Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 28
Filter
Add more filters










Publication year range
1.
Metabolism ; 157: 155932, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38729600

ABSTRACT

BACKGROUND: Obesity-induced hypogonadism (OIH) is a prevalent, but often neglected condition in men, which aggravates the metabolic complications of overweight. While hypothalamic suppression of Kiss1-encoded kisspeptin has been suggested to contribute to OIH, the molecular mechanisms for such repression in obesity, and the therapeutic implications thereof, remain unknown. METHODS: A combination of bioinformatic, expression and functional analyses was implemented, assessing the role of the evolutionary-conserved miRNAs, miR-137 and miR-325, in mediating obesity-induced suppression of hypothalamic kisspeptin, as putative mechanism of central hypogonadism and metabolic comorbidities. The implications of such miR-137/325-kisspeptin interplay for therapeutic intervention in obesity were also explored using preclinical OIH models. RESULTS: MiR-137/325 repressed human KISS1 3'-UTR in-vitro and inhibited hypothalamic kisspeptin content in male rats, while miR-137/325 expression was up-regulated, and Kiss1/kisspeptin decreased, in the medio-basal hypothalamus of obese rats. Selective over-expression of miR-137 in Kiss1 neurons reduced Kiss1/ kisspeptin and partially replicated reproductive and metabolic alterations of OIH in lean mice. Conversely, interference of the repressive actions of miR-137/325 selectively on Kiss1 3'-UTR in vivo, using target-site blockers (TSB), enhanced kisspeptin content and reversed central hypogonadism in obese rats, together with improvement of glucose intolerance, insulin resistance and cardiovascular and inflammatory markers, despite persistent exposure to obesogenic diet. Reversal of OIH by TSB miR-137/325 was more effective than chronic kisspeptin or testosterone treatments in obese rats. CONCLUSIONS: Our data disclose that the miR-137/325-Kisspeptin repressive interaction is a major player in the pathogenesis of obesity-induced hypogonadism and a putative druggable target for improved management of this condition and its metabolic comorbidities in men suffering obesity. SIGNIFICANCE STATEMENT: Up to half of the men suffering obesity display also central hypogonadism, an often neglected complication of overweight that can aggravate the clinical course of obesity and its complications. The mechanisms for such obesity-induced hypogonadism remain poorly defined. We show here that the evolutionary conserved miR137/miR325 tandem centrally mediates obesity-induced hypogonadism via repression of the reproductive-stimulatory signal, kisspeptin; this may represent an amenable druggable target for improved management of hypogonadism and other metabolic complications of obesity.


Subject(s)
Hypogonadism , Hypothalamus , Kisspeptins , MicroRNAs , Obesity , MicroRNAs/genetics , MicroRNAs/metabolism , Hypogonadism/genetics , Hypogonadism/metabolism , Hypogonadism/complications , Kisspeptins/genetics , Kisspeptins/metabolism , Animals , Obesity/metabolism , Obesity/complications , Obesity/genetics , Male , Rats , Hypothalamus/metabolism , Humans , Mice , Rats, Wistar , Comorbidity
2.
J Pharmacol Exp Ther ; 388(2): 670-687, 2024 01 17.
Article in English | MEDLINE | ID: mdl-38129126

ABSTRACT

Inflammatory mechanisms and oxidative stress seem to contribute to the pathogenesis of hypertension. ITH13001 is a melatonin-phenyl-acrylate hybrid that moderately induces the antioxidant transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) and has a potent oxidant scavenging effect compared with other derivatives of its family. Here we investigated the effect of ITH13001 on hypertension and the associated cardiovascular alterations. Angiotensin II (AngII)-infused mice were treated with ITH13001 (1 mg/kg per day, i.p.) for 2 weeks. The ITH13001 treatment prevented: 1) the development of hypertension, cardiac hypertrophy, and increased collagen and B-type natriuretic peptide (Bnp) expression in the heart; 2) the reduction of elasticity, incremental distensibility, fenestrae area, intraluminal diameter, and endothelial cell number in mesenteric resistance arteries (MRA); 3) the endothelial dysfunction in aorta and MRA; 4) the plasma and cardiovascular oxidative stress and the reduced aortic nitric oxide (NO) bioavailability; 5) the increased cardiac levels of the cytokines interleukin (IL)-1ß, IL-6, and C-C motif chemokine ligand 2 (Ccl2), the T cell marker cluster of differentiation 3 (Cd3), the inflammasome NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), the proinflammatory enzymes inducible nitric oxide synthase (iNOS) and COX-2, the toll-like receptor 4 (TLR4) adapter protein myeloid differentiation primary response 88 (MyD88), and the nuclear factor kappa B (NF-κB) subunit p65; 6) the greater aortic expression of the cytokines tumor necrosis factor alpha (Tnf-α), Ccl2 and IL-6, Cd3, iNOS, MyD88, and NLRP3. Although ITH13001 increased nuclear Nrf2 levels and heme oxygenase 1 (HO-1) expression in vascular smooth muscle cells, both cardiac and vascular Nrf2, Ho-1, and NADPH quinone dehydrogenase 1 (Nqo1) levels remained unmodified irrespective of AngII infusion. Summarizing, ITH13001 improved hypertension-associated cardiovascular alterations independently of Nrf2 pathway activation, likely due to its direct antioxidant and anti-inflammatory properties. Therefore, ITH13001 could be a useful therapeutic strategy in patients with resistant hypertension. SIGNIFICANCE STATEMENT: Despite the existing therapeutic arsenal, only half of the patients treated for hypertension have adequately controlled blood pressure; therefore, the search for new compounds to control this pathology and the associated damage to end-target organs (cerebral, cardiac, vascular, renal) is of particular interest. The present study demonstrates that a new melatonin derivative, ITH13001, prevents hypertension development and the associated cardiovascular alterations due to its antioxidant and anti-inflammatory properties, making this compound a potential candidate for treatment of resistant hypertensive patients.


