CETP expression ameliorates endothelial function in female mice through estrogen receptor-α and endothelial nitric oxide synthase pathway.

Endothelial dysfunction is an early manifestation of atherosclerosis. The cholesteryl ester transfer protein (CETP) has been considered proatherogenic by reducing plasma HDL levels. However, CETP may exhibit cell- or tissue-specific effects. We have previously reported that male mice expressing the human CETP gene show impaired endothelium-mediated vascular relaxation associated with oxidative stress. Although sexual dimorphisms on the metabolic role of CETP have been proposed, possible sex differences in the vascular effects of CETP were not previously studied. Thus, here we investigated the endothelial function of female CETP transgenic mice as compared with nontransgenic controls (NTg). Aortas from CETP females presented preserved endothelium-dependent relaxation to acetylcholine and an endothelium-dependent reduction of phenylephrine-induced contraction. eNOS phosphorylation (Ser1177) and calcium-induced NO levels were enhanced, whereas reactive oxygen species (ROS) production and NOX2 and SOD2 expression were reduced in the CETP female aortas. Furthermore, CETP females exhibited increased aortic relaxation to 17ß-estradiol (E2) and upregulation of heat shock protein 90 (HSP90) and caveolin-1, proteins that stabilize estrogen receptor (ER) in the caveolae. Indeed, CETP females showed an increased E2-induced relaxation in a manner sensitive to estrogen receptor-α (ERα) and HSP90 inhibitors methylpiperidinopyrazole (MPP) and geldanamycin, respectively. MPP also impaired the relaxation response to acetylcholine in CETP but not in NTg females. Altogether, the study indicates that CETP expression ameliorates the anticontractile endothelial effect and relaxation to E2 in females. This was associated with less ROS production, and increased eNOS-NO and E2-ERα pathways. These results highlight the need for considering the sex-specific effects of CETP on cardiovascular risk.NEW & NOTEWORTHY Here we demonstrated that CETP expression has a sex-specific impact on the endothelium function. Contrary to what was described for males, CETP-expressing females present preserved endothelium-dependent relaxation to acetylcholine and improved relaxation response to 17ß-estradiol. This was associated with less ROS production, increased eNOS-derived NO, and increased expression of proteins that stabilize estrogen receptor-α (ERα), thus increasing E2-ERα signaling sensitivity. These results highlight the need for considering the sex-specific effects of CETP on cardiovascular risk.

Modulation of endothelium-derived nitric oxide production and activity by taurine and taurine-conjugated bile acids.

Taurine is a semiessential amino acid found at high concentrations in mammalian plasma and cells, where it regulates cellular functions such as ion flux, controls cell volume and serves as a substrate for conjugated bile acids (BAs). Exogenous administration of both taurine and taurine-conjugated BAs have also been implicated in the modulation of cardiovascular functions. This brief review summarizes the role of taurine and taurine-conjugated BAs in vascular relaxation through the modulation of endothelium-derived nitric oxide (NO). The effects of taurine on vascular health are controversial. However, in the presence of cardiometabolic risk factors, it has been proposed that taurine can increase vascular NO levels by increasing eNOS expression, eNOS phosphorylation on Ser1177, NO bioavailability, the level of antioxidative defense, and the l-arginine/NOS inhibitor asymmetric dimethylarginine (ADMA) ratio. The taurine-conjugated BA-mediated activation of Farnesoid X receptor (FXR), G protein-coupled BA receptor (TGR5) and/or muscarinic 3 receptor (M3) was also reported to increase vascular NO production. FXR activation increases eNOS expression and may reduce ADMA formation, while TGR5 increases mobilization of Ca2+ and phosphorylation of eNOS and Akt in endothelial cells. Furthermore, taurine and taurine-conjugated BAs might regulate NO synthesis and activity by enhancing H2S generation. Several studies have demonstrated the beneficial effects of both taurine and taurine-conjugated BAs in reversing the endothelial dysfunction associated with diabetes, atherosclerosis, hypertension, obesity, malnutrition, and smoking. In addition, taurine-conjugated BAs have emerged as a potential treatment for portal hypertension. Despite these favorable findings, there is a need to further explore the mechanisms and signaling pathways underlying the endothelial effects of taurine and taurine-conjugated BAs. Here, we summarize the main findings regarding the effects of taurine and taurine-conjugated BAs on the endothelial dysfunction associated with altered NO metabolism in cardiovascular diseases.

