Acute Increase in O-GlcNAc Improves Survival in Mice With LPS-Induced Systemic Inflammatory Response Syndrome.

Sepsis is a systemic inflammatory response syndrome (SIRS) resulting from a severe infection that is characterized by immune dysregulation, cardiovascular derangements, and end-organ dysfunction. The modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation) influences many of the key processes that are altered during sepsis, including the production of inflammatory mediators and vascular contractility. Here, we investigated whether O-GlcNAc affects the inflammatory response and cardiovascular dysfunction associated with sepsis. Mice received an intraperitoneal injection of lipopolysaccharide (LPS, 20 mg/Kg) to induce endotoxic shock and systemic inflammation, resembling sepsis-induced SIRS. The effects of an acute increase in O-GlcNAcylation, by treatment of mice with glucosamine (GlcN, 300 mg/Kg, i.v.) or thiamet-G (ThG, 150 µg/Kg, i.v.), on LPS-associated mortality, production and release of cytokines by macrophages and vascular cells, vascular responsiveness to constrictors and blood pressure were then determined. Mice under LPS-induced SIRS exhibited a systemic and local inflammatory response with increased levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), as well as severe hypotension and vascular hyporesponsiveness, characterized by reduced vasoconstriction to phenylephrine. In addition, LPS increased neutrophil infiltration in lungs and produced significant lethality. Treatment with GlcN and ThG reduced systemic inflammation and attenuated hypotension and the vascular refractoriness to phenylephrine, improving survival. GlcN and ThG also decreased LPS-induced production of inflammatory cytokines by bone marrow-derived macrophages and nuclear transcription factor-kappa B (NF-κB) activation in RAW 264.7 NF-κB promoter macrophages. Treatment of mice with ThG increased O-glycosylation of NF-κB p65 subunit in mesenteric arteries, which was associated with reduced Ser536 phosphorylation of NF-κB p65. Finally, GlcN also increased survival rates in mice submitted to cecal ligation and puncture (CLP), a sepsis model. In conclusion, increased O-GlcNAc reduces systemic inflammation and cardiovascular disfunction in experimental sepsis models, pointing this pathway as a potential target for therapeutic intervention.

Mechanisms underlying sodium nitroprusside-induced tolerance in the mouse aorta: Role of ROS and cyclooxygenase-derived prostanoids.

AIMS: To determine the role of reactive oxygen species (ROS) on sodium nitroprusside (SNP)-induced tolerance. Additionally, we evaluated the role of ROS on NF-κB activation and pro-inflammatory cytokines production during SNP-induced tolerance. MAIN METHODS: To induce in vitro tolerance, endothelium-intact or -denuded aortic rings isolated from male Balb-c mice were incubated for 15, 30, 45 or 60min with SNP (10nmol/L). KEY FINDINGS: Tolerance to SNP was observed after incubation of endothelium-denuded, but not endothelium-intact aortas for 60min with this inorganic nitrate. Pre-incubation of denuded rings with tiron (superoxide anion (O2-) scavenger), and the NADPH oxidase inhibitors apocynin and atorvastatin reversed SNP-induced tolerance. l-NAME (non-selective NOS inhibitor) and l-arginine (NOS substrate) also prevented SNP-induced tolerance. Similarly, ibuprofen (non-selective cyclooxygenase (COX) inhibitor), nimesulide (selective COX-2 inhibitor), AH6809 (prostaglandin PGF2α receptor antagonist) or SQ29584 [PGH2/thromboxane TXA2 receptor antagonist] reversed SNP-induced tolerance. Increased ROS generation was detected in tolerant arteries and both tiron and atorvastatin reversed this response. Tiron prevented tolerance-induced increase on O2- and hydrogen peroxide (H2O2) levels. The increase onp65/NF-κB expression and TNF-α production in tolerant arteries was prevented by tiron. The major new finding of our study is that SNP-induced tolerance is mediated by NADPH-oxidase derived ROS and vasoconstrictor prostanoids derived from COX-2, which are capable of reducing the vasorelaxation induced by SNP. Additionally, we found that ROS mediate the activation of NF-κB and the production of TNF-α in tolerant arteries. SIGNIFICANCE: These findings identify putative molecular mechanisms whereby SNP induces tolerance in the vasculature.

Functional and structural changes in internal pudendal arteries underlie erectile dysfunction induced by androgen deprivation.

