Amiloride Relaxes Rat Corpus Cavernosum Relaxation In Vitro and Increases Intracavernous Pressure In Vivo.

INTRODUCTION: The antihypertensive effects of thiazide diuretics such as hydrochlorothiazide are commonly associated with erectile dysfunction. The association of hydrochlorothiazide/amiloride is not associated with erectile dysfunction. The hypothesis is that amiloride has beneficial effect in penile erection and, therefore, counterbalances the hydrochlorothiazide-induced disruptive effect. AIM: To investigate the effects of amiloride and its analogues hexamethylamiloride and benzamil on rat isolated corpus cavernosa (CC) and intracavernous pressure (ICP) in anaesthetized rats. METHODS: Rat isolated CC were incubated with amiloride, hexamethylamiloride, and benzamil (10 and 100 µmol/L each), followed by phenylephrine, potassium chloride, and electrical field stimulation (EFS). Their effect on the relaxant responses to EFS and sodium nitroprusside were also determined. Oral (30 mg/kg) and intraperitoneal (3 mg/kg) treatments with amiloride were also investigated on nerve-evoked ICP. MAIN OUTCOME MEASURES: In vitro functional studies and in vivo ICP measurement on rat CC were performed. Additionally, phosphodiesterase type V isoform A1 activity and the mRNA expressions of Na+/H+ pump, epithelial sodium channel exchangers (ENaC) channels (α-, ß- and γ subunits) and Na+/Ca2+ exchangers were evaluated in CC tissues. RESULTS: Amiloride and its analogues significantly reduced the phenylephrine-, potassium chloride-, and EFS-induced CC contractions, which were not changed by nitro-L-arginine methyl ester (100 µmol/L) or indomethacin (6 µmol/L). In phenylephrine-precontracted CC tissues, amiloride itself caused concentration-dependent relaxation and significantly increased the EFS-induced relaxation. Oral and intraperitoneal treatment with amiloride significantly increased the ICP. Phosphodiesterase type V isoform A1 activity was not affected by amiloride. Na+/H+ pump, ENaC, and Na+/Ca2+ exchanger mRNA expressions were all detected in rat CC tissues. CLINICAL IMPLICATION: Amiloride analogues may have therapeutic potential for erectile dysfunction. STRENGTH & LIMITATIONS: The interesting effect of amiloride in penile erection was observed in both in vitro and in vivo methods. The evidence at the moment is restricted to rat CC. CONCLUSION: Amiloride reduces in vitro CC contractility and enhances erectile function after oral and intraperitoneal administration, possibly via inhibition of ENaC. Campos R, Claudino MA, de Oliveira MG, et al. Amiloride Relaxes Rat Corpus Cavernosum Relaxation In Vitro and Increases Intracavernous Pressure In Vivo. J Sex Med 2019;16:500-511.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea.

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.

Histological and functional renal alterations caused by Bothrops alternatus snake venom: expression and activity of Na+/K+-ATPase.

BACKGROUND: Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na+/K+-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na+/K+-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. METHODS: Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na+/K+-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. RESULTS: Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na+/K+-ATPase α1 subunit were increased 6h post-venom, whereas Na+/K+-ATPase activity increased 6 h and 24 h post-venom. CONCLUSIONS: Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na+/K+-ATPase expression and activity in the early phase of renal damage. GENERAL SIGNIFICANCE: Enhanced Na+/K+-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.

Reduced blood pressure in sickle cell disease is associated with decreased angiotensin converting enzyme (ACE) activity and is not modulated by ACE inhibition.