New roles of aldosterone and mineralocorticoid receptors in cardiovascular disease: translational and sex-specific effects.

Recent advances in the field of mineralocorticoid receptor (MR) and its ligand aldosterone expanded the role of this hormone and its receptor far beyond their initial function as a regulator of Na+ and K+ homeostasis in epithelial cells. The symposium "New Roles of Aldosterone and Mineralocorticoid Receptors in Cardiovascular Disease: Translational and Sex-Specific Effects" presented at the 38th World Congress of the International Union of Physiological Sciences (Rio de Janeiro, Brazil) highlighted the contribution of extrarenal MRs to cardiovascular disease. This symposium showcased how MRs expressed in endothelial, vascular smooth muscle, and immune cells plays a critical role in the development of vascular disease associated with aging, obesity, and chronic aldosterone stimulation and demonstrated that MR antagonism prevents the acute renal dysfunction and tubular injury induced by ischemia-reperfusion injury. It was also shown that the adipocyte-derived hormone leptin is a new direct regulator of aldosterone secretion and that leptin-mediated aldosterone production is a major contributor to obesity-associated hypertension in women. Sex differences in the role of aldosterone and of endothelial MR in the cardiovascular outcomes of obesity were highlighted. This review summarizes these important emerging concepts regarding the contribution of aldosterone and cell-specific MR to cardiovascular disease in male and female subjects and further supports sex-specific benefits of MR antagonist drugs to be tested in additional populations.

Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum.

Impairment of nitric oxide (NO)-mediated cavernosal relaxations in middle age contributes to erectile dysfunction. However, little information is available about the alterations of sympathetic neurotransmission and contraction in erectile tissue at middle age. This study aimed to evaluate the alterations of the contractile machinery associated with tyrosine hydroxylase (TH) in rat corpus cavernosum (RCC) at middle age, focusing on the role of superoxide anion. Male Wistar young (3.5-mo) and middle-aged (10-mo) rats were used. Electrical-field stimulation (EFS)- and phenylephrine-induced contractions were obtained in RCC strips. Levels of reactive-oxygen species (ROS) and TH mRNA expression, as well as protein expressions for α1/ß1-subunits of soluble guanylyl cyclase (sGC), in RCC were evaluated. The neurogenic contractile responses elicited by EFS (4-32 Hz) were greater in RCC from the middle-aged group that was accompanied by elevated TH mRNA expression (P < 0.01). Phenylephrine-induced contractions were also greater in the middle-aged group. A 62% increase in ROS generation in RCC from middle-aged rats was observed. The mRNA expression for the α1A-adrenoceptor remained unchanged among groups. Protein levels of α1/ß1-sGC subunits were decreased in RCC from the middle-aged compared with young group. The NADPH oxidase inhibitor apocynin (85 mg·rat(-1)·day(-1), 4 wk) fully restored the enhanced ROS production, TH mRNA expressions, and α1/ß1-subunit sGC expression, indicating that excess of superoxide anion plays a major role in the sympathetic hyperactivity and hypercontractility in erectile tissue at middle age. Reduction of oxidative stress by dietary antioxidants may be an interesting approach to treat erectile dysfunction in aging population.

Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus.

BACKGROUND: Ouabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats. METHODS: Adult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 µg/kg) or saline 20 minutes before LPS (200 µg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured. RESULTS: OUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1ß, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain. CONCLUSION: Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

Soluble guanylyl cyclase (sGC) degradation and impairment of nitric oxide-mediated responses in urethra from obese mice: reversal by the sGC activator BAY 60-2770.

Obesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study investigated the USM relaxant machinery in obese mice and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 [acid 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4-(trifluoromethyl) biphenyl-4-yl] methoxy} phenyl) ethyl] amino} methyl) benzoic] rescues the urethral reactivity through improvement of sGC-cGMP (cyclic guanosine monophosphate) signaling. Male C57BL/6 mice were fed for 12 weeks with a high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg per day for 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS), and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO donors (S-nitrosoglutathione and glyceryl trinitrate), and BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine] (sGC stimulator) were markedly reduced in obese compared with lean mice. In contrast, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice (P < 0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (10 µM) potentiated BAY 60-2770-induced USM responses in the lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactive-oxygen species (ROS) production was enhanced, but protein expression of ß1 second guanylate cyclase subunit was reduced in USM from obese mice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and down-regulation of sGC-cGMP signaling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese mice.

The soluble guanylyl cyclase activator BAY 60-2770 ameliorates overactive bladder in obese mice.

PURPOSE: Activators of soluble guanylyl cyclase are of potential interest as treatment for cardiovascular diseases but to our knowledge they have never been proposed to treat overactive bladder. We evaluated the effects of the soluble guanylyl cyclase activator BAY 60-2270 on voiding dysfunction and detrusor overactivity in a mouse model of obesity associated overactive bladder. MATERIALS AND METHODS: C57BL/6 male mice fed for 10 weeks with standard chow or a high fat diet were treated with 1 mg/kg BAY 60-2770 per day for 2 weeks via gavage. Cystometric evaluations were done and responses to contractile agents in isolated bladders were determined. RESULTS: Obese mice showed an irregular micturition pattern characterized by significant increases in voiding and nonvoiding contractions, which were normalized by BAY 60-2770. Carbachol, KCl and CaCl2 produced concentration dependent contractions in isolated bladder strips, which were markedly greater in obese than in lean mice. BAY 60-2770 normalized bladder contractions in the obese group. A 78% increase in reactive oxygen species generation in the bladder tissue of obese mice was observed, which was unaffected by BAY 60-2770. Treatment with BAY 60-2770 generated a tenfold increase in cyclic guanosine monophosphate in the bladders of obese mice without affecting the nucleotide level in the lean group. Protein expression of the soluble guanylyl cyclase α1 and ß1 subunits was decreased 40% in the bladder tissue of obese mice but restored by BAY 60-2770. CONCLUSIONS: Two-week BAY 60-2770 therapy increased cyclic guanosine monophosphate and rescued expression of the soluble guanylyl cyclase α1 and ß1 subunits in bladder tissue, resulting in great amelioration of bladder dysfunction.

Prolonged therapy with the soluble guanylyl cyclase activator BAY 60-2770 restores the erectile function in obese mice.

INTRODUCTION: Cardiovascular and endocrine-metabolic diseases associated with increased oxidative stress such as obesity lead to erectile dysfunction (ED). Activators of soluble guanylyl cyclase (sGC) such as BAY 60-2770 reactivate the heme-oxidized sGC in vascular diseases. AIM: This study aimed to evaluate the effects of 2-week oral intake with BAY 60-2270 on a murine model of obesity-associated ED. METHODS: C57BL/6 male mice were fed for 12 weeks with standard chow or high-fat diet. Lean and obese mice were treated with BAY 60-2770 (1 mg/kg/day, 2 weeks). MAIN OUTCOME MEASURES: Measurements of intracavernosal pressure (ICP), along with acetylcholine (10(-9) to 10(-5) M) and electrical field stimulation (EFS; 4-10 Hz)-induced corpus cavernosum relaxations in vitro, were obtained. Levels of cyclic guanosine monophosphate (cGMP), reactive oxygen species (ROS), and sGC protein expressions in cavernosal tissues were measured. RESULTS: Cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly lower in obese compared with lean mice (P < 0.05). Two-week therapy with BAY 60-2770 fully reversed the decreased ICP in obese group. Acetylcholine-induced cavernosal relaxations were 45% lower (P < 0.001) in obese mice, which were fully restored by BAY 60-2770 treatment. Likewise, the EFS-induced relaxations in obese mice were restored by BAY 60-2770. Basal cGMP content in erectile tissue was 68% lower (P < 0.05) in obese mice, an effect normalized by BAY 60-2770. Levels of ROS were 52% higher (P < 0.05) whereas protein expression of α1 sGC subunit was reduced in cavernosal tissue of obese mice, both of which were normalized by BAY 60-2770. In lean group, BAY 60-2770 did not significantly affect any functional, biochemical, or molecular parameter analyzed. CONCLUSIONS: Two-week therapy with BAY 60-2770 restores the erectile function in obese mice that is associated with reduced ROS levels, up-regulation of α1 sGC subunit, and increased cGMP levels in the erectile tissue.