Androgen deficiency is strongly associated with erectile dysfunction (ED). Inadequate penile arterial blood flow is one of the major causes of ED. The blood flow to the corpus cavernosum is mainly derived from the internal pudendal arteries (IPAs); however, no study has evaluated the effects of androgen deprivation on IPA's function. We hypothesized that castration impairs IPAs reactivity and structure, contributing to ED. In our study, Wistar male rats, 8-week-old, were castrated and studied 30 days after orchiectomy. Functional and structural properties of rat IPAs were determined using wire and pressure myograph systems, respectively. Protein expression was determined by Western blot and immunohistochemistry. Plasma testosterone levels were determined using the IMMULITE 1000 Immunoassay System. Castrated rats exhibited impaired erectile function, represented by decreased intracavernosal pressure/mean arterial pressure ratio. IPAs from castrated rats exhibited decreased phenylephrine- and electrical field stimulation (EFS)-induced contraction and decreased acetylcholine- and EFS-induced vasodilatation. IPAs from castrated rats exhibited decreased internal diameter, external diameter, thickness of the arterial wall, and cross-sectional area. Castration decreased nNOS and α-actin expression and increased collagen expression, p38 (Thr180/Tyr182) phosphorylation, as well as caspase 3 cleavage. In conclusion, androgen deficiency is associated with impairment of IPA reactivity and structure and increased apoptosis signaling markers. Our findings suggest that androgen deficiency-induced vascular dysfunction is an event involving hypotrophic vascular remodeling of IPAs.

NLRP3 Inflammasome Mediates Aldosterone-Induced Vascular Damage.

BACKGROUND: Inflammation is a key feature of aldosterone-induced vascular damage and dysfunction, but molecular mechanisms by which aldosterone triggers inflammation remain unclear. The NLRP3 inflammasome is a pivotal immune sensor that recognizes endogenous danger signals triggering sterile inflammation. METHODS: We analyzed vascular function and inflammatory profile of wild-type (WT), NLRP3 knockout (NLRP3-/-), caspase-1 knockout (Casp-1-/-), and interleukin-1 receptor knockout (IL-1R-/-) mice treated with vehicle or aldosterone (600 µg·kg-1·d-1 for 14 days through osmotic mini-pump) while receiving 1% saline to drink. RESULTS: Here, we show that NLRP3 inflammasome plays a central role in aldosterone-induced vascular dysfunction. Long-term infusion of aldosterone in mice resulted in elevation of plasma interleukin-1ß levels and vascular abnormalities. Mice lacking the IL-1R or the inflammasome components NLRP3 and caspase-1 were protected from aldosterone-induced vascular damage. In vitro, aldosterone stimulated NLRP3-dependent interleukin-1ß secretion by bone marrow-derived macrophages by activating nuclear factor-κB signaling and reactive oxygen species generation. Moreover, chimeric mice reconstituted with NLRP3-deficient hematopoietic cells showed that NLRP3 in immune cells mediates aldosterone-induced vascular damage. In addition, aldosterone increased the expression of NLRP3, active caspase-1, and mature interleukin-1ß in human peripheral blood mononuclear cells. Hypertensive patients with hyperaldosteronism or normal levels of aldosterone exhibited increased activity of NLRP3 inflammasome, suggesting that the effect of hyperaldosteronism on the inflammasome may be mediated through high blood pressure. CONCLUSIONS: Together, these data demonstrate that NLRP3 inflammasome, through activation of IL-1R, is critically involved in the deleterious vascular effects of aldosterone, placing NLRP3 as a potential target for therapeutic interventions in conditions with high aldosterone levels.

Renoprotective Effects of Atorvastatin in Diabetic Mice: Downregulation of RhoA and Upregulation of Akt/GSK3.

Potential benefits of statins in the treatment of chronic kidney disease beyond lipid-lowering effects have been described. However, molecular mechanisms involved in renoprotective actions of statins have not been fully elucidated. We questioned whether statins influence development of diabetic nephropathy through reactive oxygen species, RhoA and Akt/GSK3 pathway, known to be important in renal pathology. Diabetic mice (db/db) and their control counterparts (db/+) were treated with atorvastatin (10 mg/Kg/day, p.o., for 2 weeks). Diabetes-associated renal injury was characterized by albuminuria (albumin:creatinine ratio, db/+: 3.2 ± 0.6 vs. db/db: 12.5 ± 3.1*; *P<0.05), increased glomerular/mesangial surface area, and kidney hypertrophy. Renal injury was attenuated in atorvastatin-treated db/db mice. Increased ROS generation in the renal cortex of db/db mice was also inhibited by atorvastatin. ERK1/2 phosphorylation was increased in the renal cortex of db/db mice. Increased renal expression of Nox4 and proliferating cell nuclear antigen, observed in db/db mice, were abrogated by statin treatment. Atorvastatin also upregulated Akt/GSK3ß phosphorylation in the renal cortex of db/db mice. Our findings suggest that atorvastatin attenuates diabetes-associated renal injury by reducing ROS generation, RhoA activity and normalizing Akt/GSK3ß signaling pathways. The present study provides some new insights into molecular mechanisms whereby statins may protect against renal injury in diabetes.