BACKGROUND: Sickle cell disease (SCD) incurs vaso-occlusive episodes and organ damage, including nephropathy. Despite displaying characteristics of vascular dysfunction, SCD patients tend to present relatively lower systemic blood pressure (BP), via an unknown mechanism. We investigated associations between BP and renin-angiotensin-system (RAS) components in SCD and determined whether an inhibitor of angiotensin converting enzyme (ACE; often used to slow SCD glomerulopathy) further modulates BP and RAS components in a murine model of SCD. METHODS: BP was compared in human subjects and mice with/without SCD. Plasma angiotensin II, ACE and renin were measured by immunoassay. BP was reevaluated after treating mice with enalapril (25 mg/kg, 5x/week) for 5 weeks; plasma and organs were stored for angiotensin II and ACE activity measurement, and quantitative real-time PCR. RESULTS: Diastolic BP and systolic BP were significantly lower in patients and mice with SCD, respectively, compared to controls. Reduced BP was associated with increased plasma renin and markers of kidney damage (mice) in SCD, as well as significantly decreased plasma ACE concentrations and ACE enzyme activity. As expected, enalapril administration lowered BP, plasma angiotensin II and organ ACE activity in control mice. In contrast, enalapril did not further reduce BP or organ ACE activity in SCD mice; however, plasma angiotensin II and renin levels were found to be significantly higher in enalapril-treated SCD mice than those of treated control mice. CONCLUSION: Relative hypotension was confirmed in a murine model of SCD, in association with decreased ACE concentrations in both human and murine disease. Given that ACE inhibition has an accepted role in decreasing BP, further studies should investigate mechanisms by which ACE depletion, via both Ang II-dependent and alternative pathways, could contribute to reduce BP in SCD and understand how ACE inhibition confers Ang II-independent benefits on kidney function in SCD.

Participation of Mac-1, LFA-1 and VLA-4 integrins in the in vitro adhesion of sickle cell disease neutrophils to endothelial layers, and reversal of adhesion by simvastatin.

BACKGROUND: Pharmacological approaches to inhibit increased leukocyte adhesive interactions in sickle cell disease may represent important strategies for the prevention of vaso-occlusion in patients with this disorder. We investigated, in vitro, the adhesion molecules involved in endothelial-sickle cell disease neutrophil interactions and the effect of simvastatin on sickle cell disease neutrophil adhesion to tumor necrosis factor-α-activated endothelial monolayers (human umbilical vein endothelial cells), and neutrophil chemotaxis. DESIGN AND METHODS: Sickle cell disease patients in steady state and not on hydroxyurea were included in the study. Endothelial cells treated, or not, with tumor necrosis factor-α and simvastatin were used for neutrophil adhesion assays. Neutrophils treated with simvastatin were submitted to interleukin 8-stimulated chemotaxis assays. RESULTS: Sickle cell disease neutrophils showed greater adhesion to endothelial cells than control neutrophils. Adhesion of control neutrophils to endothelial cells was mediated by Mac-1 under basal conditions and by the Mac-1 and LFA-1 integrins under inflammatory conditions. In contrast, adhesion of sickle cell disease neutrophils to endothelium, under both basal and tumor necrosis factor-α-stimulated conditions, was mediated by Mac-1 and LFA-1 integrins and also by VLA-4. Under stimulated inflammatory conditions, simvastatin significantly reduced sickle cell disease neutrophil adhesion, and this effect was reversed by inhibition of nitric oxide synthase. Furthermore, intercellular adhesion molecule-1 expression was significantly abrogated on tumor necrosis factor-α-stimulated endothelium incubated with simvastatin, and statin treatment inhibited the interleukin-8-stimulated migration of both control and sickle cell disease neutrophils. CONCLUSIONS: The integrins Mac-1, LFA-1 and, interestingly, VLA-4 mediate the adhesion of sickle cell disease leukocytes to activated endothelial cell layers, in vitro. Our data indicate that simvastatin may be able to reduce endothelial activation and consequent leukocyte adhesion in this in vitro model; future experiments and clinical trials may determine whether simvastatin therapy could be employed in patients with sickle cell disease, with beneficial effects on vaso-occlusion.

Characterization of the urinary bladder dysfunction in renovascular hypertensive rats.