ß-Adrenergic Stimulation-Induced PVAT Dysfunction in Male Sex: A Role for 11ß-Hydroxysteroid Dehydrogenase-1.

Long-term ß-adrenoceptor (ß-AR) stimulation is a pathological mechanism associated with cardiovascular diseases resulting in endothelial and perivascular adipose tissue (PVAT) dysfunction. In this study, we aimed to identify whether ß-adrenergic signaling has a direct effect on PVAT. Thoracic aorta PVAT was obtained from male Wistar rats and cultured ex vivo with the ß-AR agonist isoproterenol (Iso; 1 µM) or vehicle for 24 hours. Conditioned culture medium (CCM) from Iso-treated PVAT induced a marked increase in aorta contractile response, induced oxidative stress, and reduced nitric oxide production in PVAT compared to vehicle. In addition, Iso-treated PVAT and PVAT-derived differentiated adipocytes exhibited higher corticosterone release and protein expression of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), an enzyme responsible for de novo synthesis of corticosterone. Macrophages exposed to Iso also exhibited increased corticosterone release in response to ß-AR stimulation. Incubation of Iso-treated PVAT and PVAT-derived differentiated adipocytes with ß3-AR antagonist restored aorta contractile function modulated by Iso-CCM and normalized 11ß-HSD1 protein expression. These results show that ß3-AR signaling leads to upregulation of 11ß-HSD1 in PVAT, thus increasing corticosterone release and contributing to impair the anticontractile function of this tissue.

Insights by which TUDCA is a potential therapy against adiposity.

Adipose tissue is an organ with metabolic and endocrine activity. White, brown and ectopic adipose tissues have different structure, location, and function. Adipose tissue regulates energy homeostasis, providing energy in nutrient-deficient conditions and storing it in high-supply conditions. To attend to the high demand for energy storage during obesity, the adipose tissue undergoes morphological, functional and molecular changes. Endoplasmic reticulum (ER) stress has been evidenced as a molecular hallmark of metabolic disorders. In this sense, the ER stress inhibitor tauroursodeoxycholic acid (TUDCA), a bile acid conjugated to taurine with chemical chaperone activity, has emerged as a therapeutic strategy to minimize adipose tissue dysfunction and metabolic alterations associated with obesity. In this review, we highlight the effects of TUDCA and receptors TGR5 and FXR on adipose tissue in the setting of obesity. TUDCA has been demonstrated to limit metabolic disturbs associated to obesity by inhibiting ER stress, inflammation, and apoptosis in adipocytes. The beneficial effect of TUDCA on perivascular adipose tissue (PVAT) function and adiponectin release may be related to cardiovascular protection in obesity, although more studies are needed to clarify the mechanisms. Therefore, TUDCA has emerged as a potential therapeutic strategy for obesity and comorbidities.

Spironolactone prevents alterations associated with cardiac hypertrophy produced by isoproterenol in rats: involvement of serum- and glucocorticoid-regulated kinase type 1.

