preproET-1 mRNA Research Articles

Endothelin converting enzyme-1 (ECE-1) deletion in association with Endothelin-1 downregulation ameliorates kidney fibrosis in mice

Kidney fibrosis is a common final pathway of chronic kidney diseases, which are characterized by renal architecture damage, inflammation, fibroblast expansion and myofibroblast formation. Endothelin converting enzyme-1 (ECE-1) contributes to activation of Endothelin-1 (ET-1), a potent vasoconstrictor and pro-fibrotic substance. This study elucidated the effect of ECE-1 knockout in kidney fibrosis model in mice in association of ET-1 downregulation. Kidney fibrosis was performed in ECE-1 knockout (ECE-1 KO) and vascular endothelial derived ET-1 KO (VEETKO) mice (2 months, 20–30 g, n = 30) and their wild type (WT) littermates using unilateral ureteral obstruction (UUO) procedure. Mice were euthanized on day-7 and day-14 after UUO. Histopathological analysis was conducted for fibrosis and tubular injury. Immunostainings were done to quantify macrophages (F4/80), fibroblasts (FSP-1) and myofibroblasts (α-SMA). Monocyte Chemoattractant Protein-1 (MCP-1), ECE-1 and preproET-1 (ppET-1) mRNA expression were quantified with qRT-PCR, while Transforming Growth Factor-β1 (TGF-β1) and α-SMA protein level were quantified with Western blot. ECE-1 KO mice demonstrated reduction of ECE-1 and ppET-1 mRNA expression, attenuation of kidney fibrosis, tubular injury, MCP-1 mRNA expression and macrophage number compared to WT. Double immunostaining revealed fibroblast to myofibroblast formation after UUO, while ECE-1 KO mice had significantly lower fibroblast number and myofibroblast formation compared to WT, which were associated with significantly lower TGF-β1 and α-SMA protein levels in day-14 of UUO. VEETKO mice also demonstrated attenuation of ET-1 protein level, fibrosis and myofibroblast formation. In conclusion, ECE-1 knockout and ET-1 downregulation attenuated kidney fibrosis.

Inhibition of Endothelin system during the postnatal nephrogenic period in the rat. Its relationship with hypertension and renal disease in adulthood.

The aim of this work was to study the effect of a high sodium (HS) diet on blood pressure and renal function in male adult rats that have been treated with a dual Endothelin receptor antagonist (ERA) during their early postnatal period (day 1 to 20 of life). Male Sprague-Dawley rats were divided in four groups: CNS: control rats with normosodic diet; ERANS: ERA-treated rats with normosodic diet; CHS: control rats with high sodium diet; ERAHS: ERA-treated rats with HS diet. Systolic blood pressure (SBP) was recorded before and after the diet and 24-hour metabolic cage studies were performed. AQP2 and α-ENac expressions were measured by western blot and real time PCR in the renal medulla. Vasopressin (AVP) pathway was evaluated by measuring V2 receptor and adenylyl cyclase 6 (AC6) expression and cAMP production in the renal medulla. Pre-pro ET-1mRNA was also evaluated in the renal medulla. Only rats that had been treated with an ERA during their postnatal period increased their SBP after consumption of a HS diet, showing an impaired capacity to excrete sodium and water, i.e. developing salt sensitivity. This salt sensitivity would be mediated by an increase in renomedullary expression and activity of AQP2 and α-ENaC as a consequence of increased AC6 expression and cAMP production and/or a decreased ET-1 production in the renal medulla. The knowledge of the molecular mechanisms underlying the perinatal programming of salt sensitive hypertension will allow the development of reprogramming strategies in order to avoid this pathology.

Mechanical Forces Alter Endothelin-1 Signaling: Comparative Ovine Models of Congenital Heart Disease