Impaired Ca(2+) Homeostasis and Decreased Orai1 Expression Modulates Arterial Hyporeactivity to Vasoconstrictors During Endotoxemia.

We hypothesized that SIRS/endotoxemia-associated hyporesponsiveness to vasoconstrictors is mediated by smaller increases in intracellular Ca(2+) levels due to reduced signaling via the STIM/Orai. Male Wistar rats were injected either with saline or bacterial LPS (i.p.; 10 mg/kg), and experiments were performed 24 h later. LPS-injected rats exhibited decreased systolic blood pressure, increased heart rate, neutrophils' migration into the peritoneal cavity, and elevated alanine aminotransferase levels. Additionally, second-order mesenteric arteries from endotoxemic rats displayed hyporeactivity to contractile agents such as phenylephrine and potassium chloride; decreased contractile responses to Ca(2+); reduced contraction during Ca(2+) loading; and smaller intracellular Ca(2+) stores. Decreased Orai1, but not STIM1, expression was found in resistance mesenteric arteries from LPS-treated rats. Additionally, cultured vascular smooth muscle cell (VSMC) treated with LPS resulted in increased TLR-4 expression, but Myd-88 and STIM-1 expression were not changed. Our data suggest that in endotoxemia, Ca(2+) homeostasis is disrupted in VSMC, with decreased Ca(2+) influx, smaller concentrations of Ca(2+) in the sarcoplasmic reticulum, and decreased activation of Orai1. Abnormal Ca(2+) handling contributes to LPS-associated vascular hyporeactivity.

Enhancement on reactive oxygen species and COX-1 mRNA levels modulate the vascular relaxation induced by sodium nitroprusside in denuded mice aorta.

This study aimed to investigate the modulation of nitric oxide/reactive oxygen species in sodium nitroprusside relaxation in mice aorta. Sodium nitroprusside induced relaxation in endothelium-intact (e+) and endothelium-denuded (e-) aortas with greater potency in e+ than in e-. The nitric oxide synthase inhibitor did not alter the sodium nitroprusside relaxation in both e+ and e- aortas. However, the superoxide anion scavenger abolished the difference in sodium nitroprusside potency between e+ and e-. Sodium nitroprusside reduced dihydroethidium-derived fluorescent products in both groups; however, the difference between intact and denuded mice aorta remains. The glutathione levels and basal antioxidant activity of superoxide dismutase were reduced in e- aorta when compared with e+, and these values were not altered by sodium nitroprusside. Confirming these results, the levels of lipid peroxidation in e+ were significantly lower when compared to e-, and these values were not altered by sodium nitroprusside. The sodium nitroprusside potency in the presence of a nonselective COX inhibitor or the EP/DP prostaglandin receptor antagonist in endothelium denuded was similar to that in intact mice aorta. Based on these results, we performed the COX-1 and COX-2 mRNA level studies, and in denuded mice aorta, there was an upregulation in COX-1 mRNA levels. Taken together, our findings show that in the absence of endothelium, there is an enhancement of superoxide levels, leading to GSH consumption and higher levels of lipid peroxidation, showing an intense redox status. Furthermore, in denuded mice aorta, there was an upregulation of COX-1 mRNA expression, leading to vasoconstrictor prostanoids synthesis. The interaction of vasoconstrictor prostanoids with its receptors EP/DP negatively modulates the vascular relaxation induced by SNP in denuded mice aorta.

Impairment of α1-adrenoceptor-mediated calcium influx in contralateral carotids following balloon injury: beneficial effect of superoxide anions.