Persistent ß-adrenergic receptor stimulation with isoproterenol is associated with cardiac hypertrophy as well as cardiac synthesis of angiotensin II. Serum- and glucocorticoid-regulated kinase type 1 (SGK-1) is a key mediator in structural, functional and molecular cardiac effects of aldosterone in rats. This study was designed to investigate the cardiac effects of the mineralocorticoid receptor antagonist spironolactone on the response to isoproterenol treatment in rats, as well as the involvement of the main mediator of cellular aldosterone action, SGK-1, in the heart. Male Wistar rats received isoproterenol (3 mg kg(-1) day(-1)) or vehicle for 15 days. Half of the animals in each group were simultaneously treated with spironolactone (200 mg kg(-1) day(-1)). Systolic and diastolic blood pressures were not significantly different among groups. Treatment with spironolactone normalized the increased left ventricular end-diastolic pressure observed in isoproterenol-treated rats. Isoproterenol treatment induced cardiac hypertrophy and increased collagen content, both of which were normalized by spironolactone treatment. The mRNA levels of transforming growth factor ß, connective tissue growth factor, matrix metalloprotease 2, matrix metalloprotease inhibitor 2, tumour necrosis factor α, interleukin 1ß, p22phox and xanthine dehydrogenase were increased (P < 0.05) in isoproterenol-treated rats, and this effect was prevented by spironolactone (P < 0.05). Spironolactone also reduced the elevated SGK-1 expression in isoproterenol-treated rats. The observed reduction of the principal mediator of aldosterone cellular actions, SGK-1, by spironolactone in hearts from isoproterenol-treated rats suggests a role of mineralocorticoids in the cardiac hypertrophy, fibrosis, inflammation, oxidation and diastolic dysfunction induced by isoproterenol treatment in rats.

Simvastatin protects against intestinal ischemia/reperfusion-induced pulmonary artery dysfunction.

AIMS: The lung is an important target organ damage in intestinal ischemia/reperfusion (II/R), but mechanisms involved in II/R-induced pulmonary artery (PA) dysfunction, as well as its treatment, are not clear. The present study aimed to investigate the mechanisms involved in the II/R-induced PA dysfunction and a possible protective role of acute simvastatin pretreatment. MAIN METHODS: Male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min followed by 2 h reperfusion (II/R) or sham-operated surgery (sham). In some rats, simvastatin (20 mg/kg, oral gavage) was administrated 1 h before II/R. KEY FINDINGS: II/R reduced acetylcholine-induced relaxation and phenylephrine-induced contraction of PA segments, which were prevented by acute simvastatin pretreatment in vivo or restored by inducible nitric oxide synthase (iNOS) inhibition in situ with 1400 W. Elevated reactive oxygen species (ROS) levels and higher nuclear translocation of nuclear factor kappa B (NFκB) subunit p65 were observed in PA of II/R rats and prevented by simvastatin. Moreover, simvastatin increased superoxide dismutase (SOD) activity and endothelial nitric oxide synthase (eNOS) expression in PA of the II/R group as well as prevented the increased levels of interleukin (IL)-1ß and IL-6 in lung explants following II/R. SIGNIFICANCE: The study suggests that pretreatment with a single dose of simvastatin prevents the II/R-induced increase of inflammatory factors and oxidative stress, as well as PA endothelial dysfunction and adrenergic hyporreactivity. Therefore, acute simvastatin administration could be therapeutic for pulmonary vascular disease in patients suffering from intestinal ischemic events.

Selective Pharmacological Inhibition of NOX2 by GSK2795039 Improves Bladder Dysfunction in Cyclophosphamide-Induced Cystitis in Mice.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disease without consistently effective treatment. Among the many mediators implicated in cystitis, the overproduction of reactive oxygen species (ROS) seems to play a key role, although the main source of ROS remains unclear. This study aimed to investigate the contribution of NADPH oxidase (NOX) isoforms in ROS generation and the voiding dysfunction of cyclophosphamide (CYP, 300 mg/Kg, ip, 24 h)-induced cystitis in adult female mice, a well-recognized animal model to study IC/BPS, by using GKT137831 (5 mg/Kg, ip, three times in a 24 h period) or GSK2795039 (5 mg/Kg, ip, three times in a 24 h period) to inhibit NOX1/4 or NOX2, respectively. Our results showed that treatment with GSK2795039 improved the dysfunctional voiding behavior induced by CYP, reduced bladder edema and inflammation, and preserved the urothelial barrier integrity and tight junction occludin expression, besides inhibiting the characteristic vesical pain and bladder superoxide anion generation. In contrast, the NOX1/4 inhibitor GKT137831 had no significant protective effects. Taken together, our in vivo and ex vivo data demonstrate that NOX2 is possibly the main source of ROS observed in cystitis-induced CYP in mice. Therefore, selective inhibition of NOX2 by GSK2795039 may be a promising target for future therapies for IC/BPS.

