week-1) for 3 weeks. Aortic rings were used to evaluate the vasoconstrictor response to phenylephrine (PHE) and the relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP). Tissue reactive oxygen species (ROS) production and SOD, NADPH oxidase and eNOS protein expression were analysed. PHE-induced contraction was increased in aortic rings from OVX compared to SHAM, associated with a reduction in NO bioavailability. Furthermore, the relaxation induced by ACh was reduced in arteries from OVX, while SNP relaxation did not change. The incubation of aortic rings with SOD or apocynin restored the enhanced PHE-contraction and the impaired ACh-relaxation only in OVX. DHEA treatment corrected the increased PHE contraction and the impaired ACh-induced relaxation observed in OVX by an increment in NO bioavailability and decrease in ROS production. Besides, DHEA treatment restores the reduced Cu/Zn-SOD protein expression and eNOS phosphorylation and the increased NADPH oxidase protein expression in the aorta of OVX rats. The present results suggest an important action of DHEA, improving endothelial function in OVX rats by acting as an antioxidant and enhancing the NO bioavailability.