Subject(s)
Hypertension , Melatonin , Humans , Mice , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Angiotensin II , Melatonin/pharmacology , Melatonin/therapeutic use , Antioxidants/pharmacology , Antioxidants/therapeutic use , Antioxidants/metabolism , Interleukin-6/metabolism , NF-E2-Related Factor 2/metabolism , Myeloid Differentiation Factor 88/metabolism , Hypertension/chemically induced , Hypertension/drug therapy , Hypertension/prevention & control , NF-kappa B/metabolism , Cytokines/metabolism , Anti-Inflammatory Agents/pharmacology
3.
Int J Mol Sci ; 24(9)2023 May 08.
Article in English | MEDLINE | ID: mdl-37176139

ABSTRACT

Diabetic macroangiopathy is characterized by increased extracellular matrix deposition, including excessive hyaluronan accumulation, vessel thickening and stiffness, and endothelial dysfunction in large arteries. We hypothesized that the overexpression of hyaluronan in the tunica media also led to endothelial cell (EC) dysfunction. To address this hypothesis, we investigated the following in the aortas of mice with excessive hyaluronan accumulation in the tunica media (HAS-2) and wild-type mice: EC dysfunction via myograph studies, nitric oxide (NO) bioavailability via diaminofluorescence, superoxide formation via dihydroethidium fluorescence, and the distances between ECs via stereological methods. EC dysfunction, characterized by blunted relaxations in response to acetylcholine and decreased NO bioavailability, was found in the aortas of male HAS-2 mice, while it was unaltered in the aortas of female HAS-2 mice. Superoxide levels increased and extracellular superoxide dismutase (ecSOD) expression decreased in the aortas of male and female HAS-2 mice. The EC-EC distances and LDL receptor expression were markedly increased in the HAS-2 aortas of male mice. Our findings suggest hyaluronan increases oxidative stress in the vascular wall and that together with increased EC distance, it is associated with a sex-specific decrease in NO levels and endothelial dysfunction in the aorta of male HAS-2 transgenic mice.


Subject(s)
Hyaluronic Acid , Vascular Diseases , Mice , Male , Female , Animals , Hyaluronic Acid/metabolism , Superoxides/metabolism , Vasodilation , Endothelium, Vascular/metabolism , Aorta/metabolism , Mice, Transgenic , Vascular Diseases/metabolism , Tunica Media/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism
4.
Front Pharmacol ; 14: 1058488, 2023.
Article in English | MEDLINE | ID: mdl-36937865

ABSTRACT

Introduction: Vascular oxidative stress and inflammation play an important role in the pathogenesis of cardiovascular diseases (CVDs). The proinflammatory cytokine Interleukin-1ß (IL-1ß) participates in the vascular inflammatory and oxidative responses and influences vascular smooth muscle cells (VSMC) phenotype and function, as well as vascular remodelling in cardiovascular diseases. The Toll-like receptor 4 (TLR4) is also involved in the inflammatory response in cardiovascular diseases. A relationship between Interleukin-1ß and Toll-like receptor 4 pathway has been described, although the exact mechanism of this interaction remains still unknown. Moreover, the oxidative stress sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) promotes the transcription of several antioxidant and anti-inflammatory genes. Nuclear factor-erythroid 2-related factor 2 activators have shown to possess beneficial effects in cardiovascular diseases in which oxidative stress and inflammation are involved, such as hypertension and atherosclerosis; however, the molecular mechanisms are not fully understood. Here, we analysed the role of Toll-like receptor 4 in the oxidative and inflammatory effects of Interleukin-1ß as well as whether nuclear factor-erythroid 2-related factor 2 activation contributes to vascular alterations by modulating these effects. Materials: For this purpose, vascular smooth muscle cells and mice aortic segments stimulated with Interleukin-1ß were used. Results: Interleukin-1ß induces MyD88 expression while the Toll-like receptor 4 inhibitor CLI-095 reduces the Interleukin-1ß-elicited COX-2 protein expression, reactive oxygen species (ROS) production, vascular smooth muscle cells migration and endothelial dysfunction. Additionally, Interleukin-1ß increases nuclear factor-erythroid 2-related factor 2 nuclear translocation and expression of its downstream proteins heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and superoxide dismutase-2, by an oxidative stress-dependent mechanism; moreover, Interleukin-1ß reduces the expression of the nuclear factor-erythroid 2-related factor 2 inhibitor Keap1. The nuclear factor-erythroid 2-related factor 2 activator tert-butylhydroquinone (tBHQ) reduces the effects of Interleukin-1ß on the increased reactive oxygen species production and the expression of the proinflammatory markers (p-p38, p-JNK, p-c-Jun, COX-2), the increased cell proliferation and migration and prevents the Interleukin-1ß-induced endothelial dysfunction in mice aortas. Additionally, tert-butylhydroquinone also reduces the increased MyD88 expression, NADPHoxidase activity and cell migration induced by lipopolysaccharide. Conclusions: In summary, this study reveals that Toll-like receptor 4 pathway contributes to the prooxidant and proinflammatory Interleukin-1ß-induced effects. Moreover, activation of nuclear factor-erythroid 2-related factor 2 prevents the deleterious effects of Interleukin-1ß, likely by reducing Toll-like receptor 4-dependent pathway. Although further research is needed, the results are promising as they suggest that nuclear factor-erythroid 2-related factor 2 activators might protect against the oxidative stress and inflammation characteristic of cardiovascular diseases.