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 (10mg 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.

Long-term ouabain treatment impairs vascular function in resistance arteries.

BACKGROUND/AIMS: The purpose of this study was to examine the cardiovascular effects of long-term ouabain treatment at different time points. METHODS: Systolic blood pressure (SBP) was measured by tail-cuff method in male Wistar rats treated with ouabain (approx. 8.0 µg·day(-1)) or vehicle for 5, 10 and 20 weeks. Afterwards, vascular function was assessed in mesenteric resistance arteries (MRA) using a wire myograph. ROS production and COX-1 and COX-2, TNF-α, and IL-6 protein expression were investigated. RESULTS: SBP was increased by ouabain treatment up to the 6th week and remained stable until the 20th week. However, noradrenaline-induced contraction increased only in MRA in rats treated with ouabain for 20 weeks. NOS inhibition and endothelium removal increased the noradrenaline response, but to a smaller magnitude in MRA in the ouabain group. Moreover, inhibition of COX-2 or incubation with superoxide dismutase restores noradrenaline-induced contraction in the 20-week ouabain group to control levels. ROS production as well as COX-2, IL-6 and TNF-α protein expression increased in MRA in this group. CONCLUSION: Although ouabain treatment induced hypertension in all groups, a larger noradrenaline induced contraction was observed over 20 weeks of treatment. This vascular dysfunction was related to COX-2-derived prostanoids and oxidative stress, increased pro- inflammatory cytokines and reduced NO bioavailability.

Chronic cyclooxygenase-2 inhibition prevents the worsening of hypertension and endothelial dysfunction induced by ouabain in resistance arteries of spontaneously hypertensive rats.

AIM: Previous studies raise cyclooxygenase (COX) activation as a possible mechanism involved in the pathophysiology of ouabain-induced hypertension. We hypothesized that inhibition of COX-2 activity might prevent ouabain-induced vascular dysfunction and worsening of hypertension in spontaneously hypertensive rats (SHR). METHODS: SHR were exposed to ouabain or vehicle and treated or not with the selective COX-2 inhibitor nimesulide for 5 weeks. Systolic blood pressure was measured by plethysmography. Vascular reactivity by wire myograph and protein expression by Western-blot were assessed in mesenteric resistance arteries (MRA) of groups. Thromboxane A2 (TXA2) production by ELISA was evaluated in MRA supernatants of groups. RESULTS: Noradrenaline-induced maximal contraction (Emax) was greater in MRA from SHR receiving ouabain than those of vehicle group. In situ inhibition of COX-2, TXA2 synthase, or TP receptor reduced the Emax to noradrenaline in MRA of ouabain to vehicle levels. TXA2 production was higher in ouabain than in vehicle group. Ouabain enhanced expression of cytoplasmic tyrosine kinase Src (c-Src)/ERK1/2/COX-2/TXA2 synthase/TP receptor in SHR MRA, but did not change NFkB/iKB ratio. Anticontractile effect of nitric oxide (NO) was smaller in MRA from ouabain- than vehicle-treated SHR, as well as eNOS and nNOS expression. Nimesulide co-treatment prevented the ouabain-induced worsening of hypertension and noradrenaline MRA hypercontractility in SHR. CONCLUSION: Ouabain worsen hypertension and induce MRA hypercontractility in SHR associated with upregulated c-Src/ERK1/2/COX-2/TXA2 synthase/TXA2/TP receptor axis. These effects were prevented by COX-2 inhibition.

The Presence of Cholesteryl Ester Transfer Protein (CETP) in Endothelial Cells Generates Vascular Oxidative Stress and Endothelial Dysfunction.