The risk and progression of pulmonary vascular disease (PVD) in patients with congenital heart disease (CHD) dependent on the hemodynamics associated with different lesions. However, the underlying mechanisms are not understood. Endothelin-1 (ET-1) is a potent vasoconstrictor known to play a role in the pathobiology of PVD. We investigate the hypothesis that the combined effect of high pressure (P) and flow shear stresses (FSS), but not FSS alone, upregulates ET-1 signaling. We used 2 ovine models of CHD: (1) Fetal aortopulmonary graft placement (shunt), resulting in increased FSS and P; and (2) Fetal ligation of the left pulmonary artery (LPA) resulting in increased FSS but normal P to the right lung. Pulmonary arterial endothelial cells (PAEC) and peripheral lung were harvested from control, shunt, and LPA lambs at 3-7 weeks of age. PAECs from control lambs were exposed to high FSS=20dyn/cm2, cyclic stretch CS=18%, and P from 20-50mmHg at constant shear conditions FSS=5dyn/cm2, utilizing a microfluidic device. Lung preproET-1 mRNA were increased in shunt lambs compared to controls. PreproET-1 mRNA expression was unchanged in LPA lambs. In both shunt and LPA lambs, Lung ECE-1 mRNA and protein expression were increased. These changes resulted in increased lung ET-1 levels in shunt lambs, while LPA levels were similar to controls. PAECs exposed to increased FSS decreased ET-1 by 5-fold, while CS increased levels by 1.5-fold. Under physiological FSS=5dyn/cm, the additive effects of P increased preproET-1 mRNA expression, while ECE-1 expression was unchanged. These data suggest that P or the additive effect of P+FSS, rather than increased FSS alone, is the principal driver of increased ET-1 in CHD. Defining the molecular drivers of the pathobiology of PVD resulting from the different mechanical stimuli will allow for a more targeted therapeutic approach.

The citrus flavanone naringenin attenuates zymosan-induced mouse joint inflammation: induction of Nrf2 expression in recruited CD45+ hematopoietic cells.

Naringenin is a biologically active analgesic, anti-inflammatory, and antioxidant flavonoid. Naringenin targets in inflammation-induced articular pain remain poorly explored. The present study investigated the cellular and molecular mechanisms involved in the analgesic/anti-inflammatory effects of naringenin in zymosan-induced arthritis. Mice were pre-treated orally with naringenin (16.7-150mg/kg), followed by intra-articular injection of zymosan. Articular mechanical hyperalgesia and oedema, leucocyte recruitment to synovial cavity, histopathology, expression/production of pro- and anti-inflammatory mediators and NFκB activation, inflammasome component expression, and oxidative stress were evaluated. Naringenin inhibited articular pain and oedema in a dose-dependent manner. The dose of 50mg/kg inhibited leucocyte recruitment, histopathological alterations, NFκB activation, and NFκB-dependent pro-inflammatory cytokines (TNF-α, IL-1β, and IL-33), and preproET-1 mRNA expression, but increased anti-inflammatory IL-10. Naringenin also inhibited inflammasome upregulation (reduced Nlrp3, ASC, caspase-1, and pro-IL-1β mRNA expression) and oxidative stress (reduced gp91phox mRNA expression and superoxide anion production, increased GSH levels, induced Nrf2 protein in CD45+ hematopoietic recruited cells, and induced Nrf2 and HO-1 mRNA expression). Naringenin presents analgesic and anti-inflammatory effects in zymosan-induced arthritis by targeting its main physiopathological mechanisms. These data highlight this flavonoid as an interesting therapeutic compound to treat joint inflammation, deserving additional pre-clinical and clinical studies.

Erratum to: The Sesquiterpene Lactone, Budlein A, Inhibits Antigen-Induced Arthritis in Mice: Role of NF-κB and Cytokines.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by debilitating pain, cartilage destruction, and loss of joint function. Management of RA includes drugs that target NF-κB and downstream cytokine production. Therefore, molecules that act by inhibiting this signaling pathway without the severe side effects of, for instance, corticoids would be suitable therapeutic strategies. Budlein A is a sesquiterpene lactone with antinociceptive and anti-inflammatory properties related to the inhibition of pro-inflammatory cytokines and neutrophil recruitment. In this study, the effect of budlein A was evaluated in antigen-induced arthritis (AIA) in mice. At the 26th day, leukocyte recruitment to the knee joint, knee contents of proteoglycans, blood levels of ALT and AST, stomach tissue myeloperoxidase activity, and RT-qPCR for pro-inflammatory gene mRNA expression in knee joint samples was performed. NF-κB luciferase activity was evaluated in RAW 264.7 macrophages. Budlein A treatment dose-dependently inhibited AIA-induced mechanical hyperalgesia, edema, total leukocytes and neutrophil recruitment, and proteoglycan degradation. Budlein A did not induce gastric or liver damage. Budlein also inhibited AIA-induced Il-33, Tnf, Il-1β, preproET-1, and Cox-2 mRNA expression. In vitro, budlein reduced TNF- and IL-1β-induced NF-κB activity in RAW 264.7 macrophages. Altogether, we demonstrate that budlein A ameliorates AIA-induced inflammation and pain by targeting NF-κB. Importantly, budlein A does not induce in vivo side effects, suggesting that it possesses a favorable pre-clinical profile as analgesic and it is a prosperous molecule to be further investigated for the treatment of RA.