There are many evidences indicating a compensatory mechanism in contralateral carotids following balloon injury. Previously it was observed α1-adrenoceptor-mediated hyper-reactivity and impairment of calcium influx in contralateral carotids 4 days after injury. At a later stage, α1-adrenoceptor-mediated contraction is similar to the control and we hypothesized that downstream signaling was normal. In the present study, we aimed to evaluate α1-adrenoceptor-mediated calcium influx in contralateral carotids 15 days after balloon injury. Concentration-response curves for CaCl2 in presence of the α1-adrenoceptor agonist (phenylephrine), measurement of the intracellular calcium transient and the levels of reactive oxygen species using fluorescent dyes were performed in control and contralateral carotids. Phenylephrine-induced intracellular calcium mobilization in contralateral carotids was not altered, while phenylephrine-induced calcium influx was reduced in the contralateral artery. Nitric oxide synthase inhibitors, L-NAME or L-NNA, restored this response, but nitrite and nitrate levels were decreased in contralateral carotids. Additionally, a rise in oxygen free radicals was observed in contralateral carotids. Furthermore, Tiron, a superoxide anion scavenger, restored α1-adrenoceptor-mediated calcium influx in contralateral carotids to the control level. Similar results were observed with the selective potassium channels blockers 4-aminopyridine and charybdotoxin. In conclusion, data showed that balloon catheter injury resulted in increased superoxide anions levels, activation of potassium channels (Kv and BKCa), inhibition of calcium channels (Cav) and preservation of α1-adrenoceptor-mediated contraction at a later stage after injury.

Arginase inhibition prevents the low shear stress-induced development of vulnerable atherosclerotic plaques in ApoE-/- mice.

AIMS: Wall shear stress differentially regulates the arginase pathway in carotid arteries perfused ex vivo. Specific patterns of wall shear stress can locally determine atherosclerotic plaque size and composition in vivo. The present work investigates the effects of arginase inhibition on shear stress induced plaque composition. METHODS AND RESULTS: Carotid arteries of apolipoprotein E deficient mice were exposed to high (HSS), low (LSS) and oscillatory (OSS) shear stress conditions by the placement of a local shear stress modifier device for 9 weeks with or without the administration of the arginase inhibitor N-ω-Hydroxy-nor-L-arginine (nor-Noha) (10 mg/kg, i.p., 5 days/week). Carotid arginase activity was measured by colorimetric determination of urea. Atherosclerotic plaque size and composition, arginase expression and cellular localization were assessed by immunohistochemistry. Arginase activity was significantly increased in both LSS and OSS regions as compared to HSS. In the lesions, arginase II isoform co-localized preferentially with EC. Inhibition of arginase by nor-Noha decreased arginase activity and reduced plaque size in both LSS and OSS regions. Arginase inhibition affected mainly the composition of plaques developed in LSS regions by decreasing the total vascular ROS, the number of macrophages, apoptosis rate, lipid and collagen contents. CONCLUSIONS: Arginase activity is modulated by patterns of wall shear stress in vivo. Chronic inhibition of vascular arginase decreased the size of atherosclerotic lesions in both OSS and LSS regions, whereas changes on plaque composition were more pronounced in plaques induced by LSS. We identified wall shear stress as a key biomechanical regulator of arginase during plaque formation and stability.

Swimming training improves the vasodilator effect of angiotensin-(1-7) in the aorta of spontaneously hypertensive rat.

INTRODUCTION: endothelial dysfunction plays a critical role in the pathogenesis of hypertension. It is well established that physical training has beneficial effects on the cardiovascular system. We recently reported that angiotensin-(1-7) [Ang-(1-7)] concentration and the Mas receptor expression is increased in the left ventricle of trained spontaneous hypertensive rats (SHR). The vascular effects of Ang-(1-7) in trained animals remain so far unknown. In the present study we investigated the effects of physical training on the vasodilator effect of Ang-(1-7) in the aorta of SHR. METHODOLOGY: normotensive Wistar rats and SHR were subjected to an 8-wk period of 5% overload of body weight swimming training. Changes in isometric tension were recorded on myograph. Western blot was used to investigate Ang-(1-7) receptors expression. RESULTS: in aortas from normotensive rats Ang-(1-7) and ACh induced a concentration-dependent vasodilator effect, which was not modified by the physical training. Vessels from SHR had an impaired vasodilator response to Ang-(1-7) and ACh. The swimming training strongly potentiated the vasodilator effect induced by Ang-(1-7) in SHR, but did not modify the effect of ACh. Interestingly, Mas receptor protein expression was substantially increased by physical training in SHR. In trained SHR, the vasodilator effect of Ang-(1-7) was abrogated by removal of the endothelium and by the selective Ang-(1-7) receptor antagonists A-779 and d-Pro(7)-Ang-(1-7). l-NAME decreased Ang-(1-7) vasodilator response and indomethacin abolished the remaining dilatory response. CONCLUSION: physical training increased Mas receptors expression in SHR aortas, thereby improving the vasodilator effect of Ang-(1-7) through an endothelium-dependent mechanism involving nitric oxide and prostacyclin.

Impaired calcium influx despite hyper-reactivity in contralateral carotid following balloon injury: eNOS involvement.

Balloon catheter injury results in hyper-reactivity to phenylephrine in contralateral carotids. Decreased nitric oxide (NO) modulation and/or increased intracellular calcium concentration triggers vascular smooth muscle contraction. Therefore, this study explores the participation of NO signaling pathway and calcium mobilization on hyper-reactivity to phenylephrine in contralateral carotids. Concentration-response curves for calcium (CaCl(2)) and phenylephrine were obtained in control and contralateral carotids four days after balloon injury, in the presence and absence of the inhibitors (L-NAME, L-NNA, 1400W, 7-NI, Oxyhemoglobin, ODQ or Tiron). Confocal microscopy using Fluo-3AM or DHE was performed to detect the intracellular levels of calcium and reactive oxygen species, respectively. The modulation of NO on phenylephrine-induced contraction was absent in the contralateral carotid. Phenylephrine-induced intracellular calcium mobilization was not altered in contralateral carotids. However, extracellular calcium mobilization by phenylephrine was reduced in the contralateral carotid compared to control arteries, and this result was confirmed by confocal microscopy. L-NAME increased phenylephrine-induced extracellular calcium mobilization in the contralateral carotid to the control levels. Results obtained with L-NNA, 1400W, 7-NI, OxyHb, ODQ or Tiron showed that this response was mediated by products from endothelial NOS (eNOS) different from NO and without soluble guanylate cyclase activation, but it involved superoxide anions. Furthermore, Tiron or L-NNA reduced the levels of reactive oxygen species in contralateral carotids. Data suggest that balloon catheter injury promoted eNOS uncoupling in contralateral carotids, which generates superoxide rather than NO, and reduces phenylephrine-induced extracellular calcium mobilization, despite the hyper-reactivity to phenylephrine in contralateral carotids.

An apparent paradox: attenuation of phenylephrine-mediated calcium mobilization and hyperreactivity to phenylephrine in contralateral carotid after balloon injury.

Balloon catheter injury promotes hyperreactivity to phenylephrine (Phe) in the contralateral carotid. Phe-induced contraction involves calcium mobilization, a process that may be sensitive to reactive oxygen species. In this study, we investigated whether increased reactivity to Phe in the contralateral carotid is due to alterations in calcium mobilization by Phe and reactive oxygen species signaling. Concentration-response curves to Phe were obtained in control and contralateral arteries 4 days after balloon injury. Tiron did not modify Emax to Phe in control arteries but reduced this parameter in the contralateral carotid to control levels. Moreover, immunofluorescence to dihydroethydine showed increased basal oxidative stress in the contralateral artery compared with control artery. Intracellular calcium mobilization by Phe in the contralateral artery was not different from control, but Phe-induced extracellular calcium mobilization was reduced in the contralateral artery compared with that in the control. These data were confirmed by confocal microscopy using Fluo 3-AM. Tiron and SC-236 increased Phe-induced calcium influx in the contralateral artery, which was similar to controls in the same conditions. However, catalase did not modify this response. Taken together, our results suggest that superoxide anions and prostanoids from cyclooxygenase-2 alter pathways downstream of alpha1-adrenoceptor activation in the contralateral carotid in response to injury. This results in reduced Phe-induced calcium influx, despite hyperreactivity to Phe.

Diabetes and vascular disease: basic concepts of nitric oxide physiology, endothelial dysfunction, oxidative stress and therapeutic possibilities.

The vascular manifestations associated with diabetes mellitus (DM) result from the dysfunction of several vascular physiology components mainly involving the endothelium, vascular smooth muscle and platelets. It is also known that hyperglycemia-induced oxidative stress plays a role in the development of this dysfunction. This review considers the basic physiology of the endothelium, especially related to the synthesis and function of nitric oxide. We also discuss the pathophysiology of vascular disease associated with DM. This includes the role of hyperglycemia in the induction of oxidative stress and the role of advanced glycation end-products. We also consider therapeutic strategies.