Endothelial dysfunction precedes atherosclerosis and is an independent predictor of cardiovascular events. Cholesterol levels and oxidative stress are key contributors to endothelial damage, whereas high levels of plasma high-density lipoproteins (HDL) could prevent it. Cholesteryl ester transfer protein (CETP) is one of the most potent endogenous negative regulators of HDL-cholesterol. However, whether and to what degree CETP expression impacts endothelial function, and the molecular mechanisms underlying the vascular effects of CETP on endothelial cells, have not been addressed. Acetylcholine-induced endothelium-dependent relaxation of aortic rings was impaired in human CETP-expressing transgenic mice, compared to their non-transgenic littermates. However, endothelial nitric oxide synthase (eNOS) activation was enhanced. The generation of superoxide and hydrogen peroxide was increased in aortas from CETP transgenic mice, while silencing CETP in cultured human aortic endothelial cells effectively decreased oxidative stress promoted by all major sources of ROS: mitochondria and NOX2. The endoplasmic reticulum stress markers, known as GADD153, PERK, and ARF6, and unfolded protein response effectors, were also diminished. Silencing CETP reduced endothelial tumor necrosis factor (TNF) α levels, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression, diminishing monocyte adhesion. These results support the notion that CETP expression negatively impacts endothelial cell function, revealing a new mechanism that might contribute to atherosclerosis.

Ventricular Fibrosis and Coronary Remodeling Following Short-Term Exposure of Healthy and Malnourished Mice to Bisphenol A.

Bisphenol-A (BPA) is an endocrine disruptor associated with higher risk of insulin resistance, type 2 diabetes, and cardiovascular diseases especially in susceptible populations. Because malnutrition is a nutritional disorder associated with high cardiovascular risk, we sought to compare the effects of short-term BPA exposure on cardiovascular parameters of healthy and protein-malnourished mice. Postweaned male mice were fed a normo- (control) or low-protein (LP) diet for 8 weeks and then exposed or not to BPA (50 µg kg-1 day-1) for the last 9 days. Systolic blood pressure was higher in BPA or LP groups compared with the control group. However, diastolic blood pressure was enhanced by BPA only in malnourished mice. Left ventricle (LV) end diastolic pressure (EDP), collagen deposition, and CTGF mRNA expression were higher in the control or malnourished mice exposed to BPA than in the respective nonexposed groups. Nevertheless, mice fed LP diet exposed to BPA exhibited higher angiotensinogen and cardiac TGF-ß1 mRNA expression than mice treated with LP or BPA alone. Wall:lumen ratio and cross-sectional area of intramyocardial arteries were higher either in the LP or BPA group compared with the control mice. Taken together, our data suggest that short-term BPA exposure results in LV diastolic dysfunction and fibrosis, and intramyocardial arteries inward remodeling, besides potentiate protein malnutrition-induced hypertension and cardiovascular risk.

Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR.

Abnormalities in renal proximal tubular (PT) sodium transport play an important role in the pathophysiology of essential hypertension. The Na(+)/H(+) exchanger isoform 3 (NHE3) represents the major route for sodium entry across the apical membrane of renal PT cells. We therefore aimed to assess in vivo NHE3 transport activity and to define the molecular mechanisms underlying NHE3 regulation before and after development of hypertension in the spontaneously hypertensive rat (SHR). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo stationary microperfusion in PT from young prehypertensive SHR (Y-SHR; 5-wk-old), adult SHR (A-SHR; 14-wk-old), and age-matched Wistar Kyoto (WKY) rats. We found that NHE3-mediated PT bicarbonate reabsorption was reduced with age in the SHR (1.08 ± 0.10 vs. 0.41 ± 0.04 nmol/cm(2)×s), while it was increased in the transition from youth to adulthood in the WKY rat (0.59 ± 0.05 vs. 1.26 ± 0.11 nmol/cm(2)×s). Higher NHE3 activity in the Y-SHR compared with A-SHR was associated with a predominant microvilli confinement and a lower ratio of phosphorylated NHE3 at serine-552 to total NHE3 (P-NHE3/total). After development of hypertension, P-NHE3/total increased and NHE3 was retracted out of the microvillar microdomain along with the regulator dipeptidyl peptidase IV (DPPIV). Collectively, our data suggest that the PT is playing a role in adapting to the hypertension in the SHR. The molecular mechanisms of this adaptation possibly include an increase of P-NHE3/total and a redistribution of the NHE3-DPPIV complex from the body to the base of the PT microvilli, both predicted to decrease sodium reabsorption.