The Sesquiterpene Lactone, Budlein A, Inhibits Antigen-Induced Arthritis in Mice: Role of NF-κB and Cytokines.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by debilitating pain, cartilage destruction, and loss of joint function. Management of RA includes drugs that target NF-κB and downstream cytokine production. Therefore, molecules that act by inhibiting this signaling pathway without the severe side effects of, for instance, corticoids would be suitable therapeutic strategies. Budlein A is a sesquiterpene lactone with antinociceptive and anti-inflammatory properties related to the inhibition of pro-inflammatory cytokines and neutrophil recruitment. In this study, the effect of budlein A was evaluated in antigen-induced arthritis (AIA) in mice. At the 26th day, leukocyte recruitment to the knee joint, knee contents of proteoglycans, blood levels of ALT and AST, stomach tissue myeloperoxidase activity, and RT-qPCR for pro-inflammatory gene mRNA expression in knee joint samples was performed. NF-κB luciferase activity was evaluated in RAW 264.7 macrophages. Budlein A treatment dose-dependently inhibited AIA-induced mechanical hyperalgesia, edema, total leukocytes and neutrophil recruitment, and proteoglycan degradation. Budlein A did not induce gastric or liver damage. Budlein also inhibited AIA-induced Il-33, Tnf, Il-1β, preproET-1, and Cox-2 mRNA expression. In vitro, budlein reduced TNF- and IL-1β-induced NF-κB activity in RAW 264.7 macrophages. Altogether, we demonstrate that budlein A ameliorates AIA-induced inflammation and pain by targeting NF-κB. Importantly, budlein A does not induce in vivo side effects, suggesting that it possesses a favorable pre-clinical profile as analgesic and it is a prosperous molecule to be further investigated for the treatment of RA.

The Endothelin Receptor Etb Plays a Crucial Role for Recruitment of Neutrophils to the Vascular Wall in Sickle Cell Disease

Introduction: Although Sickle Cell Disease (SCD) is due to abnormal hemoglobin, many cell types, including endothelial cells and polymorphonuclear neutrophils (PMNs), play a key role in the pathophysiology of the disease, particularly in the vaso-occlusive events. Adhesion of PMNs to activated endothelium is critical in SCD and might contribute to vaso-occlusion; thus targeting PMN interactions with the endothelium may represent a good opportunity for new therapeutics. We looked for candidate mediators that may be involved in PMN activation and recruitment in tissues. We focused on endothelin-1 (ET-1) since (i) high levels of cytokines have been reported in SCD patients; (ii) we previously demonstrated that ET-1 receptors blockade in the SAD mouse model of SCD leads to reversal or prevention of vaso-occlusive events, nitrative stress, kidney and lung damage, and even death. While demonstrating that ET receptor (ETR) antagonism inhibited a tonic ET-1-dependent vasoconstriction during experimental vaso-occlusive crisis, we observed unexpected inhibition of PMN recruitment in lungs and kidneys. We postulated that activation of ETRs might stimulate a pathogenic proinflammatory role for PMNs in SCD.Methods: In the present study, we combinedintravital videomicroscopy of the cremaster muscle microcirculation in SAD mice and quantitative microfluidic fluorescence microscopy of human blood to investigate the involvement of the ETRs in the interaction between neutrophils and endothelial cells.Results: Experiments performed both on SAD mice and SCD patients indicate that blocking ETRs hinders PMN recruitment to endothelial cells in inflammatory conditions (Fig.1). In SAD mice, we showed that the ETRs are involved at several steps of PMN microvascular recruitment. Rolling adhesion involves the ETB receptor only; firm adhesion and post-adhesive dynamic events with transmigration involve both the ETA and the ETB receptors. Inhibition experiments performed with the highly selective BQ788 antagonist (specific for ETB receptor) provide potent anti-inflammatory action in SAD mice. In SCD patients and healthy volunteers, we evidenced that human PMNs display functional ETB receptors that trigger a downstream calcium signaling leading to enhanced adhesion to endothelial cells. Furthermore, we investigated the expression of preproET-1 mRNA and the secretion of ET-1 in human PMNs from SCD patients and healthy controls and we highlighted the ability of PMNs to produce and secrete ET-1. We also found an unexpected stimulatory role for the ETB receptor in PMN adhesion to endothelial cells in laminar flow conditions. Last, we showed that this abnormal adhesion involves ETB receptors on both endothelial cells and PMNs.Conclusion: SAD mice and SCD patients both provide consistent evidence for a powerful anti-adhesion role of ETB blockade. We emphasize that our work is the first to unravel the impact of the ET receptors on the different phases of PMN-endothelial interaction, further complementing early evidence that endothelins are chemoattractants for neutrophils in vitro. Our findings involving a potent contribution of the ETB receptor to vascular inflammation are novel as well as the fact that this pathogenic phenomenon is found in sickle but not in normal mice. Overall, the aforementioned findings indicated that human neutrophils display functional ETB receptors with calcium signaling capability, and we confirmed that human PMNs synthesize ET-1 that may be involved in autocrine and paracrine pathophysiological actions. Thus, the ET-1- ETB axis should be considered a cytokine-like potent proinflammatory pathway in SCD. If endothelin receptors antagonists prove safe and effective for preventing or treating acute vasoocclusive events in the clinical setting, they should include anti- ETB potency and may provide major benefits for lung and renal integrity, quality of life, and survival of SCD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