AIMS: Association between arterial hypertension and urinary bladder dysfunction has been reported in humans and spontaneously hypertensive rats. However, no study exists evaluating the bladder dysfunction in conditions of renovascular hypertension. The purpose of this study was to characterize the bladder dysfunction in two kidney-one clip (2K-1C) hypertensive rats. METHODS: A silver clip was placed around the renal artery of male Wistar rats. After 8 weeks, cystometric study, concentration-response curves to contractile and relaxant agents, frequency-dependent contractions, histomorphometry, muscarinic M(2) /M(3) mRNA expression and cyclic AMP measurements were performed. RESULTS: 2K-1C rats showed enhanced bladder volume, wall thickness and smooth muscle density. 2K-1C rats also exhibited increases in bladder capacity and non-void contractions, and decreases in the inter-contraction intervals. In isolated detrusor smooth muscle (DSM), contractions to carbachol and electrical-field stimulation (EFS) were significantly greater in 2K-1C rats. The Rho-kinase inhibitor Y27632 (10 µM) significantly reduced the carbachol-induced contractions in SHAM and 2K-1C rats, but DSM remained overactive in 2K-1C rats in presence of Y27632. Concentration-dependent contractions to the P2X receptor agonist α,ß-methylene ATP, KCl and extracellular Ca(2+) did not change between SHAM and 2K-1C groups. In 2K-1C rats, isoproterenol, metaproterenol and BRL 37-344 (non-selective, ß(2) - and ß(3) -selective adrenoceptor agonists, respectively) produced significantly lower relaxations and decreased cAMP levels, whereas relaxant responses to sodium nitroprusside and BAY 41-2272 remained unchanged. Muscarinic M(3) mRNA expression receptors were higher in 2K-1C group. CONCLUSIONS: Renovascular hypertensive rats exhibit bladder dysfunction that involves tissue remodeling and enhanced muscarinic M(3) -mediated contractions associated with reduced ß-adrenoceptor-mediated signal transduction.

Increased adhesive properties of platelets in sickle cell disease: roles for alphaIIb beta3-mediated ligand binding, diminished cAMP signalling and increased phosphodiesterase 3A activity.

Whilst high pro-coagulant activity is reported in sickle cell disease (SCD), the precise role of platelets (PLTs) in SCD inflammatory and vaso-occlusive processes is unclear. Adhesion of PLTs from healthy controls (CON), SCD individuals (SCD) and SCD patients on hydroxycarbamide (SCDHC) to fibrinogen (FB) was compared using static adhesion assays. PLT adhesion molecules and intraplatelet cyclic adenosine monophosphate (icAMP) were observed by flow cytometry and enzyme-linked immunosorbent assay. SCD-PLTs demonstrated significantly greater adhesion than CON-PLTs to FB. Participation of the alpha(IIb)beta(3)-integrin in SCD-PLT adhesion was implicated by increased alpha(IIb)beta(3) activation and data showing that an alpha(IIb)beta(3)-function-inhibiting antibody significantly diminished SCD-PLT adhesion to FB. Platelet activation was potentiated by reductions in icAMP; cAMP levels were decreased in SCD-PLTs, being comparable to those of thrombin-stimulated CON-PLTs. Furthermore, SCD-PLT adhesion to FB was significantly reduced by cilostazol, an inhibitor of cAMP-hydrolyzing phosphodiesterase 3A (PDE3A). Both alpha(IIb)beta(3)-integrin activation and icAMP correlated significantly with fetal haemoglobin in SCD. Accordingly, hydroxycarbamide therapy was associated with lower PLT adhesion and higher icAMP. SCD-PLTs may be capable of adhering to proteins encountered on the inflamed vascular wall and, potentially, participate in vaso-occlusive processes. Hydroxycarbamide and, speculatively, nitric oxide donor or cyclic-nucleotide-targeted therapies may aid in the reversal of PLT adhesive properties in SCD.

Women with TT genotype for eNOS gene are more responsive in lowering blood pressure in response to exercise.

INTRODUCTION: The aim of this study was to investigate whether -786T>C endothelial nitric oxide synthase (eNOS) gene polymorphism might influence the effect of long-term exercise training (ET) on the blood pressure and its relationship with NO production in healthy postmenopausal women. DESIGN: Longitudinal study. METHOD: Fifty-five postmenopausal women were studied in a double-blinded design. ET was performed for 3 days a week, each session consisting of 60 min during 6 months, in an intensity of 50-70% VO2max. After that, eNOS genotype analysis was performed and women were divided into two groups: TC+CC (n=41) and TT (n=14) genotype. RESULTS: No changes were found in the anthropometric parameters after ET in both the groups. Systolic and diastolic BP values were significantly reduced in both the groups, but women with TT genotype were more responsive in lowering BP as compared with those with TC+CC genotype. Plasma nitrite/nitrate concentrations were similar at baseline in both the groups, but the magnitude of increment in NO production in response to ET was higher in women with TT genotype as compared with those with TC+CC genotype. CONCLUSION: Our study shows clearly that women with or without eNOS gene polymorphism had no differences in NO production at basal conditions, but when physical exercise is applied an evident difference is detected showing that the presence of -786T>C eNOS gene polymorphism had a significant impact in the health-promoting effect of aerobic physical training on the blood pressure in postmenopausal women.