5.
Front Physiol ; 12: 643435, 2021.
Article in English | MEDLINE | ID: mdl-33716792

ABSTRACT

Hypertension is one predictive factor for stroke and heart ischemic disease. Nowadays, it is considered an inflammatory disease with elevated cytokine levels, oxidative stress, and infiltration of immune cells in several organs including heart, kidney, and vessels, which contribute to the hypertension-associated cardiovascular damage. Macrophages, the most abundant immune cells in tissues, have a high degree of plasticity that is manifested by polarization in different phenotypes, with the most well-known being M1 (proinflammatory) and M2 (anti-inflammatory). In hypertension, M1 phenotype predominates, producing inflammatory cytokines and oxidative stress, and mediating many mechanisms involved in the pathogenesis of this disease. The increase in the renin-angiotensin system and sympathetic activity contributes to the macrophage mobilization and to its polarization to the pro-inflammatory phenotype. Heme oxygenase-1 (HO-1), a phase II detoxification enzyme responsible for heme catabolism, is induced by oxidative stress, among others. HO-1 has been shown to protect against oxidative and inflammatory insults in hypertension, reducing end organ damage and blood pressure, not only by its expression at the vascular level, but also by shifting macrophages toward the anti-inflammatory phenotype. The regulatory role of heme availability for the synthesis of enzymes involved in hypertension development, such as cyclooxygenase or nitric oxide synthase, seems to be responsible for many of the beneficial HO-1 effects; additionally, the antioxidant, anti-inflammatory, antiapoptotic, and antiproliferative effects of the end products of its reaction, carbon monoxide, biliverdin/bilirubin, and Fe2+, would also contribute. In this review, we analyze the role of HO-1 in hypertensive pathology, focusing on its expression in macrophages.

6.
Br J Pharmacol ; 178(8): 1836-1854, 2021 04.
Article in English | MEDLINE | ID: mdl-33556997

ABSTRACT

BACKGROUND AND PURPOSE: KV 1.3 channels are expressed in vascular smooth muscle cells (VSMCs), where they contribute to proliferation rather than contraction and participate in vascular remodelling. KV 1.3 channels are also expressed in macrophages, where they assemble with KV 1.5 channels (KV 1.3/KV 1.5), whose activation generates a KV current. In macrophages, the KV 1.3/KV 1.5 ratio is increased by classical activation (M1). Whether these channels are involved in angiotensin II (AngII)-induced vascular remodelling, and whether they can modulate the macrophage phenotype in hypertension, remains unknown. We characterized the role of KV 1.3 channels in vascular damage in hypertension. EXPERIMENTAL APPROACH: We used AngII-infused mice treated with two selective KV 1.3 channel inhibitors (HsTX[R14A] and [EWSS]ShK). Vascular function and structure were measured using wire and pressure myography, respectively. VSMC and macrophage electrophysiology were studied using the patch-clamp technique; gene expression was analysed using RT-PCR. KEY RESULTS: AngII increased KV 1.3 channel expression in mice aorta and peritoneal macrophages which was abolished by HsTX[R14A] treatment. KV 1.3 inhibition did not prevent hypertension, vascular remodelling, or stiffness but corrected AngII-induced macrophage infiltration and endothelial dysfunction in the small mesenteric arteries and/or aorta, via a mechanism independent of electrophysiological changes in VSMCs. AngII modified the electrophysiological properties of peritoneal macrophages, indicating an M1-like activated state, with enhanced expression of proinflammatory cytokines that induced endothelial dysfunction. These effects were prevented by KV 1.3 blockade. CONCLUSIONS AND IMPLICATIONS: We unravelled a new role for KV 1.3 channels in the macrophage-dependent endothelial dysfunction induced by AngII in mice which might be due to modulation of macrophage phenotype.


Subject(s)
Angiotensin II , Hypertension , Angiotensin II/toxicity , Animals , Hypertension/chemically induced , Macrophages , Mice , Myocytes, Smooth Muscle , Vascular Remodeling
7.
Front Physiol ; 11: 667, 2020.
Article in English | MEDLINE | ID: mdl-32655412

ABSTRACT

Endothelial cell dysfunction and vessel stiffening are associated with a worsened prognosis in diabetic patients with cardiovascular diseases. The present study hypothesized that sex impacts endothelial dysfunction and structural changes in arteries from diabetic mice. In diabetic (db/db) and normoglycaemic (db/db+) mice, the mechanical properties were investigated in pressurized isolated left anterior descending coronary arteries and aorta segments that were subjected to tensile testing. Functional studies were performed on wire-mounted vascular segments. The male and female db/db mice were hyperglycaemic and had markedly increased body weight. In isolated aorta segments without the contribution of smooth muscle cells, load to rupture, viscoelasticity, and collagen content were decreased suggesting larger distensibility of the arterial wall in both male and female db/db mice. In male db/db aorta segments with smooth muscle cell contribution, lumen diameter was smaller and the passive stretch-tension curve was leftward-shifted, while they were unaltered in female db/db aorta segments versus control db/db+ mice. In contrast to female db/db mice, coronary arteries from male db/db mice had altered stress-strain relationships and increased distensibility. Transthoracic echocardiography revealed a dilated left ventricle with unaltered cardiac output, while aortic flow velocity was decreased in male db/db mice. Impairment of acetylcholine relaxation was aggravated in aorta from female db/db compared to control and male db/db mice, while impairment of sodium nitroprusside relaxations was only observed in aorta from male db/db mice. The remodeling in the coronary arteries and aorta suggests an adaptation of the arterial wall to the reduced flow velocity with sex-specific differences in the passive properties of aorta and coronary arteries. The findings of less distensible arteries and more pronounced endothelial dysfunction in female compared to male diabetic mice may have implications for the observed higher incidence of macrovascular complications in diabetic women.