JS ISH-ECCR-2 ANG-(1–7) AND ET-1, A NEW PARTNERSHIP

ACE2 and Ang-1–7 have been shown to protect against pulmonary hypertension (PH). Mechanisms for this remain unclear. Considering the important role of ET-1 in the pathophysiology of PH and endothelial dysfunction, we questioned whether Ang-(1–7) influences ET-1 signaling in endothelial cells and whether Ang-(1–7) treatment influences the ET-1 system in PH. Human microvascular endothelial cells (HMEC) were stimulated with ET-1 in the absence/presence of Ang 1–7 and showed that Ang 1–7 increased preproET-1 mRNA levels, ET-1 release, and ETBR protein levels. ET-1 increases in e-selectin mRNA, VCAM-1 protein and TNFα production were blocked by Ang 1–7. Pro-inflammatory effects were dependent on NO production. Ang 1–7 increased NO production in a Mas and ETBR-dependent manner. An interaction between Mas and ETBR was observed by immunoprecipitation. To further characterise a physical interaction between Mas/ETBR, we utilised novel technology, employing a library of overlapping peptides scanning the entirety of the MasR sequence, to define the interaction sites for ETBR binding. By substitution or sequence truncation we identified two distinct regions on the MasR that confer specificity for ETBR binding. Peptides that disrupt each of these regions to prevent Mas/ETBR interaction were developed for in vitro validation. To investigate the pathophysiological significance of our findings, we investigated whether Ang-(1–7) treatment ameliorates PH and whether this is associated with changes in ET-1 status. Hypobaric hypoxia was used to induce PH in mice, which were divided in 4 groups: normoxic controls (NC), hypoxic PH (HP), normoxic (NA) and hypoxic PH (HA) treated with orally active Ang 1–7 30 μg/kg/day for 14 days. In HP mice, RVSP, RVH and ET-1 levels were increased and blocked by Ang 1–7 treatment. Hyper-contractility and endothelial dysfunction in pulmonary arteries of HP mice compared to NC was attenuated by Ang 1–7. These findings indicate that vasoprotective effects of Ang-(1–7) may be mediated through dimerization of MAS:ETBR. In vivo studies support a relationship between the Ang-(1–7)/MAS and ET-1 systems. In conclusion we have identified a novel link between Ang-(1–7) and ET-1 through physical interactions between MAS and ETBR.

Impact of Superoxide Dismutase Mimetic AEOL 10150 on the Endothelin System of Fischer 344 Rats.

Endothelin-1 is a potent vasoconstrictor and mitogenic peptide involved in the regulation of vasomotor tone and maintenance of blood pressure. Oxidative stress activates the endothelin system, and is implicated in pulmonary and cardiovascular diseases including hypertension, congestive heart failure, and atherosclerosis. Superoxide dismutase mimetics designed with the aim of treating diseases that involve reactive oxygen species in their pathophysiology may exert a hypotensive effect, but effects on the endothelin system are unknown. Our objective was to determine the effect of the superoxide dismutase mimetic AEOL 10150 on the basal endothelin system in vivo. Male Fischer-344 rats were injected subcutaneously with 0, 2 or 5 mg/kg body weight of AEOL 10150 in saline. Plasma oxidative stress markers and endothelins (bigET-1, ET-1, ET-2, ET-3) as well as lung and heart endothelin/nitric oxide system gene expressions were measured using HPLC-Coularray, HPLC-Fluorescence and RT-PCR respectively. AEOL 10150 reduced (p<0.05) the circulating levels of isoprostane (-25%) and 3-nitrotyrosine (-50%) measured in plasma 2h and 24h after treatment, confirming delivery of a physiologically-relevant dose and the potent antioxidant activity of the drug. The reduction in markers of oxidative stress coincided with sustained 24h decrease (p<0.05) of plasma levels of ET-1 (-50%) and ET-3 (-10%). Expression of preproET-1 and endothelin converting enzyme-1 mRNA were not altered significantly in the lungs. However preproET-1 (not significant) and ECE-1 mRNA (p<0.05) were increased (10–25%) in the heart. Changes in the lungs included decrease (p<0.05) of mRNA for the ET-1 clearance receptor ETB and the vasoconstriction-signaling ETA receptor (-30%), and an early surge of inducible nitric oxide synthase expression followed by sustained decrease (-40% after 24 hours). The results indicate that interception of the endogenous physiological flux of reactive nitrogen species and reactive oxygen species in rats impacts the endothelin/nitric oxide system, supporting a homeostatic relationship between those systems.