Newer aspects of the pathophysiology of sickle cell disease vaso-occlusion.

Sickle cell disease is an inherited disorder of hemoglobin (Hb) synthesis, caused by a single nucleotide substitution (GTG>GAG) at the sixth codon of the beta-globin gene, leading to the production of a defective form of Hb, Hb S. When deoxygenated, Hb S polymerizes, damaging the sickle erythrocyte and it is this polymerization that is the primary indispensable event in the molecular pathogenesis of sickle cell disease. Hb S polymerization results in a series of cellular alterations in red cell morphology and function that shorten the red cell life span and leads to vascular occlusion. Sickle cell disease vaso-occlusion is now known to constitute a complex multifactorial process characterized by recurrent vaso-occlusion, ischemia-reperfusion injury, and oxidative stress with consequent vascular endothelial cell activation that induces a chronic inflammatory state in sickle cell disease individual and is propagated by elevated levels of circulating inflammatory cytokines. Activation of the endothelium results in the induction of endothelial adhesion molecule expression that mediates red and white cell adhesion to the vessel wall and the formation of heterocellular aggregates, followed by secondary red cell trapping, all of which contribute to reduced blood flow and eventually obstruction of the micro-circulation. Reduced nitric oxide bioavailability, caused principally by its consumption by cell-free Hb, liberated during intravascular hemolysis, contributes to this process by facilitating vasoconstriction and adhesion molecule activity.

Increased adhesive properties of neutrophils in sickle cell disease may be reversed by pharmacological nitric oxide donation.

Increased leukocyte adhesion to vascular endothelium contributes to vaso-occlusion in sickle cell disease. Since nitric oxide bioavailability is decreased in sickle cell disease and nitric oxide may inhibit leukocyte adhesion, we investigated whether stimulation of NO-signaling pathways can reduce the adhesive properties of neutrophils from sickle cell disease individuals (sickle cell diseaseneu). sickle cell diseaseneu presented greater adhesion in vitro to both fibronectin and ICAM-1 than control neutrophils. Co-incubation of sickle cell diseaseneu with the nitric oxide-donor agents, sodium nitroprusside and dietheylamine NONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272, all significantly reduced the increased adhesion to fibronectin/ICAM-1. Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, reversed sodium nitroprusside/DEANO-diminished adhesion to fibronectin, implicating cGMP-dependent signaling in this mechanism. Interestingly, intracellular cGMP was significantly higher in neutrophils from sickle cell disease individuals on hydroxyurea (sickle cell diseaseHUneu). Accordingly, sickle cell diseaseHUneu adhesion to fibronectin/ICAM-1 was significantly lower than that of sickle cell diseaseneu. Agents that stimulate the nitric oxide/cGMP-dependent pathway may have beneficial effects on leukocyte function if used in these subjects.

Ablation of primary afferent neurons by neonatal capsaicin treatment reduces the susceptibility of the portal hypertensive gastric mucosa to ethanol-induced injury in cirrhotic rats.

Primary sensory afferent neurons modulate the hyperdynamic circulation in cirrhotic rats with portal hypertension. The stomach of cirrhotic rats is prone to damage induced by ethanol, a phenomenon associated with reduced gastric hyperemic response to acid-back diffusion. The aim of this study was to examine the impact of ablation of capsaicin-sensitive neurons and the tachykinin NK(1) receptor antagonist A5330 on the susceptibility of the portal hypertensive gastric mucosa to ethanol-induced injury and its effects on gastric cyclooxygenase (COX) and nitric oxide synthase (NOS) mRNA expression. Capsaicin was administered to neonatal, male, Wistar rats and the animals were allowed to grow. Cirrhosis was then induced by bile duct ligation in adult rats while controls had sham operation. Ethanol-induced gastric damage was assessed using ex vivo gastric chamber experiments. Gastric blood flow was measured as well as COX/NOS mRNA expression. Topical application of ethanol produced significant gastric damage in cirrhotic rats compared to controls, which was reversed in capsaicin- and A5330-treated animals. Mean arterial and portal pressure was normalized in capsaicin-treated cirrhotic rats. Capsaicin and A5330 administration restored gastric blood flow responses to topical application of ethanol followed by acid in cirrhotic rats. Differential COX and NOS mRNA expression was noted in bile duct ligated rats relative to controls. Capsaicin treatment significantly modified gastric eNOS/iNOS/COX-2 mRNA expression in cirrhotic rats. Capsaicin-sensitive neurons modulate the susceptibility of the portal hypertensive gastric mucosa to injury induced by ethanol via tachykinin NK(1) receptors and signalling of prostaglandin and NO production/release.