8.
Front Pharmacol ; 11: 619152, 2020.
Article in English | MEDLINE | ID: mdl-33643042

ABSTRACT

Endothelial cell dysfunction and fibrosis are associated with worsening of the prognosis in patients with cardiovascular disease. Pirfenidone has a direct antifibrotic effect, but vasodilatation may also contribute to the effects of pirfenidone. Therefore, in a first study we investigated the mechanisms involved in the relaxant effect of pirfenidone in rat intrapulmonary arteries and coronary arteries from normal mice. Then in a second study, we investigated whether pirfenidone restores endothelial function in the aorta and mesenteric arteries from diabetic animals. From 16-18-week old normal male C57BL/6 mice and normoglycemic (db/db+), and type 2 diabetic (db/db) male and female mice, arteries were mounted in microvascular isometric myographs for functional studies, and immunoblotting was performed. In rat pulmonary arteries and mouse coronary arteries, pirfenidone induced relaxations, which were inhibited in preparations without endothelium. In mouse coronary arteries, pirfenidone relaxation was inhibited in the presence of a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine (L-NOARG), a blocker of large-conductance calcium-activated potassium channels (BKCa), iberiotoxin, and a blocker of KV7 channels, XE991. Patch clamp studies in vascular smooth muscle revealed pirfenidone increased iberiotoxin-sensitive current. In the aorta and mesenteric small arteries from diabetic db/db mice relaxations induced by the endothelium-dependent vasodilator, acetylcholine, were markedly reduced compared to db/db + mice. Pirfenidone enhanced the relaxations induced by acetylcholine in the aorta from diabetic male and female db/db mice. An opener of KV7 channels, flupirtine, had the same effect as pirfenidone. XE991 reduced the effect of pirfenidone and flupirtine and further reduced acetylcholine relaxations in the aorta. In the presence of iberiotoxin, pirfenidone still increased acetylcholine relaxation in aorta from db/db mice. Immunoblotting for KV7.4, KV7.5, and BKCa channel subunits were unaltered in aorta from db/db mice. Pirfenidone failed to improve acetylcholine relaxation in mesenteric arteries, and neither changed acetylcholine-induced transient decreases in blood pressure in db/db+ and db/db mice. In conclusion, pirfenidone vasodilates pulmonary and coronary arteries. In coronary arteries from normal mice, pirfenidone induces NO-dependent vasodilatation involving BKCa and KV7 channels. Pirfenidone improves endothelium-dependent vasodilatation in aorta from diabetic animals by a mechanism involving voltage-gated KV7 channels, a mechanism that may contribute to the antifibrotic effect of pirfenidone.

9.
Sci Rep ; 9(1): 16461, 2019 11 11.
Article in English | MEDLINE | ID: mdl-31712626

ABSTRACT

Endothelin-1 (ET-1) is an important modulator of the vascular tone and a proinflammatory molecule that contributes to the vascular damage observed in hypertension. Peroxisome-proliferator activated receptors-γ (PPARγ) agonists show cardioprotective properties by decreasing inflammatory molecules such as COX-2 and reactive oxygen species (ROS), among others. We investigated the possible modulatory effect of PPARγ activation on the vascular effects of ET-1 in hypertension. In spontaneously hypertensive rats (SHR), but not in normotensive rats, ET-1 enhanced phenylephrine-induced contraction through ETA by a mechanism dependent on activation of TP receptors by COX-2-derived prostacyclin and reduction in NO bioavailability due to enhanced ROS production. In SHR, the PPARγ agonist pioglitazone (2.5 mg/Kg·day, 28 days) reduced the increased ETA levels and increased those of ETB. After pioglitazone treatment of SHR, ET-1 through ETB decreased ROS levels that resulted in increased NO bioavailability and diminished phenylephrine contraction. In vascular smooth muscle cells from SHR, ET-1 increased ROS production through AP-1 and NFκB activation, leading to enhanced COX-2 expression. These effects were blocked by pioglitazone. In summary, in hypertension, pioglitazone shifts the vascular ETA/ETB ratio, reduces ROS/COX-2 activation and increases NO availability; these changes explain the effect of ET-1 decreasing phenylephrine-induced contraction.


Subject(s)
Endothelin-1/metabolism , Hypertension/drug therapy , Hypoglycemic Agents/pharmacology , Muscle Contraction/drug effects , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Pioglitazone/pharmacology , Animals , Hypertension/metabolism , Hypertension/pathology , Male , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Oxidative Stress/drug effects , PPAR gamma/metabolism , Rats , Rats, Inbred SHR , Rats, Wistar , Reactive Oxygen Species/metabolism
10.
J Food Biochem ; 43(12): e13062, 2019 12.
Article in English | MEDLINE | ID: mdl-31571257

ABSTRACT

Metabolic Syndrome (MS) is related to increased risk of early death due to cardiovascular complications, among others. Dietary intervention has been suggested as the safest and most cost-effective alternative for treatment of those alterations in patients with MS. The aim of this study was to investigate the effects of different egg white hydrolysates (HEW1 and HEW2) in obese Zucker rats, focus on the development of cardiovascular complications. Blood pressure, heart rate, basal cardiac function and vascular reactivity in aorta and mesenteric resistance arteries were evaluated. Reactive oxygen species production by dihydroethidium-emitted fluorescence, NOX-1 mRNA levels by qRT-PCR, angiotensin-converting enzyme activity by fluorimetry and kidney histopathology were also analysed. Both hydrolysates improve the endothelial dysfunction occurring in resistance arteries. Additionally, HEW2 reduced vascular oxidative stress. PRACTICAL APPLICATIONS: Egg white is a good source of bioactive peptides, some of them with high antioxidant activity. They may be used as functional foods ingredients and could serve as an alternative therapeutic option to decrease some Metabolic Syndrome-related complications. This study suggests that these hydrolysates could be an interesting non-pharmacological tool to control cardiovascular complications related to Metabolic Syndrome.


Subject(s)
Antioxidants/metabolism , Aorta/drug effects , Blood Pressure/drug effects , Egg White/chemistry , Mesenteric Arteries/drug effects , Obesity/complications , Oxidative Stress/drug effects , Protein Hydrolysates/metabolism , Animals , Rats , Rats, Zucker
11.
Environ Res ; 155: 182-192, 2017 05.
Article in English | MEDLINE | ID: mdl-28222365

ABSTRACT

This study aimed to verify whether a prolonged exposure to low-level mercury promotes haemodynamic disorders and studied the reversibility of this vascular damage. Rats were divided into seven groups: three control groups received saline solution (im) for 30, 60 or 90 days; two groups received HgCl2 (im, first dose, 4.6µg/kg, subsequent doses 0.07µg/kg/day) for 30 or 60 days; two groups received HgCl2 for 30 or 60 days (im, same doses) followed by a 30-day washout period. Systolic blood pressure (SBP) was measured, along with analysis of vascular response to acetylcholine (ACh) and phenylephrine (Phe) in the absence and presence of endothelium, a nitric oxide (NO) synthase inhibitor, an NADPH oxidase inhibitor, superoxide dismutase, a non-selective cyclooxygenase (COX) inhibitor and an AT1 receptor blocker. Reactive oxygen species (ROS) levels and antioxidant power were measured in plasma. HgCl2 exposure for 30 and 60 days: a) reduced the endothelium-dependent relaxation; b) increased the Phe-induced contraction and the contribution of ROS, COX-derived vasoconstrictor prostanoids and angiotensin II acting on AT1 receptors to this response while the NO participation was reduced; c) increased the oxidative stress in plasma; d) increased the SBP only after 60 days of exposure. After the cessation of HgCl2 exposure, SBP, endothelium-dependent relaxation, Phe-induced contraction and the oxidative stress were normalised, despite the persistence of the increased COX-derived prostanoids. These results demonstrated that long-term HgCl2 exposure increases SBP as a consequence of vascular dysfunction; however, after HgCl2 removal from the environment the vascular function ameliorates.


Subject(s)
Environmental Pollutants/toxicity , Mercury/toxicity , Acetylcholine/pharmacology , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiology , Blood Pressure/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Environmental Pollutants/blood , Environmental Pollutants/pharmacokinetics , In Vitro Techniques , Male , Mercury/blood , Mercury/pharmacokinetics , Oxidative Stress/drug effects , Phenylephrine/pharmacology , Rats, Wistar , Reactive Oxygen Species/metabolism , Vasoconstrictor Agents/pharmacology , Vasodilator Agents/pharmacology
12.
J Hypertens ; 34(2): 253-65, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26682942

ABSTRACT

OBJECTIVE: NOX-1 and NOX-4 are key enzymes responsible for reactive oxygen species (ROS) generation in vascular smooth muscle cells (VSMC). The RNA-binding protein Hu antigen R (HuR) is implicated in posttranscriptional regulation of gene expression; however, its role regulating NOX is unknown. We investigated transcriptional and posttranscriptional mechanisms underlying angiotensin II (AngII) and IL-1ß regulation of NOX-1 and NOX-4 in VSMC and their implications in cell migration. METHODS: Rat and human VSMC were stimulated with AngII (0.1 µmol/l) and/or IL-1ß (10 ng/ml). NOX-1 and NOX-4 mRNA and protein levels, NOX-1 and NOX-4 promoter and 3'UTR activities, NADPH oxidase activity, ROS production, and cell migration were studied. RESULTS: IL-1ß increased NOX-1 expression, NADPH oxidase activity and ROS production, and decreased NOX-4 expression and H2O2 production in VSMC. AngII potentiated the IL-1ß-mediated induction of NOX-1 expression, NADPH oxidase activity, ROS production, and cell migration. However, AngII did not influence IL-1ß-induced NOX-4 downregulation. AngII + IL-1ß interfered with the decay of NOX-1 mRNA and promoted HuR binding to NOX-1 mRNA. Moreover, HuR blockade reduced NOX-1 mRNA stability and AngII + IL-1ß-induced NOX-1 mRNA levels. IL-1ß decreased NOX-4 expression through a transcriptional mechanism that involved response elements situated in the proximal promoter. AngII and/or IL-1ß-induced cell migration were prevented by NOX-1 and HuR blockade and were augmented by NOX-4 overexpression. CONCLUSION: In VSMC HuR-mediated mRNA stabilization is partially responsible for AngII + IL-1ß-dependent NOX-1 expression, whereas transcriptional mechanisms are involved in decreased NOX-4 expression induced by IL-1ß. NOX4 and HuR regulation of NOX-1 contributes to VSMC migration, important in vascular inflammation and remodeling.


Subject(s)
Angiotensin II/pharmacology , ELAV-Like Protein 1/metabolism , Interleukin-1beta/pharmacology , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Animals , Cell Movement/drug effects , Cell Movement/genetics , Cells, Cultured , ELAV-Like Protein 1/antagonists & inhibitors , Gene Expression Regulation , Humans , Hydrogen Peroxide/metabolism , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , NADH, NADPH Oxidoreductases/drug effects , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , NADPH Oxidase 1 , NADPH Oxidase 4 , NADPH Oxidases/drug effects , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , RNA Stability/drug effects , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Reactive Oxygen Species/metabolism
13.
PLoS One ; 9(8): e104020, 2014.
Article in English | MEDLINE | ID: mdl-25093580

ABSTRACT

Hypertension is considered as a low-grade inflammatory disease, with adaptive immunity being an important mediator of this pathology. TLR4 may have a role in the development of several cardiovascular diseases; however, little is known about its participation in hypertension. We aimed to investigate whether TLR4 activation due to increased activity of the renin-angiotensin system (RAS) contributes to hypertension and its associated endothelial dysfunction. For this, we used aortic segments from Wistar rats treated with a non-specific IgG (1 µg/day) and SHRs treated with losartan (15 mg/kg·day), the non-specific IgG or the neutralizing antibody anti-TLR4 (1 µg/day), as well as cultured vascular smooth muscle cells (VSMC) from Wistar and SHRs. TLR4 mRNA levels were greater in the VSMC and aortas from SHRs compared with Wistar rats; losartan treatment reduced those levels in the SHRs. Treatment of the SHRs with the anti-TLR4 antibody: 1) reduced the increased blood pressure, heart rate and phenylephrine-induced contraction while it improved the impaired acetylcholine-induced relaxation; 2) increased the potentiation of phenylephrine contraction after endothelium removal; and 3) abolished the inhibitory effects of tiron, apocynin and catalase on the phenylephrine-induced response as well as its enhancing effect of acetylcholine-induced relaxation. In SHR VSMCs, angiotensin II increased TLR4 mRNA levels, and losartan reduced that increase. CLI-095, a TLR4 inhibitor, mitigated the increases in NAD(P)H oxidase activity, superoxide anion production, migration and proliferation that were induced by angiotensin II. In conclusion, TLR4 pathway activation due to increased RAS activity is involved in hypertension, and by inducing oxidative stress, this pathway contributes to the endothelial dysfunction associated with this pathology. These results suggest that TLR4 and innate immunity may play a role in hypertension and its associated end-organ damage.


Subject(s)
Angiotensin II/pharmacology , Aorta/physiopathology , Hypertension/physiopathology , Reactive Oxygen Species/metabolism , Toll-Like Receptor 4/metabolism , Up-Regulation/drug effects , Acetylcholine/pharmacology , Animals , Antioxidants/pharmacology , Aorta/drug effects , Aorta/pathology , Blood Pressure/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Gene Expression Regulation/drug effects , Heart Rate/drug effects , Hypertension/genetics , Hypertension/pathology , In Vitro Techniques , Male , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , NADPH Oxidases/metabolism , Oxidative Stress/drug effects , Phenylephrine/pharmacology , Rats, Inbred SHR , Rats, Wistar , Superoxides/metabolism , Toll-Like Receptor 4/antagonists & inhibitors , Toll-Like Receptor 4/genetics , Vasoconstriction/drug effects , Vasodilation/drug effects
14.
Am J Physiol Heart Circ Physiol ; 306(11): H1582-93, 2014 Jun 01.
Article in English | MEDLINE | ID: mdl-24727493

ABSTRACT

Glitazones have anti-inflammatory properties by interfering with the transcription of proinflammatory genes, such as cyclooxygenase (COX)-2, and with ROS production, which are increased in hypertension. This study analyzed whether pioglitazone modulates COX-2 expression in hypertension by interfering with ROS and endothelin (ET)-1. In vivo, pioglitazone (2.5 mg·kg(-1)·day(-1), 28 days) reduced the greater levels of COX-2, pre-pro-ET-1, and NADPH oxidase (NOX) expression and activity as well as O2 (·-) production found in aortas from spontaneously hypertensive rats (SHRs). ANG II increased COX-2 and pre-pro-ET-1 levels more in cultured vascular smooth muscle cells from hypertensive rats compared with normotensive rats. The ETA receptor antagonist BQ-123 reduced ANG II-induced COX-2 expression in SHR cells. ANG II also increased NOX-1 expression, NOX activity, and superoxide production in SHR cells; the selective NOX-1 inhibitor ML-171 and catalase reduced ANG II-induced COX-2 and ET-1 transcription. ANG II also increased c-Jun transcription and phospho-JNK1/2, phospho-c-Jun, and p65 NF-κB subunit nuclear protein expression. SP-600125 and lactacystin, JNK and NF-κB inhibitors, respectively, reduced ANG II-induced ET-1, COX-2, and NOX-1 levels and NOX activity. Pioglitazone reduced the effects of ANG II on NOX activity, NOX-1, pre-pro-ET-1, COX-2, and c-Jun mRNA levels, JNK activation, and nuclear phospho-c-Jun and p65 expression. In conclusion, ROS production and ET-1 are involved in ANG II-induced COX-2 expression in SHRs, explaining the greater COX-2 expression observed in this strain. Furthermore, pioglitazone inhibits ANG II-induced COX-2 expression likely by interfering with NF-κB and activator protein-1 proinflammatory pathways and downregulating ROS production and ET-1 transcription, thus contributing to the anti-inflammatory properties of glitazones.


Subject(s)
Angiotensin II/pharmacology , Cyclooxygenase 2/metabolism , Endothelin-1/metabolism , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Reactive Oxygen Species/metabolism , Thiazolidinediones/pharmacology , Transcription, Genetic/drug effects , Animals , Aorta/cytology , Aorta/drug effects , Aorta/metabolism , Endothelin-1/genetics , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , NADPH Oxidases/metabolism , Pioglitazone , Rats , Rats, Inbred SHR , Rats, Inbred WKY
15.
Clin Sci (Lond) ; 126(2): 111-21, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24059588

ABSTRACT

Elevated production of prostanoids from the constitutive (COX-1) or inducible (COX-2) cyclo-oxygenases has been involved in the alterations in vascular function, structure and mechanical properties observed in cardiovascular diseases, including hypertension. In addition, it is well known that production of ROS (reactive oxygen species) plays an important role in the impaired contractile and vasodilator responses, vascular remodelling and altered vascular mechanics of hypertension. Of particular interest is the cross-talk between NADPH oxidase and mitochondria, the main ROS sources in hypertension, which may represent a vicious feed-forward cycle of ROS production. In recent years, there is experimental evidence showing a relationship between ROS and COX-derived products. Thus ROS can activate COX and the COX/PG (prostaglandin) synthase pathways can induce ROS production through effects on different ROS generating enzymes. Additionally, recent evidence suggests that the COX-ROS axis might constitute a vicious circle of self-perpetuating vasoactive products that have a pathophysiological role in altered vascular contractile and dilator responses and hypertension development. The present review discusses the current knowledge on the role of oxidative stress and COX-derived prostanoids in the vascular alterations observed in hypertension, highlighting new findings indicating that these two pathways act in concert to induce vascular dysfunction.


Subject(s)
Hypertension/physiopathology , Prostaglandin-Endoperoxide Synthases/metabolism , Reactive Oxygen Species/metabolism , Animals , Cardiovascular System/physiopathology , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/metabolism , Humans , Mitochondria/metabolism , NADPH Oxidases/physiology , Prostaglandins/physiology
16.
Curr Hypertens Rep ; 15(3): 204-14, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23519745

ABSTRACT

The regular practice of physical activity is a well-recommended strategy for the prevention and treatment of several cardiovascular and metabolic diseases. Physical exercise prevents the progression of vascular diseases and reduces cardiovascular morbidity and mortality. Exercise training also ameliorates vascular changes including endothelial dysfunction and arterial remodeling and stiffness, usually present in type 2 diabetes, obesity, hypertension and metabolic syndrome. Common to these diseases is excessive oxidative stress, which plays an important role in the processes underlying vascular changes. At the vascular level, exercise training improves the redox state and consequently NO availability. Moreover, growing evidence indicates that other mediators such as prostanoids might be involved in the beneficial effects of exercise. The purpose of this review is to update recent findings describing the adaptation response induced by exercise in cardiovascular and metabolic diseases, focusing more specifically on the beneficial effects of exercise in the vasculature and the underlying mechanisms.


Subject(s)
Cardiovascular Diseases/metabolism , Cardiovascular System/metabolism , Diabetes Mellitus, Type 2/metabolism , Exercise/physiology , Metabolic Syndrome/metabolism , Cardiovascular Diseases/prevention & control , Diabetes Mellitus, Type 2/prevention & control , Humans , Metabolic Syndrome/prevention & control , Obesity/metabolism , Obesity/prevention & control
17.
Br J Pharmacol ; 166(4): 1303-19, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22220498

ABSTRACT

BACKGROUND AND PURPOSE: PPARγ agonists, glitazones, have cardioprotective and anti-inflammatory actions associated with gene transcription interference. In this study, we determined whether chronic treatment of adult spontaneously hypertensive rats (SHR) with pioglitazone alters BP and vascular structure and function, and the possible mechanisms involved. EXPERIMENTAL APPROACH: Mesenteric resistance arteries from untreated or pioglitazone-treated (2.5 mg·kg⁻¹ ·day⁻¹ , 28 days) SHR and normotensive [Wistar Kyoto (WKY)] rats were used. Vascular structure was studied by pressure myography, vascular function by wire myography, protein expression by Western blot and immunohistochemistry, mRNA levels by RT-PCR, prostanoid levels by commercial kits and reactive oxygen species (ROS) production by dihydroethidium-emitted fluorescence. KEY RESULTS: In SHR, pioglitazone did not modify either BP or vascular structural and mechanical alterations or phenylephrine-induced contraction, but it increased vascular COX-2 levels, prostacyclin (PGI2) production and the inhibitory effects of NS 398, SQ 29,548 and tranylcypromine on phenylephrine responses. The contractile phase of the iloprost response, which was reduced by SQ 29,548, was greater in pioglitazone-treated and pioglitazone-untreated SHR than WKY. In addition, pioglitazone abolished the increased vascular ROS production, NOX-1 levels and the inhibitory effect of apocynin and allopurinol on phenylephrine contraction, whereas it did not modify eNOS expression but restored the potentiating effect of N-nitro-L-arginine methyl ester on phenylephrine responses. CONCLUSIONS AND IMPLICATIONS: Although pioglitazone did not reduce BP in SHR, it increased COX-2-derived PGI2 production, reduced oxidative stress, and increased NO bioavailability, which are all involved in vasoconstrictor responses in resistance arteries. These effects would contribute to the cardioprotective effect of glitazones reported in several pathologies.


Subject(s)
Cyclooxygenase 2/metabolism , Epoprostenol/metabolism , Hypertension/drug therapy , Mesenteric Arteries/drug effects , Oxidative Stress/drug effects , PPAR gamma/agonists , Thiazolidinediones/therapeutic use , Animals , Antihypertensive Agents/therapeutic use , Cyclooxygenase 2/genetics , Cyclooxygenase Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Hypertension/metabolism , Hypertension/physiopathology , Male , Mesenteric Arteries/metabolism , Mesenteric Arteries/physiopathology , Nitric Oxide/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , Pioglitazone , RNA, Messenger/metabolism , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Reactive Oxygen Species/metabolism , Up-Regulation/drug effects , Vasodilation/drug effects
18.
J Hypertens ; 30(2): 315-26, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22179086

ABSTRACT

AIMS: Hypertension is associated with increased plasma inflammatory markers such as cytokines and increased vascular cyclooxygenase-2 (COX-2) expression. The ability of peroxisome proliferator-activated receptor-γ (PPARγ) agonists to reduce oxidative stress seems to contribute to their anti-inflammatory properties. This study analyzes the effect of pioglitazone, a PPARγ agonist, on interleukin-1ß-induced COX-2 expression and the role of reactive oxygen species (ROS) on this effect. METHODS AND RESULTS: Vascular smooth muscle cells from hypertensive rats stimulated with interleukin-1ß (10 ng/ml, 24 h) were used. Interleukin-1ß increased: 1) COX-2 protein and mRNA levels; 2) protein and mRNA levels of the NADPH oxidase subunit NOX-1, NADPH oxidase activity and ROS production; and 3) phosphorylation of inhibitor of nuclear factor kappa B (IκB) kinase (IKK) nuclear expression of the p65 nuclear factor kappa B (NF-κB) subunit and cell proliferation, all of which were reduced by apocynin (30 µmol/l). Interleukin-1ß-induced COX-2 expression was reduced by apocynin, tempol (10 µmol/l), catalase (1000 U/ml) and lactacystin (5 µmol/l). Moreover, H2O2 (50 µmol/l, 90 min) induced COX-2 expression, which was reduced by lactacystin. Pioglitazone (10 µmol/l) reduced the effects of interleukin-1ß on: 1) COX-2 protein and mRNA levels; 2) NOX-1 protein and mRNA levels, NADPH oxidase activity and ROS production; and 3) p-IKK, p65 expressions and cell proliferation. Pioglitazone also reduced the H2O2-induced COX-2 expression and increased Cu/Zn and Mn-superoxide dismutase protein expression. PPARγ small interfering RNA (5 nmol/l) further increased interleukin-1ß-induced COX-2 and NOX-1 mRNA levels. In addition, pioglitazone increased the interleukin-1ß-induced PPARγ mRNA levels. CONCLUSION: PPARγ activation with pioglitazone reduces interleukin-1ß-induced COX-2 expression by interference with the redox-sensitive transcription factor NF-κB.


Subject(s)
Cyclooxygenase 2/metabolism , Muscle, Smooth, Vascular/enzymology , Oxidative Stress , PPAR gamma/agonists , Animals , Blotting, Western , Cells, Cultured , Interleukin-1beta/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , NF-kappa B/metabolism , Pioglitazone , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , Thiazolidinediones/pharmacology
19.
J Hypertens ; 27(9): 1814-22, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19491705

ABSTRACT

OBJECTIVE: To analyse the role of angiotensin II, via AT1 receptors, and oxidative stress in the mechanisms underlying the increased response to hydrogen peroxide (H2O2) of mesenteric resistance arteries from spontaneously hypertensive rats (SHRs). METHODS: Arteries from normotensive and SHRs untreated or treated with the AT1 receptor antagonist, losartan (15 mg/kg per day, 12 weeks), or with the superoxide dismutase analogue, tempol (1 mmol/l, 17 days), were used. Arteries were mounted in microvascular myographs for isometric tension recording; superoxide anion (O2(*-)) production was evaluated by dihydroethidium fluorescence, thromboxane A2 production by enzyme immunoassay and plasma nitrite levels by the Griess method. RESULTS: H2O2 (1-100 micromol/l) induced higher contractile responses in mesenteric resistance arteries from hypertensive than normotensive rats. In SHRs, losartan and tempol treatments induced the following effects: normalized the increased H2O2 contractile responses observed; modified neither the inhibitory effects of the cyclooxygenase inhibitor, indomethacin [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3-acetic acid] (1 micromol/l), and the thromboxane A2/prostaglandin H2 receptor antagonist, SQ 29 548 (1 micromol/l), on H2O2 contraction, nor the increase in thromboxane A2 production in response to H2O2; abolished the increased vascular O2(*-) production; increased both the potentiatory effect of the nitric oxide inhibitor, N(G)-nitro-L-arginine methyl ester (100 micromol/l), on H2O2 responses and the acetylcholine-induced relaxation. Moreover, losartan treatment abolished the effect of the O2(*-) scavenger, tiron (1 mmol/l), on H2O2 responses and increased plasma nitrite levels. CONCLUSION: Nitric oxide removal by an excessive O2(*-) production, probably from an upregulated renin-angiotensin system, participates in the increased response to H2O2 in mesenteric resistance arteries from SHRs.


Subject(s)
Hydrogen Peroxide/metabolism , Hypertension/physiopathology , Mesenteric Arteries/physiopathology , Oxidative Stress , Renin-Angiotensin System , Vasoconstriction , Acetylcholine , Animals , Antihypertensive Agents/pharmacology , Antioxidants/pharmacology , Cyclic N-Oxides/pharmacology , In Vitro Techniques , Losartan/pharmacology , Male , Prostaglandins/metabolism , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Reactive Oxygen Species/metabolism , Spin Labels , Vasodilation , Vasodilator Agents/pharmacology
20.
Hypertension ; 54(1): 142-9, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19451411

ABSTRACT

Angiotensin II (Ang II) modulates vasomotor tone, cell growth, and extracellular matrix deposition. This study analyzed the effect of atorvastatin in the possible alterations induced by Ang II on structure and mechanics of mesenteric resistance arteries and the signaling mechanisms involved. Wistar rats were infused with Ang II (100 ng/kg per day, SC minipumps, 2 weeks) with or without atorvastatin (5 mg/kg per day). Ang II increased blood pressure and plasmatic malondialdehyde levels. Compared with controls, mesenteric resistance arteries from Ang II-treated rats showed the following: (1) decreased lumen diameter; (2) increased wall/lumen; (3) decreased number of adventitial, smooth muscle, and endothelial cells; (4) increased stiffness; (5) increased collagen deposition; and (6) diminished fenestrae area and number in the internal elastic lamina. Atorvastatin did not alter blood pressure but reversed all of the structural and mechanical alterations of mesenteric arteries, including collagen and elastin alterations. In mesenteric resistance arteries, Ang II increased vascular O(2)(.-) production and diminished endothelial NO synthase and CuZn/superoxide dismutase but did not modify extracellular-superoxide dismutase expression. Atorvastatin improved plasmatic and vascular oxidative stress, normalized endothelial NO synthase and CuZn/superoxide dismutase expression, and increased extracellular-superoxide dismutase expression, showing antioxidant properties. Atorvastatin also diminished extracellular signal-regulated kinase 1/2 activation caused by Ang II in these vessels, indicating an interaction with Ang II-induced intracellular responses. In vascular smooth muscle cells, collagen type I release mediated by Ang II was reduced by different antioxidants and statins. Moreover, atorvastatin downregulated the Ang II-induced NADPH oxidase subunit, Nox1, expression. Our results suggest that statins might exert beneficial effects on hypertension-induced vascular remodeling by improving vascular structure, extracellular matrix alterations, and vascular stiffness. These effects might be mediated by their antioxidant properties.


Subject(s)
Angiotensin II/pharmacology , Heptanoic Acids/pharmacology , Mesenteric Arteries/drug effects , Oxidative Stress/drug effects , Pyrroles/pharmacology , Animals , Atorvastatin , Blood Pressure/drug effects , Blotting, Western , Cells, Cultured , Collagen/metabolism , Elasticity/drug effects , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Female , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , In Vitro Techniques , Malondialdehyde/blood , Mesenteric Arteries/metabolism , Mesenteric Arteries/physiology , Microscopy, Confocal , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Nitric Oxide Synthase Type III/metabolism , Rats , Rats, Sprague-Dawley , Rats, Wistar , Superoxide Dismutase/metabolism , Superoxides/metabolism , Vasoconstrictor Agents/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL