Alessandra Simão Padilha

Chronic Cadmium Treatment Promotes Oxidative Stress and Endothelial Damage in Isolated Rat Aorta

Cadmium is a highly toxic metal that is present in phosphate fertilizers, and the incidence of cadmium poisoning in the general population has increased, mainly due to cigarette smoking. Once absorbed, cadmium accumulates in the tissues, causing harmful effects including high blood pressure, endothelial damage and oxidative stress. Oxidative stress is known to efficiently produce oxidized low-density lipoprotein and consequently atherosclerosis, mainly in the aorta. However, the mechanisms through which endothelial damage is induced by cadmium have not been elucidated. Thus, the aim of this study was to investigate the effects of this metal in the isolated aorta and the possible role of oxidative stress. Rats received 100 mg.L−1 cadmium chloride (CdCl2) in the drinking water or distilled water alone for four weeks. The pressor effect of cadmium was followed throughout the exposure period by tail plethysmography. At the end of the fourth week, the blood cadmium content was established, and the vascular reactivity of the isolated aorta to phenylephrine, acetylcholine and sodium nitroprusside was analyzed in the context of endothelium denudation and incubation with L-NAME, apocynin, losartan, enalapril, superoxide dismutase (SOD) or catalase. We observed an increased response to phenylephrine in cadmium-treated rats. This increase was abolished by catalase and SOD incubation. Apocynin treatment reduced the phenylephrine response in both treatment groups, but its effect was greater in cadmium-treated rats, and NOX2 expression was greater in the cadmium group. These results suggested that cadmium in blood concentrations similar to those found in occupationally exposed populations is able to stimulate NOX2 expression, contributing to oxidative stress and reducing NO bioavailability, despite enhanced eNOS expression. These findings suggest that cadmium exposure promotes endothelial damage that might contribute to inflammation, vascular injury and the development of atherosclerosis.

Low-level lead exposure increases systolic arterial pressure and endothelium-derived vasodilator factors in rat aortas

Chronic lead exposure induces hypertension and alters endothelial function. However, treatment with low lead concentrations was not yet explored. We analyzed the effects of 7 day exposure to low lead concentrations on endothelium-dependent responses. Wistar rats were treated with lead (1st dose 4 µg/100 g, subsequent dose 0.05 µg/100 g, i.m. to cover daily loss) or vehicle; blood levels attained at the end of treatment were 9.98 µg/dL. Lead treatment had the following effects: increase in systolic blood pressure (SBP); reduction of contractile response to phenylephrine (1 nM–100 µM) of aortic rings; unaffected relaxation induced by acetylcholine (0.1 nM–300 µM) or sodium nitroprusside (0.01 nM–0.3 µM). Endothelium removal, N G-nitro-L-arginine methyl ester (100 µM) and tetraethylammonium (2 mM) increased the response to phenylephrine in treated rats more than in untreated rats. Aminoguanidine (50 µM) increased but losartan (10 µM) and enalapril (10 µM) reduced the response to phenylephrine in treated rats. Lead treatment also increased aortic Na+/K+-ATPase functional activity, plasma angiotensin-converting enzyme (ACE) activity, protein expression of the Na+/K+-ATPase alpha-1 subunit, phosphorylated endothelial nitric oxide synthase (p-eNOS), and inducible nitric oxide synthase (iNOS). Our results suggest that on initial stages of lead exposure, increased SBP is caused by the increase in plasma ACE activity. This effect is accompanied by increased p-eNOS, iNOS protein expression and Na+/K+-ATPase functional activity. These factors might be a compensatory mechanism to the increase in SBP.

Low-dose chronic lead exposure increases systolic arterial pressure and vascular reactivity of rat aortas

Chronic lead exposure induces hypertension affecting endothelial function. We investigated whether low-concentration lead exposure alters blood pressure and vascular reactivity, focusing on the roles of NO, oxidative stress, cyclooxygenase-derived vasoconstrictor prostanoids, and the local angiotensin-renin system. Aortic rings from 3-month-old Wistar rats were treated daily with lead acetate (first dose 4mg/100g, subsequent doses 0.05mg/100g, im) or vehicle for 30 days. Treatment increased lead blood levels (12μg/dl), blood pressure, and aortic ring contractile response to phenylephrine (1nM-100mM). Contractile response after L-NAME administration increased in both groups but was higher after lead treatment. Lead effects on Rmax decreased more after apocynin and superoxide dismutase administration compared to control. Indomethacin reduced phenylephrine response more after lead treatment than in controls. The selective COX-2 inhibitor NS398, thromboxane A2/prostaglandin H2 receptor antagonist SQ 29,548, TXA2 synthase inhibitor furegrelate, EP1 receptor antagonist SC 19220, and ACE inhibitor and AT1 receptor antagonist losartan reduced phenylephrine responses only in vessels from lead-treated rats. Basal and stimulated NO release was reduced and local O2(-) liberation increased in the lead-treated group compared to controls. eNOS, iNOS, and AT1 receptor protein expression increased with lead exposure, but COX-2 protein expression decreased. This is the first demonstration that blood Pb(2+) (12µg/dl) concentrations below the WHO-established values increased systolic blood pressure and vascular phenylephrine reactivity. This effect was associated with reduced NO bioavailability, increased reactive oxygen species production, increased participation of COX-derived contractile prostanoids, and increased renin-angiotensin system activity.

Low Mercury Concentration Produces Vasoconstriction, Decreases Nitric Oxide Bioavailability and Increases Oxidative Stress in Rat Conductance Artery

Mercury is an environmental pollutant that reduces nitric oxide (NO) bioavailability and increases oxidative stress, having a close link with cardiovascular diseases, as carotid atherosclerosis, myocardial infarction, coronary heart disease and hypertension. One of the main sites affected by oxidative stress, which develops atherosclerosis, is the aorta. Under acute exposure to low mercury concentrations reactive oxygen species (ROS) production were only reported for resistance vessels but if low concentrations of mercury also affect conductance arteries it is still unclear. We investigated the acute effects of 6 nM HgCl2 on endothelial function of aortic rings measuring the reactivity to phenylephrine in rings incubated, or not, with HgCl2 for 45 min, the protein expression for cyclooxygenase 2 (COX-2) and the AT1 receptor. HgCl2 increased Rmax and pD2 to phenylephrine without changing the vasorelaxation induced by acetylcholine and sodium nitroprusside. Endothelial damage abolished the increased reactivity to phenylephrine. The increase of Rmax and pD2 produced by L-NAME was smaller in the presence of HgCl2. Enalapril, losartan, indomethacin, furegrelate, the selective COX-2 inhibitor NS 398, superoxide dismutase and the NADPH oxidase inhibitor apocynin reverted HgCl2 effects on the reactivity to phenylephrine, COX-2 protein expression was increased, and AT1 expression reduced. At low concentration, below the reference values, HgCl2 increased vasoconstrictor activity by reducing NO bioavailability due to increased ROS production by NADPH oxidase activity. Results suggest that this is due to local release of angiotensin II and prostanoid vasoconstrictors. Results also suggest that acute low concentration mercury exposure, occurring time to time could induce vascular injury due to endothelial oxidative stress and contributing to increase peripheral resistance, being a high risk factor for public health.

Acute Lead Exposure Increases Arterial Pressure: Role of the Renin-Angiotensin System

Background Chronic lead exposure causes hypertension and cardiovascular disease. Our purpose was to evaluate the effects of acute exposure to lead on arterial pressure and elucidate the early mechanisms involved in the development of lead-induced hypertension. Methodology/Principal Findings Wistar rats were treated with lead acetate (i.v. bolus dose of 320 µg/Kg), and systolic arterial pressure, diastolic arterial pressure and heart rate were measured during 120 min. An increase in arterial pressure was found, and potential roles of the renin-angiotensin system, Na+,K+-ATPase and the autonomic reflexes in this change in the increase of arterial pressure found were evaluated. In anesthetized rats, lead exposure: 1) produced blood lead levels of 37±1.7 µg/dL, which is below the reference blood concentration (60 µg/dL); 2) increased systolic arterial pressure (Ct: 109±3 mmHg vs Pb: 120±4 mmHg); 3) increased ACE activity (27% compared to Ct) and Na+,K+-ATPase activity (125% compared to Ct); and 4) did not change the protein expression of the α1-subunit of Na+,K+-ATPase, AT1 and AT2. Pre-treatment with an AT1 receptor blocker (losartan, 10 mg/Kg) or an ACE inhibitor (enalapril, 5 mg/Kg) blocked the lead-induced increase of arterial pressure. However, a ganglionic blockade (hexamethonium, 20 mg/Kg) did not prevent leads hypertensive effect. Conclusion Acute exposure to lead below the reference blood concentration increases systolic arterial pressure by increasing angiotensin II levels due to ACE activation. These findings offer further evidence that acute exposure to lead can trigger early mechanisms of hypertension development and might be an environmental risk factor for cardiovascular disease.

Cadmium exposure induces vascular injury due to endothelial oxidative stress: the role of local angiotensin II and COX-2

Cadmium is an environmental pollutant that is closely linked with cardiovascular diseases, such as atherosclerosis and hypertension. Moreover, cadmium can induce an increase in oxidative stress. One of the main sites affected by oxidative stress is the aorta, which consequently develops atherosclerosis. However, there are few reports demonstrating aortic effects induced by small concentrations of cadmium that are similar to those found in the blood resulting from occupational exposure. Furthermore, several studies have reported on chronic cadmium exposure, and the results of these studies may have been influenced by the secondary effects induced by this metal, such as hypertension. Therefore, we investigated the effects of acute cadmium exposure on the vascular reactivity to phenylephrine of aortic rings isolated from male Wistar rats. Cadmium increased phenylephrine reactivity without changing the vasorelaxation induced by acetylcholine and sodium nitroprusside. Endothelial damage or incubation with L-NAME shifted the phenylephrine concentration-response curves leftward in arteries incubated with or without cadmium, but the curves were shifted to a lesser degree after cadmium incubation. Enalapril, losartan, the nonselective COX inhibitor indomethacin, the TXA(2) synthase inhibitor furegrelate, the selective COX-2 inhibitor NS 398, the TP receptor antagonist SQ 29.548, the EP1 receptor antagonist SC 19.220, superoxide dismutase, and the NADPH oxidase inhibitor apocynin partially reverted the cadmium-induced effects on the reactivity to phenylephrine. Cadmium exposure increased vasoconstrictor activity by reducing NO bioavailability owing to the increased production of ROS by NADPH oxidase. The results of the tested cadmium concentration, which is below the reference values, suggest that acute cadmium exposure may induce vascular injury through endothelial oxidative stress. These data contribute to the evidence indicating that cadmium is a high risk to public health.

Ouabain treatment changes the role of endothelial factors in rat resistance arteries.

This study investigates the participation of the endothelial factors in the alpha-adrenoceptor contractile responses in mesenteric resistance arteries from 15 days ouabain-treated (25 microg/kg/day) and untreated rats. Ouabain treatment increased blood pressure and heart rate without changing the contractile response to phenylephrine (3 nM-30 microM). Endothelium removal or N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), increased the responses to phenylephrine. The endothelial modulation was similar in both rat groups, but the L-NAME effects were bigger in arteries from ouabain-treated rats. However, the endothelial NOS expression and the relaxation to acetylcholine (0.1 nM-10 microM) remained unaltered after ouabain treatment. The coincubation with L-NAME and indomethacin (100 microM) leftward shifted the concentration-response curves to phenylephrine in arteries from untreated rats similarly to the displacement after incubation only with L-NAME. However, in mesenteric arteries from treated rats, the co-incubation with indomethacin and L-NAME did not alter the response to phenylephrine. The addition of the inhibitor of calcium activated potassium channels tetraethylammonium (2 mM) further leftward shifted the phenylephrine curves only in arteries from untreated rats. Cyclooxygenase-2 (COX-2) expression was greater in vessels from ouabain-treated rats. In conclusion, the chronic ouabain treatment for 15 days modified the participation of endothelial factors in response to phenylephrine in mesenteric resistance arteries, by increasing the release of NO and prostanoids and impairment the endothelium-derived hyperpolarizing factor (EDHF) release. This was accompanied by an increased COX-2 expression. Although this balance avoids changes in the phenylephrine concentration-response curves, these vascular changes might contribute to maintain the ouabain-induced hypertension.

Ouabain at Nanomolar Concentration Promotes Synthesis and Release of Angiotensin II from the Endothelium of the Tail Vascular Bed of Spontaneously Hypertensive Rats

The effects of 1 nM ouabain (OUA) on the contractile actions of phenylephrine (PHE, 0.001–100 μg) and functional activity of the sodium pump (NKA) in isolated-perfused tail vascular beds from WKY and SHR were investigated. In preparations from SHR, perfusion with OUA in the presence of endothelium (E+) increased the sensitivity (pED50) of PHE (before: 2.14 ± 0.06 versus after: 2.47 ± 0.07; P < 0.05) without altering the maximal response (Emax). After endothelial damage, OUA reduced the Emax of PHE in SHR (before: 350 ± 29 versus after: 293 ± 25 mm Hg; P < 0.05). In SHR/E+, pretreatment with losartan (10 μM) or enalaprilat (1 μM) prevented the increased sensitivity to PHE induced by OUA. OUA increased NKA activity in SHR/E+ (before: 45 ± 6 versus after: 58 ± 5%, P < 0.05). Losartan (10 mg/Kg, i.v.) also abolished the increment in systolic and diastolic blood pressure induced by OUA (0.18 μg/Kg, i.v.) in anesthetized SHR. OUA did not alter the actions of PHE in either anesthetized WKY rats or vascular preparations. Results suggest that 1 nM OUA increased the vascular reactivity to PHE only in SHR/E+. This effect is mediated by OUA-induced activation of endothelial angiotensin converting enzyme that promotes the local formation of angiotensin II, which sensitizes the vascular smooth muscle to the actions of PHE.

Activation of BKCa channels by nitric oxide prevents coronary artery endothelial dysfunction in ouabain-induced hypertensive rats

Objective Chronic-ouabain administration to rats induces hypertension and increases the endothelial modulation of vasoconstrictor responses. The aim of this study was to analyze whether ouabain-treatment affects the mechanisms involved in endothelium-dependent relaxation of coronary arteries. Methods Coronary arteries from control and ouabain-treated rats (∼8.0 μg/day, 5 weeks) were used. Vascular reactivity was analyzed by isometric tension recording and membrane currents were measured using the whole-cell configuration of the patch-clamp technique. Results In 5-hydroxytryptamine (5-HT) precontracted arteries, acetylcholine (ACh, 1 nmol/l–10 μmol/l) induced a similar relaxant response in coronary arteries from both groups that was abolished by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (100 μmol/l). However, when arteries were contracted with high KCl (60 mmol/l) or preincubated with the large-conductance Ca2+-activated K+ (BKCa) channels-blocker iberiotoxin (0.1 μmol/l), the relaxation elicited by ACh was more reduced in ouabain-treated than control rats. After iberiotoxin preincubation, the relaxant response of the nitric-oxide donor, DEA-NO (10 nmol/l–100 μmol/l) was significantly inhibited in ouabain-treated coronary arteries but not in control vessels. The soluble guanylyl cyclase activator BAY 41-2272 (10 nmol/l–30 μmol/l) induced relaxant responses that were inhibited by iberiotoxin. In coronary-artery myocytes isolated from ouabain-treated rats DEA-NO (1 μmol/l) markedly increased the amplitude of the iberiotoxin-sensitive current in the whole range of test potentials, compared with nontreated rats. Conclusion Our results indicate that chronic ouabain treatment increases activation of BKCa currents by nitric oxide and this effect might contribute to preserve the endothelial function in coronary arteries in this hypertension model.

Post-Weaning Protein Malnutrition Increases Blood Pressure and Induces Endothelial Dysfunctions in Rats

Malnutrition during critical periods in early life may increase the subsequent risk of hypertension and metabolic diseases in adulthood, but the underlying mechanisms are still unclear. We aimed to evaluate the effects of post-weaning protein malnutrition on blood pressure and vascular reactivity in aortic rings (conductance artery) and isolated-perfused tail arteries (resistance artery) from control (fed with Labina®) and post-weaning protein malnutrition rats (offspring that received a diet with low protein content for three months). Systolic and diastolic blood pressure and heart rate increased in the post-weaning protein malnutrition rats. In the aortic rings, reactivity to phenylephrine (10−10–3.10−4 M) was similar in both groups. Endothelium removal or L-NAME (10−4 M) incubation increased the response to phenylephrine, but the L-NAME effect was greater in the aortic rings from the post-weaning protein malnutrition rats. The protein expression of the endothelial nitric oxide isoform increased in the aortic rings from the post-weaning protein malnutrition rats. Incubation with apocynin (0.3 mM) reduced the response to phenylephrine in both groups, but this effect was higher in the post-weaning protein malnutrition rats, suggesting an increase of superoxide anion release. In the tail artery of the post-weaning protein malnutrition rats, the vascular reactivity to phenylephrine (0.001–300 µg) and the relaxation to acetylcholine (10−10–10−3 M) were increased. Post-weaning protein malnutrition increases blood pressure and induces vascular dysfunction. Although the vascular reactivity in the aortic rings did not change, an increase in superoxide anion and nitric oxide was observed in the post-weaning protein malnutrition rats. However, in the resistance arteries, the increased vascular reactivity may be a potential mechanism underlying the increased blood pressure observed in this model.