Abstract 017: Ang-(1-7) Influences ET-1 Signaling Through MAS: ETBR Interactions: Implications in Pulmonary Hypertension

ACE2 and Ang 1-7 have been shown to protect against pulmonary hypertension (PH). Mechanisms remain unclear. Considering the important role of ET-1 in PH pathophysiology and endothelial dysfunction, we asked whether Ang 1-7 influences ET-1 signaling in endothelial cells and whether Ang 1-7 treatment influences the ET-1 system in PH. Human microvascular endothelial cells (HMEC) were stimulated with ET-1 in absence/presence of Ang 1-7 and showed that Ang 1-7 increased preproET-1 mRNA (250%), ET-1 release (125%) and ETBR protein (50%), p&lt;0.05. ET-1 increases in e-selectin mRNA (400%), VCAM-1 protein (38%) and TNFα production (30%) were blocked by Ang 1-7, p&lt;0.05. Pro-inflammatory effects were dependent on NO. Ang 1-7 increased NO production (257%) in a Mas and ETBR-dependent manner. Mas and ETBR interaction was observed by immunoprecipitation. To characterise physical interactions between Mas/ETBR, we utilised novel technology, employing a library of peptides scanning the MasR sequence, to define sites of ETBR binding. Substitution and truncation identified regions on MasR that confer specificity for ETBR binding. Peptide disruptors to prevent Mas/ETBR interaction were used for in vitro validation. We previously demonstrated in HMEC that Ang 1-7 stimulates Akt phosphorylation (180%), an effect inhibited by pre-incubation with peptide disruptors, p&lt;0.05. To investigate pathophysiological significance of our findings, we investigated whether Ang 1-7 treatment ameliorates PH and whether this is associated with altered ET-1 status. Hypoxia was used to induce PH in mice: normoxic controls (NC), hypoxic PH (HP), normoxic (NA) and hypoxic PH (HA) treated with Ang 1-7 30μg/kg/day. In HP mice, RVSP (18.7 NC vs. 47.6mmHg HP, p&lt;0.05) RVH (0.19 NC vs. 0.28 HP, p&lt;0.01) and ET-1 levels (0.8 NC vs 2.4pg/ml HP, p&lt;0.05) were increased and blocked by Ang 1-7. Hypercontractility and endothelial dysfunction in pulmonary arteries of HP mice was attenuated by Ang 1-7. These findings indicate that vasoprotective effects of Ang 1-7 may be mediated through MAS:ETBR dimerization. In vivo studies support a relationship between Ang 1-7/MAS and ET-1 systems. In conclusion we have identified a novel link between Ang 1-7 and ET-1 through physical interactions between MAS and ETBR.

Effect of gedunin on acute articular inflammation and hypernociception in mice.

Gedunin, a natural limonoid from Meliaceae species, has been previously described as an antiinflammatory compound in experimental models of allergic inflammation. Here, we report the antiinflammatory and antinociceptive effects of gedunin in an acute model of articular inflammation induced by zymosan (500 μg/cavity; intra-articular) in C57BL/6 mice. Intraperitoneal (i.p.) pretreatment with gedunin (0.005–5 mg/kg) impaired zymosan-induced edema formation, neutrophil accumulation and hypernociception in mouse knee joints, due to decreased expression of preproET-1 mRNA and production of LTB4, PGE2, TNF-α and IL-6. Mouse post-treatment with gedunin (0.05 mg/kg; i.p.) 1 and 6 h after stimulation also impaired articular inflammation, by reverting edema formation, neutrophil accumulation and the production of lipid mediators, cytokines and endothelin. In addition, gedunin directly modulated the functions of neutrophils and macrophages in vitro. The pre-incubation of neutrophil with gedunin (100 µM) impaired shape change, adhesion to endothelial cells, chemotaxis and lipid body formation triggered by different stimuli. Macrophage pretreatment with gedunin impaired intracellular calcium mobilization, nitric oxide production, inducible nitric oxide synthase expression and induced the expression of the antiinflammatory chaperone heat shock protein 70. Our results demonstrate that gedunin presents remarkable antiinflammatory and anti-nociceptive effects on zymosan-induced inflamed knee joints, modulating different cell populations.