A thalidomide-hydroxyurea hybrid increases HbF production in sickle cell mice and reduces the release of proinflammatory cytokines in cultured monocytes.

Fetal hemoglobin (HbF) induction by hydroxyurea (HU) therapy is associated with decreased morbidity and mortality in sickle cell anemia (SCA) patients, but not all patients respond to or tolerate HU. This provides a rationale for developing novel HbF inducers to treat SCA. Thalidomide analogs have the ability to induce HbF production while inhibiting the release of tumor necrosis factor-alpha. Molecular hybridization of HU and thalidomide was used to synthesize 3- (1,3-dioxoisoindolin-2-yl) benzyl nitrate (compound 4C). In this study, we show that compound 4C increases HbF production in a transgenic SCA mouse model and reduces the production of pro-inflammatory cytokines by SCA mouse monocytes cultured ex vivo. Therefore, compound 4C is a novel drug designed to treat SCA with a unique combination of HbF-inducing and anti-inflammatory properties.

Expressions of the VLA-4, LFA-1 and Mac-1 integrins in eosinophil migration in a case of chronic eosinophilic leukaemia.

Migration of eosinophil (Eo) into tissues is a hallmark of chronic eosinophilic leukaemia (CEL), but the exact mechanism involved in cell migration is unknown. We report on a patient with CEL who presented high expressions of VLA-4, LFA-1 and Mac-1 integrins on the Eo surface, increased chemotaxis of Eo to eotaxin, decreased chemotaxis of Eo after inhibition of the cyclic guanosine monophosphate (cGMP), and a high level of intracellular cGMP. These findings suggest that an upregulation of expression of these integrins on Eo surface and a high intracellular level of cGMP may be involved in the increased Eo migration observed in our CEL patient. However, the definitive roles of these molecules in the proliferation and migration of Eo in CEL disease require wider confirmation by analysis of additional patients with the disease.

Interactions between eotaxin and interleukin-5 in the chemotaxis of primed and non-primed human eosinophils.

This study was designed to understand the relationship between interleukin-5 and eotaxin in modulating the chemotaxis of eosinophils obtained from healthy subjects and subjects with allergic rhinitis. Chemotaxis of eosinophils from patients with allergic rhinitis toward interleukin-5 (0.25 ng/ml) was 78% higher than that of healthy subjects. Incubation of eosinophils with eotaxin (100 ng/ml) did not change the interleukin-5-induced chemotaxis of eosinophils from healthy subjects, but it reversed the enhanced chemotaxis seen in eosinophils from allergic patients. Chemotaxis of eosinophils from patients with allergic rhinitis toward eotaxin (100 ng/ml) was 65% higher than that of eosinophils from healthy subjects. Incubation of eosinophils with interleukin-5 (100 ng/ml) significantly increased the eotaxin-induced chemotaxis in both subject groups, but such increases were markedly higher for cells from patients with allergic rhinitis. Our finding that eotaxin inhibits the enhanced eosinophil chemotaxis toward interleukin-5 in primed cells suggests that this chemokine may downregulate eosinophil accumulation in the nasal mucosa of allergic patients.

Effects of neonatal capsaicin treatment in the neutrophil production, and expression of preprotachykinin-I and tachykinin receptors in the rat bone marrow.

Bone marrow is richly innervated with both myelinated and non-myelinated nerve fibers, but the role of this innervation on hemopoiesis is poorly understood. Therefore, the aim of this study was to investigate the role of C-fibers on hematopoiesis. Wistar rats were neonatally injected with either capsaicin or its vehicle, and used at adult ages (8-10 weeks). In capsaicin-pretreated rats, the levels of substance P (SP) in bone marrow fluid were markedly reduced in comparison with the vehicle group (13.1+/-4.5 pg/ml versus 47.3+/-5.5 pg/ml, p<0.05). In bone marrow, the number of total leukocytes was 28% higher (p<0.05) in capsaicin-pretreated group, and this accompanied by a higher number of neutrophils, particularly of the immature forms. The mononuclear cell and eosinophils counts did not differ significantly among vehicle and capsaicin groups. In peripheral blood, the number of circulating neutrophils in the capsaicin group increased by 53.8% (p<0.05), whereas the number of mononuclear cells did not change significantly among groups. Eosinophils were virtually absent in the circulating blood in both groups. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed that both preprotachykinin (PPT)-I mRNA and the tachykinin neurokinin (NK)-1 mRNA expression in bone marrow cells significantly increased in capsaicin group, whereas the NK-2 mRNA expression was unchanged after capsaicin pretreatment. In conclusion, our data show that chronic neuropeptide depletion enhance the neutrophil proliferation and differentiation in the rat bone marrow by mechanisms involving upregulation of PPT-I gene and NK-1 receptors.

Inflammatory mechanisms underlying the rat pulmonary neutrophil influx induced by airway exposure to staphylococcal enterotoxin type A.

Association between staphylococcal infection and pathogenesis of upper airways disease has been reported. This study aimed to investigate the mechanisms underlying the rat pulmonary inflammation induced by airway exposure to staphylococcal enterotoxin A (SEA). SEA (0.3-10 ng trachea(-1)) caused dose-dependent neutrophil accumulation in BAL fluid, reaching maximal responses at 4 h (25-fold increase for 3 ng trachea(-1)). Significant accumulation of both lymphocytes and macrophages in BAL fluid was also observed at 4 h (2.1- and 1.9-fold increase, respectively, for 3 ng trachea(-1)). At later times (16 h), neutrophil counts in bone marrow (immature forms) and peripheral blood increased by 63 and 81%, respectively. SEA failed to directly induce chemotaxis and adhesion of isolated neutrophils. Analysis of mRNA expression for iNOS, COX-2 and CINC-2 in lung tissue showed an upregulation of these enzymes, which paralleled elevated levels of LTB4, PGE2, TNF-alpha, IL-6 and NO2- in BAL fluid. Expression of CINC-1 was unchanged, whereas CINC-3 was reduced in SEA-treated rats. Incubation of isolated alveolar macrophages with SEA (3 microg ml(-1)) resulted in significant elevations of TNF-alpha and NO2- levels in the cell supernatants. Dexamethasone (0.5 mg kg(-1)), celecoxib (3 mg kg(-1)) and compound 1400 W (5 mg kg(-1)) markedly reduced SEA-induced lung neutrophil influx and NO2- levels in BAL fluid. The lipoxygenase inhibitor AA-861 (100 microg kg(-1)) partly inhibited the neutrophil influx in SEA-treated rats without modifying the NO2- levels. None of these treatments reduced the number of mononuclear cells in BAL fluid (except of dexamethasone, which abolished the increased lymphocyte counts). Our study shows that airways exposure to SEA results in marked neutrophil influx through mechanisms involving increased expressions of CINC-2, iNOS and COX-2, as well as enhanced production of NO, PGE2, LTB4, TNF-alpha and IL-6.

Association of Nitric Oxide Synthase and Matrix Metalloprotease Single Nucleotide Polymorphisms with Preeclampsia and Its Complications.

BACKGROUND: Preeclampsia is one of the leading causes of maternal and neonatal morbidity and mortality in the world, but its appearance is still unpredictable and its pathophysiology has not been entirely elucidated. Genetic studies have associated single nucleotide polymorphisms in genes encoding nitric oxide synthase and matrix metalloproteases with preeclampsia, but the results are largely inconclusive across different populations. OBJECTIVES: To investigate the association of single nucleotide polymorphisms (SNPs) in NOS3 (G894T, T-786C, and a variable number of tandem repetitions VNTR in intron 4), MMP2 (C-1306T), and MMP9 (C-1562T) genes with preeclampsia in patients from Southeastern Brazil. METHODS: This prospective case-control study enrolled 77 women with preeclampsia and 266 control pregnant women. Clinical data were collected to assess risk factors and the presence of severe complications, such as eclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome. RESULTS: We found a significant association between the single nucleotide polymorphism NOS3 T-786C and preeclampsia, independently from age, height, weight, or the other SNPs studied, and no association was found with the other polymorphisms. Age and history of preeclampsia were also identified as risk factors. The presence of at least one polymorphic allele for NOS3 T-786C was also associated with the occurrence of eclampsia or HELLP syndrome among preeclamptic women. CONCLUSIONS: Our data support that the NOS3 T-786C SNP is associated with preeclampsia and the severity of its complications.

Evidence for the involvement of a macrophage-derived chemotactic mediator in the neutrophil recruitment induced by staphylococcal enterotoxin B in mice.

Staphylococcus aureus secretes enterotoxins which are superantigens and the major cause of food poisoning in man. Staphylococcal enterotoxins types A and B can induce neutrophil migration into the peritoneal cavity of mice through sensory C-fiber neuropeptides, lipoxygenase or cyclooxygenase metabolites, nitric oxide, histamine, platelet-activating factor and resident macrophages. In this work, we examined the influence of macrophage-derived products on neutrophil migration during peritonitis induced by staphylococcal enterotoxin type B (SEB) in mice. Macrophages stimulated with SEB released a thermolabile neutrophil chemotactic protein with a molecular weight of 1,000-3,000 (by ultrafiltration). This release was inhibited 30% by dexamethasone (an inhibitor of cytokine synthesis and phospholipase A(2) activity), but not by indomethacin (a cyclooxygenase inhibitor) or BW755C (a dual cyclo- and lipoxygenase inhibitor). Dexamethasone also inhibited (100%) the neutrophil migration induced by the chemotactic protein. Similar inhibition occurred in mice pretreated with BWA4C (lipoxygenase inhibitor; 90%), BW755C (99%), BN52021 (platelet-activating factor-acether receptor antagonist; 93%), cimetidine (histamine H(2) receptor antagonist; 76%), capsaicin (a depletor of sensory C-fiber neuropeptides; 82%) and the neurokinin-1 receptor antagonist SR140333 (71%), but not by indomethacin or the neurokinin(2) receptor antagonist SR48968. These results confirm that macrophages are involved in the neutrophil recruitment induced by SEB, and that the chemotactic protein apparently induces neutrophil migration by a mechanism mediated by platelet-activating factor, histamine H(2) receptors, lipoxygenase products and substance P.

Reduced plasma angiotensin II levels are reversed by hydroxyurea treatment in mice with sickle cell disease.

AIMS: Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin-angiotensin system (RAS) may be altered in an animal model of SCD. MAIN METHODS: Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50-75mg/kg/day, 4weeks) treatment on these parameters were also determined. KEY FINDINGS: Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart. SIGNIFICANCE: Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.

Increased adhesive properties of neutrophils and inflammatory markers in venous thromboembolism patients with residual vein occlusion and high D-dimer levels.

BACKGROUND: Venous thromboembolism (VTE) develops via a multicellular process on the endothelial surface. Although widely recognized, the relationship between inflammation and thrombosis, this relationship has been mostly explored in clinical studies by measuring circulating levels of inflammatory cytokines. However, the role of inflammatory cells, such as neutrophils, in the pathogenesis of VTE is not clear in humans. AIMS: To evaluate the adhesive properties of neutrophils, erythrocytes and platelets in VTE patients and to correlate findings with inflammatory and hypercoagulability marker levels. METHODS: Study group consisted of twenty-nine VTE patients and controls matched according to age, gender and ethnic background. Adhesive properties of neutrophils, erythrocytes and platelets were determined using a static adhesion assay. Neutrophil adhesion molecules expressions were evaluated by flow cytometry. Inflammatory and hypercoagulability marker levels were evaluated by standard methods. Residual vein occlusion (RVO) was evaluated by Doppler ultrasound. RESULTS: No significant difference could be observed in platelet and erythrocyte adhesion between VTE patients and controls. Interestingly, VTE patients with high levels of D-dimer and RVO, demonstrated a significant increase in neutrophil adhesion, compared to controls and remaining patients. Inflammatory markers (IL-6, IL-8, TNF-α) were also significantly elevated in this subgroup, compared to other VTE patients. Adhesive properties of neutrophils correlated with IL-6 and D-dimer levels. Neutrophils adhesion molecules (CD11a, CD11b and CD18) were not altered in any of the groups. CONCLUSION: These findings not only support the hypothesis of an association between inflammation and hypercoagulability, but more importantly, highlight the role of neutrophils in this process.