Characterization and Functions of Vascular Adventitial Fibroblast Subpopulations

Background: Adventitial fibroblasts have been shown to play an important role in vascular remodeling and contribute to neointimal formation in vascular diseases. However, little is known about adventitial fibroblast subpopulations. This study explored the process of isolating rat thoracic aorta adventitial fibroblast subpopulations and characterized their properties following stimulation with angiotensin II (ANG II), a critical factor involved in cardiovascular diseases such as hypertension. Methods: Adventitial fibroblasts were isolated and cultured from rat aorta. Fibroblast subpopulations were individually expanded using cloning ring techniques. Cells were treated with ANG II (10 nM, 100 nM and 1 μM) for 0.5, 1, 1.5, 3, 6, 12, or 24 h, and ANG II-induced proliferation and migration were measured by MTT assay and Transwell. Cells were treated with ANG II (100 nM) in the presence or absence of ANG II receptor antagonists (100 μM), losartan (for AT1) and PD-123319 (for AT2). PreproET-1 mRNA and ET-1 were determined by RT-PCR and ELISA, respectively. Collagen type I was detected by western blotting. Results: Two major fibroblast subpopulations were found in the adventitia, epithelioid-like cells and spindle-like cells; Although ANG II promotes the growth of both subpopulations, epithelioid-like cell proliferation shows dose-dependency on ANG II from 10 nM to 1 μM, while proliferation of spindle-like cells reaches a peak value following 100 nM ANG II stimulation; ANG II stimulation enhanced epithelioid-like but not spindle-like cell migration; ANG II dose-dependently increased the expression of preproET-1 and collagen type I, and enhanced ET-1 secretion in epithelioid-like but not spindle-like cells, effects abolished by the AT1 receptor antagonist, but not with AT2 receptor antagonist. Conclusion: Adventitial fibroblasts are heterogeneous and epithelioid-like subpopulations with high sensitivity to ANG II stimulation may be implicated in the pathophysiological mechanisms of vascular remodeling, reparative processes and cardiovascular diseases.

Abstract 316: Angiotensin 1-7 Regulation Of Endothelin-1 System In Pulmonary Arterial Hypertension

Studies suggest that activation of ACE2-Ang-(1-7)-Mas ameliorates vascular, cardiac and lung damage observed in pulmonary hypertension (PAH); a condition where endothelin-1 (ET-1) is important. Here we assessed whether Ang 1-7 regulates ET-1 system in pulmonary hypertension and putative mechanisms. Hypobaric hypoxia was used to induce hypoxic PAH in mice, which were divided in 4 groups: normoxic controls (NC), hypoxic PAH (HP), normoxic (NA) and hypoxic PAH (HA) treated with Ang 1-7 (hydroxypropyl-βcyclodextrin-Ang(1-7) 30μg/kg/day given by oral gavage for 14 days after established hypoxia-induced PAH. In HP mice, RVSP (18.7 NC vs. 47.6mmHg HP, p&lt;0.001), RVH by Fulton Index (0.198 NC vs. 0.279 HP, p&lt;0.01) and urinary ET-1 levels (0.88 NC vs 2.48pg/ml HP, p&lt;0.05 vs NC) were increased, an effect blocked by Ang1-7 treatment. In NA, Ang 1-7 increased urinary levels of ET-1 (2.24pg/ml, p&lt;0.05 vs NC). No changes were observed on ETBR protein expression between all groups. Human microvascular endothelial cells (HMEC) were also used and stimulated with ET-1 in the absence/presence of Ang 1-7. Ang 1-7 stimulation increased preproET-1 mRNA levels (250%), ET-1 release (125%), and ETBR protein levels (50%), p&lt;0.05 vs vehicle. To examine whether the regulation of ET-1 system by Ang 1-7 is beneficial, HMEC were stimulated with ET-1 in the absence/presence of Ang 1-7. ET-1 increased e-selectin mRNA (400%) and VCAM-1 protein (38%) levels, as well as TNFα production (30%); all effects blocked by Ang 1-7 (p&lt;0.05). Ang 1-7 inhibition of ET-1 pro-inflammatory effects was dependent on NO production, since it was blocked by L-NAME and LY294002 (inhibitors of NO pathway). Interestingly, Ang 1-7 increased NO production (257%) through Mas and ETBR-dependent manner. An interaction between Mas and ETbR was observed in HMEC by immunoprecipitation and peptide array protocols. In conclusion, Ang 1-7 modulates ET-1 system in pulmonary hypertension and HMEC. Our findings indicate that Ang 1-7 negatively modulates proinflammatory signalling in human endothelial cells. The protective effect may involve upregulation/crosstalk of/with ETBR. These data identify the ET-1 system as a novel molecular mechanism involved in the putative protective action of Ang 1-7 in pulmonary hypertension.

Intermittent hypoxia-induced increases in reactive oxygen species activate NFATc3 increasing endothelin-1 vasoconstrictor reactivity

Sleep apnea (SA), defined as intermittent respiratory arrest during sleep, is associated with increased incidence of hypertension, peripheral vascular disease, stroke, and sudden cardiac death. We have shown that intermittent hypoxia with CO2 supplementation (IH), a model for SA, increases blood pressure and circulating ET-1 levels, upregulates lung pre-pro ET-1 mRNA, increases vasoconstrictor reactivity to ET-1 in rat small mesenteric arteries (MA) and increases vascular reactive oxygen species (ROS). NFAT activity is increased in the aorta (AO) and MA of mice exposed to IH in an ET-1-dependent manner, and the genetic ablation of the isoform NFATc3 prevents IH-induced hypertension. We hypothesized that IH causes an increase in arterial ROS generation, which activates NFATc3 to increase vasoconstrictor reactivity to ET-1. In support of our hypothesis, we found that IH increases ROS in AO and MA. In vivo administration of the SOD mimetic tempol during IH exposure prevents IH-induced increases in NFAT activity in mouse MA and AO. We found that IH causes an NFATc3-dependent increase in vasoconstrictor reactivity to ET-1, accompanied by an increase in vessel wall [Ca2+]. Our results indicate that IH exposure causes an increase in arterial ROS to activate NFATc3, which then increases vasoconstrictor reactivity and Ca2+ response to ET-1. These studies highlight a novel regulatory pathway, and demonstrate the potential clinical relevance of NFAT inhibition to prevent hypertension in SA patients.

GW24-e1006 Effects of telmisartan on left ventricular hypertrophy and expression of endothelin-1 in spontaneously hypertensive rats

Objectives To investigate the effect of telmisartan (telmi) on blood pressure, LV morphology, plasma angiotensin II, expression of preproET-1 and ET-1 in left ventricular hypertrophy (LVH) of SHR.MethodsTwenty Wistar-Kyoto (WKY)...

IL‐33/ST2 signalling contributes to carrageenin‐induced innate inflammation and inflammatory pain: role of cytokines, endothelin‐1 and prostaglandin E2

IL-33 signals through ST2 receptors and induces adaptive and innate inflammation. IL-33/ST2 is involved in adaptive inflammation-induced pain. Here, we have investigated the contribution of IL-33/ST2-triggered mechanisms to carrageenin-induced innate inflammation. Carrageenin- and IL-33-induced inflammatory responses were assessed in BALB/c- (WT) and ST2-deficient ((-/-) ) mice as follows: oedema (plethysmometer), myeloperoxidase activity (colorimetric assay), mechanical hyperalgesia (electronic version of von Frey filaments), cytokine levels (ELISA), PGE2 (RIA), mRNA expression (quantitative PCR), drug treatments targeting leukocyte recruitment (fucoidin), TNF-α (infliximab), CXCL1 (antibody to CXCL1), IL-1 (IL-1ra), endothelin ETA (clazosentan) and ETB (BQ788) receptors and COX (indomethacin). Carrageenin injection increased ST2 and IL-33 mRNA expression and IL-33 production in paw skin samples. Carrageenin-induced paw oedema, hyperalgesia and myeloperoxidase activity were reduced in ST2(-/-) compared with WT mice, effects mimicked by IL-33 injection in the paw. Furthermore, IL-33-induced hyperalgesia was reduced by fucoidin suggesting a role for recruited leukocytes in its hyperalgesic effect. IL-33-induced hyperalgesia in naïve mice was reduced by treatments targeting TNF, CXCL1, IL-1, endothelin receptors and COX while carrageenin-induced ST2-dependent TNF-α, CXCL1, IL-1β, IL-10 and PGE2 production and preproET-1 mRNA expression. Combining IL-33 and carrageenin at doses that were ineffective as single treatment induced significant hyperalgesia, oedema, myeloperoxidase activity and cytokine production in a ST2-dependent manner. IL-33/ST2 signalling triggers the production of inflammatory mediators contributing to carrageenin-induced inflammation. These data reinforces the importance of IL-33/ST2 signalling as a target in innate inflammation and inflammatory pain.

Albumin upregulates eNOS mRNA through ETRA/B in human proximal tubular epithelial cells

Proximal tubular cells respond to proteinuria by expressing several cytokines and inflammatory molecules that induce interstitial fibrosis. Increased attention has been drawn toward the systems of endothelin (ET) and nitric oxide (NO). This work contributes to the elucidation of the interplay between these two systems in proximal tubular epithelial cells (PTECs) after exposure in proteinuric conditions. HK-2 cells, a human PTEC line, were incubated with albumin, simulating proteinuric conditions. Cells were then lysed and either total RNA was isolated or whole cell extracts were prepared. PreproET-1, ET receptors (ETRA and ETRB) and NO synthases (eNOS, iNOS) mRNA accumulation was estimated by RT-PCR, and proteins by Western blot analysis. NO production was assessed using Griess reaction. Furthermore, we treated HK-2 cells with NO donor sodium nitroprusside, NO inhibitor L-NAME, ETRA inhibitor BQ123, ETRB inhibitor BQ788 and purified ET-1, and investigated the potential interplay between albumin-induced stimulation of NO or ET-1 systems. We found that albumin upregulates preproET-1, ETRA, ETRB, eNOS and iNOS mRNA as well as protein and stimulates NO production. Additionally, we recorded an ETRA/B dependent regulation of albumin-induced eNOS expression. For the first time an in vitro albumin-induced ET-1 and NO interplay was revealed.

Statin inhibits hypoxia-induced endothelin-1 via accelerated degradation of HIF-1α in vascular smooth muscle cells

Endothelin-1 (ET-1) contributes to the pathogenesis of cardiovascular diseases with multiple properties such as vasoconstriction. Human ET-1 gene expression is up-regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) through hypoxia response element (HRE). Although previous studies suggested that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) alter HIF-1-related gene expression, it remained unclear whether statins modulate HIF-1-mediated ET-1 expression. Therefore, we investigated the effect of fluvastatin on hypoxia-induced human ET-1 expression in vascular smooth muscle cells (VSMC). Hypoxia (1% O(2)), compared with the normoxic condition (21% O(2)), significantly induced the expression of preproET-1 mRNA, ET-1 protein, and ET-1 secretion in VSMC. Hypoxia induced a 2.3-fold increase in HRE-dependent ET-1 reporter gene activation. Under concentrations of 1 µmol/L or greater, fluvastatin attenuated the hypoxia-induced ET-1 gene expression through the accelerated ubiquitin/proteasome-dependent degradation of HIF-1α, thus consequently attenuating HIF-1α binding to the HRE of the ET-1 gene. These inhibitory effects of fluvastatin were cancelled by concomitant treatment with mevalonate, farnesyl pyrophosphate, or geranylgeranyl pyrophosphate, but not squalene. The present study suggests that fluvastatin attenuates HIF-1-dependent ET-1 gene expression in conjunction with the stimulation of HIF-1α ubiquitin/proteasome-dependent degradation via isoprenoid-dependent mechanisms.

Preproendothelin-1 expression is negatively regulated by IFNγ during hepatic stellate cell activation

Endothelin-1 (ET-1), a powerful vasoconstrictor peptide, is produced by activated hepatic stellate cells (HSC) and promotes cell proliferation, fibrogenesis, and contraction, the latter of which has been thought to be mechanistically linked to portal hypertension in cirrhosis. Interferon-γ (IFNγ), a Th1 cytokine produced by T cells, inhibits stellate cell proliferation, fibrogenesis, and muscle-specific gene expression. Whether IFNγ-induced inhibitory effects are linked to regulation of ET-1 expression in activated stellate cells remains unknown. Here we examined IFNγ's effects on preproET-1 mRNA expression and the signaling pathways underlying this process. We demonstrated that preproET-1 mRNA expression in HSCs was prominently increased during cell culture-induced activation; IFNγ significantly inhibited both preproET-1 mRNA expression and ET-1 peptide production. Similar results were found in an in vivo model of liver injury and intraperitoneal administration of IFNγ. PreproET-1 promoter analysis revealed that IFNγ-induced inhibition of preproET-1 mRNA expression was closely linked to the AP-1 and Smad3 signaling pathways. Furthermore, IFNγ reduced JNK phosphorylation, which tightly was associated with decreased phosphorylation of downstream factors c-Jun and Smad3 and decreased binding activity of c-Jun and Smad3 in the preprpET-1 promoter. Importantly, IFNγ reduced both c-Jun mRNA and protein levels. Given the important role of ET-1 in wound healing, our results suggest a novel negative signaling network by which IFNγ inhibits preproET-1 expression, highlighting one potential molecular mechanism for IFNγ-induced host immunomodulation of liver fibrogenesis.

Bosentan, an endothelin receptor antagonist, ameliorates collagen-induced arthritis: the role of TNF-α in the induction of endothelin system genes

Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice. CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100mg/kg) once a day, starting from the day when arthritis was clinically detectable. CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student's t test. Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1β, TNFα and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNFα upregulated ET system genes. These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNFα.