Endothelin-1 Release Research Articles

Activation of the Mineralocorticoid Receptor Stimulates Protein Disulfide Isomerase: Role in Sickle Cell Disease

Activation of the mineralocorticoid receptor (MR), a critical component of the Renin-Angiotensin-Aldosterone (ALDO)-System (RAAS), has been shown to play an important role in inflammatory and vascular endothelial responses in addition to its well-described effects on sodium and water homeostasis. Activationof endothelial cells leads to, among other factors, increased endothelin-1 (ET-1) and protein disulfide isomerase (PDI) release. PDI and ET-1 contribute to vascular inflammation and are increased in patients with SCD and sickle transgenic mouse models. The MR is a member of the steroid family of nuclear receptors and transcription factors that upon activation binds to hormone response elements of edn1, the gene for ET-1, leading to increased ET-1 expression.In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and ET-1 mRNA expression. However, the role of MR in SCD is unclear. We hypothesized that MR blockade in sickle transgenic mice would reduce PDI activity and improve hematological parameters and inflammation. We first studied EA.hy926 (EA) cells, a human endothelial cell line that expresses MR. We incubated EA cells with ALDO (10-8 M), an MR agonist, for 24 hr and observed a rise in PDI mRNA levels by qRT-PCR (P<0.01, n=5), an event that was blocked by pre-incubation of EA cells with 1 μM canrenoic acid (CA), an MR antagonist (P<0.05, n=5). We then measured PDI activity in the supernatant of ALDO-stimulated EA cells using a Di-E-GSSH fluorescent marker and observed a rise in PDI activity following ALDO (10-8 M) when compared to vehicle treatment (P<0.05; n=5). To test the in vivo effects of MR activation, we studied Berkeley Sickle Transgenic (BERK) mice that were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR antagonist (MRA), for 14 days. We observed significantly lower plasma PDI activity in mice treated with MRA than those on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units (RFU); P<0.005, n=6 and 9, respectively). Treatment with MRA was associated with reduced plasma ET-1 and myeloperoxidase (MPO) levels in BERK mice. We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells x hr; P<0.04 n= 5 and 7 respectively). Consistent with these results, MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). We also studied gene expression by qRT-PCR in heart tissue from these mice and observed that MR blockade reduced mRNA expression of: ET-1 (0.654 ± 0.233, P<0.05, n=5 and n=7); PDI (0.546 ± 0.063, P<0.01, n=5 and n=7); and Tumor Necrosis Factor Receptor Superfamily Member 1A mRNA (0.464 ± 0.061, P<0.01, n=5 and n=7). Thus, our results suggest a novel role for RAAS and, in particular, MR activation in SCD. DisclosuresNo relevant conflicts of interest to declare.

Pathophysiological Role of Adiponectin, Leptin and Asymmetric Dimethylarginine in the Process of Atherosclerosis.

Adipose tissue is recognized as a rich source of proinflammatory mediators that may directly contribute to vascular injury, insulin resistance, and atherogenesis. Many studies have shown that adiponectin has antiatherogenic and anti-inflammatory properties. Adiponectin acts not only as a factor increasing insulin sensitivity, and the protective effect may result from its ability to suppress production of proinflammatory cytokines. It negatively regulates the expression of TNF-alpha and C-reactive protein (CRP) in adipose tissue; reduces expression of vascular and intracellular adhesion molecules (VCAM-1, ICAM-1), E-selectin, interleukin-8 (IL-8). Hyperleptinemia has been linked with the development of hypertension and endothelial dysfunction/atherosclerosis, two main pathophysiological conditions associated with cardiovascular disease development. Leptin-mediated increases in sympathetic nervous system activity may be among the principal mechanisms evoking obesity related hypertension. Leptin stimulates the secretion of proinflammatory cytokines, and increases the release of endothelin-1 (ET-1), which may promote hypertension. Increased serum levels of asymmetric dimethylarginine (ADMA), a physiological regulator of the biosynthesis of nitric oxide (NO), promote the process of atherosclerosis, leading to the occurrence of endothelial dysfunction and cardiovascular disease.

Impact of selective anti-BMP9 treatment on tumor cells and tumor angiogenesis

The role of bone morphogenic protein 9 (BMP9) signaling in angiogenesis has been controversial, with a number of studies showing that it acts either as a pro-angiogenic or, conversely, as an anti-angiogenic factor in a context-dependent manner. Notably, BMP9 was also reported to function in both pro- or anti-tumorigenic roles during tumor progression. It has therefore remained unclear, whether selective BMP9 inhibition is a useful target for antibody therapy of cancer. To shed light on these questions, we characterized BMP9 expression in plasma of patients with different cancer indications and found elevated levels of pro-domains and precursor BMP9 with a strong response in renal cell carcinoma (RCC). These studies prompted us to evaluate the potential of selective anti-BMP9 cancer therapy in RCC. We generated a novel monoclonal therapeutic antibody candidate, mAb BMP9-0093, that selectively targets all different BMP9 variants but does not bind to the closest homolog BMP10. In vitro, mAb BMP9-0093 treatment inhibited signaling, endothelin-1 (ET-1) production and spreading of endothelial cells and restored BMP9-induced decrease in pericyte migration and attachment. Furthermore, BMP9-mediated epithelial–mesenchymal transition of renal cell carcinoma cells was reversed by mAb BMP9-0093 treatment in vitro. In vivo, mAb BMP9-0093 showed significant anti-tumor activity that was associated with an increase in apoptosis as well as a decrease in tumor cell proliferation and ET-1 release. Furthermore, mAb BMP9-0093 induced mural cell coverage of endothelial cells, which was corroborated by a reduction in vascular permeability, demonstrated by a diminished penetration of omalizumab-Alexa 647 into tumor tissue. Our findings provide new evidence for a better understanding of BMP9 contribution in tumor progression and angiogenesis that may result in the development of effective targeted therapeutic interventions.

Neutrophil Extracellular Traps Promote Angiogenesis: Evidence From Vascular Pathology in Pulmonary Hypertension.

Inflammation and dysregulated angiogenesis are features of endothelial dysfunction in pulmonary hypertension. Neutrophil extracellular traps (NETs), produced by dying neutrophils, contribute to pathogenesis of numerous vascular disorders but their role in pulmonary hypertension has not been studied. We sought evidence of (NETs) formation in pulmonary hypertension and investigated the effect of NETs on endothelial function. Plasma and lung tissues of patients with pulmonary hypertension were analyzed for NET markers. The effects of NETs on endothelial function were studied in vitro and in vivo. Patients with chronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension showed elevated plasma levels of DNA, neutrophil elastase, and myeloperoxidase. NET-forming neutrophils and extensive areas of NETosis were found in the occlusive plexiform lesions and vascularized intrapulmonary thrombi. NETs induced nuclear factor κB-dependent endothelial angiogenesis in vitro and increased vascularization of matrigel plugs in vivo. Angiogenic responses were associated with increased release of matrix metalloproteinase-9, heparin-binding epidermal growth factor-like growth factor, latency-associated peptide of the transforming growth factor β1, and urokinase-type plasminogen activator, accompanied by increased endothelial permeability and cell motility. NETs-induced responses depended on myeloperoxidase/H2O2-dependent activation of Toll-like receptor 4/nuclear factor κB signaling. NETs stimulated the release of endothelin-1 in HPAECs (human pulmonary artery endothelial cells) and stimulated pulmonary smooth muscle cell proliferation in vitro. We are the first to implicate NETs in angiogenesis and provide a functional link between NETs and inflammatory angiogenesis in vitro and in vivo. We demonstrate the potential pathological relevance of this in 2 diseases of disordered vascular homeostasis, pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension.

PP.36.15] VENOUS OCCLUSION PLETHYSMOGRAPHY AND BIOMARKERS AS EVALUATION METHODS OF ENDOTHELIUM FUNCTION IN PATIENTS WITH ARTERIAL HYPERTENSION

Objective: The aim of this study was to investigate the correlation between serum markers of inflammation and microcirculatory parameters observed by Venous occlusion plethysmography (VOP) in hypertensive patients Design and method: It is a cross-sectional study. Patients with resistant (RH, 58 [50–63] years, n = 25) or mild to moderate hypertension (MMH, 56[47–64], n = 25) were compared to normotensive ones (Control Group – CG, 33 [27–52], n = 25). C-reactive protein (CRP), endothelin, adiponectin, I-CAM and V-CAM were obtained by laboratorial analysis. Flow rate and resistance in the basal period, post-ischemic 5 minutes on the forearm (endothelium-dependent phase) and post-use of sublingual nitroglycerin (endothelium-independent phase) were determined by VOP. A sub-analysis was also conducted on hypertensive patients not taking statins, with controlled/uncontrolled blood pressure. Results: RH group showed a lower percentage of maximum flow and increased post-ischemia minimal resistance compared to MMH and CG ones (p < 0,003) with worse values in patients with uncontrolled blood pressure (p < 0,05). Post-nitroglycerin phase (endothelium independent) showed no significant differences among the three groups, with higher CRP values in RH and MMH groups. An increase in endothelin was seen only in patients not taking statins in both hypertensive groups. The VOP analyzes were adjusted for confounding factors. Conclusions: Patients with severe hypertension and uncontrolled blood pressure levels presented more pronounced impairment of vascular reactivity, when analyzed by VOP, as well as higher serum values for CRP and endothelin. It is possible that the use of statins decrease endothelin release.

PP.12.14] MICROCIRCULATION AND BIOMARKERS IN PATIENTS WITH RESISTANT OR MILD TO MODERATE ARTERIAL HYPERTENSION

Objective: The aim of this study was to investigate the correlation between serum markers of inflammation and microcirculatory parameters observed by nailfold videocapillaroscopy (NVC) in hypertensive patients Design and method: It is a cross-sectional study. Patients with resistant (RH, 58 [50–63] years, n = 25) or mild to moderate hypertension (MMH, 56[47–64], n = 25) were compared to normotensive ones (Control Group – CG, 33 [27–52], n = 25). C-reactive protein (CRP), endothelin, adiponectin, I-CAM and V-CAM were obtained by laboratorial analysis. Functional capillary density (FCD, number of capillaries with flowing red blood cells by unit tissue area) and capillary diameters, maximum red blood cell velocity (RBCVmax) during the reactive hyperemia response/RBCVbaseline after 1 min arterial occlusion at the finger base and time to reach RBCVmax were determined by NVC. A sub-analysis was also conducted on hypertensive patients not taking statins, with controlled/uncontrolled blood pressure. Results: RH group showed lower RBCV and RBCVmax values and higher TRBCVmax compared to MMH and CG ones (p < 0,007) with worse values in patients with uncontrolled blood pressure (p < 0,008). FCD and diameters showed no significant differences among the three groups, with higher CRP values in RH and MMH groups. An increase in endothelin was seen only in patients not taking statins in both hypertensive groups. The NVC analyzes were adjusted for confounding factors. Conclusions: Patients with severe hypertension and uncontrolled blood pressure levels presented more pronounced microvascular dysfunction, as well as higher serum values for CRP and endothelin. It is possible that the use of statins decrease endothelin release.

Abstract 2901: Nuclear β-arrestin1 is a critical cofactor of hypoxia-inducible factor-1α signaling in endothelin-1-induced ovarian tumor progression

Abstract In epithelial ovarian cancer (EOC), endothelin-1 (ET-1)/endothelin A receptor (ETAR) axis has been reported to be involved in invasiveness, epithelial-to-mesenchymal transition, chemoresistance and metastatic dissemination and is thereby associated with poor prognosis. In these cells, ET-1 is able to induce the nuclear translocation and stability of hypoxia-inducible factor-1α (HIF-1α) the principal transcriptional factor that allows cellular adaptation to hypoxia. The importance of co-factors that trigger an epigenetic regulation of HIF-1α within tumor is not well understood. Here we elucidate that ET-1/ETAR-induced pathway physically and functionally couples the scaffold protein β-arrestin1 (β-arr1) to HIF-1α signaling. In EOC cells, ET-1 induced vascular-endothelial growth factor (VEGF), the pro-agiogenic target downstream of HIF-1α through HIF-1α nuclear accumulation. Activation of ET-1/ETAR axis, by mimicking hypoxia, promotes the nuclear association between β-arr1 and HIF-1α on the hypoxia responsive elements (HRE) binding sites. By ChIP assay, nuclear β-arr1 was able to interact with HIF-1α, by promoting the recruitment of p300 acetyltransferase. The formation of this functional complex resulted in enhanced histone acetylation, and gene transcription of HIF-1α downstream targets, such as ET-1 and VEGF, required for cell invasion. The induced release of ET-1 and VEGF by EOC cells, promoted angiogenic effects in endothelial cells, as evaluated by cell migration and tube-like structure formation assays. Interestingly ETAR/β-arr1 promoted the self-amplifying HIF-1α-mediated transcription of ET-1 that sustained a regulatory circuit required for invasiveness and angiogenic responses. These effects were abrogated by the silencing of β-arr1 or HIF-1α or by pharmacological treatment with the dual ET-1 receptor antagonist macitentan. In a murine orthotropic model of metastatic human EOC, treatment with macitentan, or silencing of β-arr1, inhibits intravasation and metastasis formation, suggesting that ET-1, together with VEGF, represents one of such factors that mediates tumor-endothelial cell communications, favoring a permissive environment for metastatic spread. Collectively these findings reveal the interplay of β-arr1 with HIF-1α in the complexity of ET-1/ETAR pathway, mediating epigenetic modifications, to an extent comparable with hypoxia, directly involved in metastatic progression. Targeting ET-1 axis by macitentan may represent a new opportunity to target the orchestration mediated by nuclear β-arr1/HIF-1α complex in EOC, demonstrating that this approved small molecule interfering with stromal compartment, expressing ETBR, and tumor cells, expressing mainly ETAR, can be used in clinical setting for improved therapeutics in EOC. Citation Format: Piera Tocci, Roberta Cianfrocca, Laura Rosanò, Valentina Caprara, Rosanna Sestito, Valeriana Di Castro, Anna Bagnato. Nuclear β-arrestin1 is a critical cofactor of hypoxia-inducible factor-1α signaling in endothelin-1-induced ovarian tumor progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2901.

Exogenous melatonin reduced blood pressure in late-term ovine fetus via MT1/MT2 receptor pathways

Melatonin is involved in the regulation of blood pressure through the receptor dependent or independent route. However, the effect of melatonin on fetal blood pressure is unknown. This study investigated the effect of melatonin on blood pressure of the late-term ovine fetus in utero. Melatonin and/or antagonists were intravenously administered into the fetuses. Mean arterial pressure and heart rate were recorded. Fetal blood samples were analyzed for biochemical parameters and hormones, including cortisol, angiotensin I, angiotensin II, aldosterone, atrial natriuretic peptide, corticotrophin-releasing hormone, adrenocorticotropic hormone, and endothelin. Fetal blood pressure was decreased following administration of melatonin, whereas it was increased following administration of luzindole, but not prazosin. Plasma level of endothelin was decreased by melatonin, which was blocked by luzindole. Our study suggested that melatonin reduced fetal blood pressure via MT1/MT2 receptors and possibly involving release of endothelin.

Immature endothelial cells initiate endothelin-mediated constriction of newborn arteries.

Endothelial expression and the release of endothelin-1 (ET-1) in levels sufficient to initiate vasoconstriction is considered to be a hallmark feature of pathological endothelial dysfunction. During the immediate postnatal period, arterial endothelial cells undergo remarkable structural and functional changes as they transition to a mature protective cell layer, which includes a marked increase in NO dilator activity. The present study demonstrates that endothelial cells lining newborn central arteries express high levels of ET-1 peptides and, in response to endothelial stimulation, rapidly release ET-1 and initiate powerful ET-1-mediated constriction. This activity is lost as the endothelium matures in the postnatal period. Heightened activity of ET-1 in the neonatal endothelium might contribute to inappropriate responses of immature arteries to stress or injury. Indeed, the immature endothelium resembles dysfunctional endothelial cells, and retention or re-emergence of this phenotype may contribute to the development of vascular disease. Endothelial cells lining fetal and newborn arteries have an unusual phenotype, including reduced NO activity, prominent actin stress fibres and poorly developed cellular junctions. Experiments were performed to determine whether the immature endothelium of newborn arteries also expresses and releases endothelin-1 (ET-1) and initiates endothelium-dependent constriction. Carotid arteries were isolated from newborn (postnatal day 1; P1), postnatal day 7 (P7) and postnatal day 21 (P21) mice and assessed in a pressure myograph system. Endothelial stimulation with A23187 or thrombin caused constriction in P1 arteries, no significant change in diameter of P7 arteries, and dilatation in P21 arteries. In P1 arteries, constriction to thrombin or A23187 was inhibited by endothelial-denudation, by ET-1 receptor antagonists (BQ123 plus BQ788) or by inhibition of endothelin-converting enzyme (phosphoramidon or SM19712). ET-1 receptor antagonism did not affect responses to thrombin or A23187 in more mature arteries. Exogenous ET-1 caused similar concentration-dependent constrictions of P1, P7 and P21 arteries. Endothelial stimulation with thrombin rapidly increased the endothelial release of ET-1 from P1 but not P21 aortas. Endothelial expression of ET-1 peptides, as assessed by immunofluorescence analysis, was increased in P1 compared to P21 arteries. Therefore, newborn endothelial cells express high levels of ET-1 peptides, rapidly release ET-1 in response to endothelial stimulation, and initiate ET-1-mediated endothelium-dependent constriction. This activity is diminished as the endothelium matures in the immediate postnatal period. Heightened activity of ET-1 in neonatal endothelium probably reflects an early developmental role of the peptide, although this might contribute to inappropriate responses of immature arteries to stress or injury.

Transforming growth factor beta 1 induced endothelin-1 release is peroxisome proliferator-activated receptor gamma dependent in A549 cells.

BackgroundEndothelin-1 (ET-1) is involved in pulmonary vascular remodeling. The aim of this study was to investigate the biochemical interactions between PPAR-γ, TGF-β1 and ET-1 in vitro.MethodsA549 cells were pre-treated with S2505 (10 μM), S2871 (10 μM) with/without SB203580 (10 μM) for 60 min following 2 h treatment with 10 ng/mL TGF-β1. A549 cells were also transfected with positive or negative PPAR-γ plasmids for comparison. RT-PCR, ELISA, western blotting and confocal laser scanning microscopy (CLSM) were used to measure the relevant expression of mRNA, protein, mediators of pathways and nuclear factor translocation.ResultsSB203580 inhibited TGF-β1 induced ET-1 expression in A549 cells. S2871 decreased PPAR-γ mRNA and increase TGF-β1-induced ET-1 expression. S2871 increased phosphorylation of p38 MAPK and Smad2. Cells transfected with PPAR-γ negative plasmid increased TGF-β1 induced ET-1 expression, and increased the expression of phospho-p38 MAPK and phospho-Smad2. S2505 increased PPAR-γ mRNA expression, suppressed the increased TGF-β1-induced expression of ET-1. S2505 inhibited TGF-β1 induced phosphorylation of p38 MAPK and Smad2, also the nuclear translocation of Smad2. Cells transfected with PPAR-γ positive plasmid reduced TGF-β1-induced ET-1 expression, and inhibited the expression of phospho-p38 MAPK and phospho-Smad2.ConclusionsTGF-β1 induced release of endothelin-1 is PPAR-γ dependent in cultured A549 cells.

The heart as an extravascular target of endothelin-1 in particulate matter-induced cardiac dysfunction.

Exposure to particulate matter air pollution has been causally linked to cardiovascular disease in humans. Several broad and overlapping hypotheses describing the biological mechanisms by which particulate matter exposure leads to cardiovascular disease have been explored, although linkage with specific factors or genes remains limited. These hypotheses may or may not also lead to particulate matter-induced cardiac dysfunction. Evidence pointing to autocrine/paracrine signaling systems as modulators of cardiac dysfunction has increased interest in the emerging role of endothelins as mediators of cardiac function following particulate matter exposure. Endothelin-1, a well-described small peptide expressed in the pulmonary and cardiovascular systems, is best known for its ability to constrict blood vessels, although it can also induce extravascular effects. Research on the role of endothelins in the context of air pollution has largely focused on vascular effects, with limited investigation of responses resulting from the direct effects of endothelins on cardiac tissue. This represents a significant knowledge gap in air pollution health effects research, given the abundance of endothelin receptors found on cardiac tissue and the ability of endothelin-1 to modulate cardiac contractility, heart rate, and rhythm. The plausibility of endothelin-1 as a mediator of particulate matter-induced cardiac dysfunction is further supported by the therapeutic utility of certain endothelin receptor antagonists. The present review examines the possibility that endothelin-1 release caused by exposure to PM directly modulates extravascular effects on the heart, deleteriously altering cardiac function.

Bisphenol A exposure alters release of immune and developmental modulators and expression of estrogen receptors in human fetal lung fibroblasts

Bisphenol A (BPA) has been shown to exert biological effects through estrogen receptor (ER)-dependent and ER-independent mechanisms. Recent studies suggest that prenatal exposure to BPA may increase the risk of childhood asthma. To investigate the underlying mechanisms in the actions of BPA, human fetal lung fibroblasts (hFLFs) were exposed to varying doses of BPA in culture for 24hr. Effects of BPA on localization and uptake of BPA, cell viability, release of immune and developmental modulators, cellular localization and expression of ERα, ERβ and G-protein coupled estrogen receptor 30 (GPR30), and effects of ERs antagonists on BPA-induced changes in endothelin-1 (ET-1) release were examined. BPA at 0.01–100μmol/L caused no changes in cell viability after 24hr of exposure. hFLFs expresses all three ERs. BPA had no effects on either cellular distribution or protein expression of ERα, however, at 100μmol/L (or 23μmol/L intracellular BPA) increased ERβ protein levels in the cytoplasmic fractions and GPR30 protein levels in the nuclear fractions. These paralleled with increased release of growth differentiation factor-15, decreased phosphorylation of nuclear factor kappa B p65 at serine 536, and decreased release of ET-1, interleukin-6, and interferon gamma-induced protein 10. ERs antagonists had no effects on BPA-induced decrease in ET-1 release. These data suggest that BPA at 100μmol/L altered the release of immune and developmental modulators in hFLFs, which may negatively influence fetal lung development, maturation, and susceptibility to environmental stressors, although the role of BPA in childhood asthma remains to be confirmed in in vivo studies.

Severe Respiratory Failure Due to Interferon Beta-Related Pulmonary Hypertension

SESSION TITLE: Pulmonary Vascular Disease SESSION TYPE: Case Report Poster PRESENTED ON: Sunday, April 17, 2016 at 11:45 AM - 12:45 PM INTRODUCTION: Pulmonary arterial hypertension (PAH) is an uncommon but devastating disease. There is increasing evidence of a correlation between interferon (IFN) use and PAH. Very few cases of PAH in patients treated with IFN are reported in literature. CASE PRESENTATION: We report a case of a 47 year old woman with previous diagnosis of multiple sclerosis (MS) treated with IFN β-1a for six years presenting severe respiratory failure (paO2/FiO2 228) due to pulmonary hypertension. The suspension of the drug along with the treatment of PAH improved the clinical picture allowing cessation of oxygen administration. DISCUSSION: Interferon beta side effects are increasing; the number of associated secondary pathologies are also on the rise [5]. Evidence of a correlation between IFN use and PAH is increasing In the literature only a case series and sporadic cases of PAH correlated with these drugs (no more than twenty) have been described. Recent experimental studies have suggested that IFN could promote endothelial dysfunction increasing the release of endothelin-1 by human vascular cells. CONCLUSIONS: With the increased use of IFNs we should expect more cases in the future. Clinicians should be aware of this possible treatable cause of PAH. Reference #1: 1. Savale L, Sattler C, Gunther S, Montani D, Chaumains MC, Perrin S et al. Pulmonary arterial hypertension in patients treated with interferon. ERJ 2014;44:1627-1634 Reference #2: Gibbons E, Promislow S, Davies RA, Chandy G, Stewart DJ, Vladimir CD et al. Reversible pulmonary arterial hypertension associated with interferon-beta treatment for multiple sclerosis Can Resp J 2015; Jun 17, pli 16820 [Epub ahead of print ] DISCLOSURE: The following authors have nothing to disclose: Antonello Nicolini No Product/Research Disclosure Information

Matrix Metalloproteinase 1 Causes Vasoconstriction and Enhances Vessel Reactivity to Angiotensin II via Protease-Activated Receptor 1.

Matrix metalloproteinase 1 (MMP-1) is an activator of protease-activated receptor 1 (PAR-1), which is known to mediate the release of endothelin 1 (ET-1) in endothelial cells and activate the RhoA kinase (ROCK) pathway. Recently, we reported increased serum and vascular MMP-1 in women with preeclampsia and hypothesized that the action of MMP-1 on PAR-1 might have vasoconstrictive effects. Resistance-sized omental arteries obtained from normal pregnant women were mounted on a myograph system and perfused with MMP-1 in a dose range of 0.025 to 25 ng/mL or with angiotensin II (Ang II) in a dose range of 0.001 to 10 µmol/L in the presence of intraluminal MMP-1 (2.5 ng/mL) perfusion. Angiotensin II dose response was also performed with omental arteries from women with preeclampsia. Matrix metalloproteinase 1 caused dose-dependent vasoconstriction in endothelium-intact, but not in endothelium-denuded, vessels from normal pregnant women, which was blocked by inhibitors of PAR-1 and ET-1 type A receptor blocker. Intraluminal perfusion with a constant amount of MMP-1 enhanced vessel reactivity to Ang II, which was blocked by inhibitors of PAR-1, ROCK, and ET-1. Enhanced vascular reactivity to Ang II was observed in endothelium-intact, but not in endothelium-denuded, arteries of women with preeclampsia. Inhibitors of PAR-1, ROCK, and ET-1 blocked enhanced vascular reactivity to Ang II in endothelium-intact preeclamptic arteries. These data demonstrate that MMP-1 has potent vasoconstrictor effects and the ability to enhance vascular reactivity to vasoconstrictor hormones, which are mediated by an endothelial PAR-1, ROCK, and ET-1 pathway. Increased circulating levels of MMP-1 and its increased expression in systemic vessels of women with preeclampsia may contribute to the development of maternal hypertension.

Induction of endothelium-dependent constriction of mesenteric arteries in endotoxemic hypotensive shock.

Effective management of hypotension refractory to vasoconstrictors in severe sepsis is limited. A new strategy to ameliorate endotoxemic hypotension by inducing endothelium-dependent constriction of large arteries was assessed. Endotoxemia in rats was induced by injection of LPS (10 mg·kg(-1), i.v.). Haemodynamics were measured in vivo, reactivity of isolated mesenteric arteries by myography and expression of proteins and enzyme activities by immunohistochemistry, biochemistry and molecular biology. Six hours after LPS, the hypotension was promptly reversed following injection (i.v. or i.p.) of oroxylin-A (OroA) . In isolated LPS-treated but not normal mesenteric arteries, OroA (1-10 μM) induced endothelium-dependent, sustained constriction, blocked by endothelin-1 (ET-1) receptor antagonists. OroA further enhanced LPS-induced expression of endothelin-converting enzyme, ET-1 mRNA and proteins and ET-1 release, OroA also enhanced phosphorylation of Rho-associated protein kinase (ROCK) and reversed LPS-induced suppression of RhoA activities in smooth muscle of arteries with endothelium. Activated- phosphorylation of smooth muscle ROCK was blocked by ET-1-receptor antagonists and ROCK inhibitors. Moreover, OroA post-treatment suppressed, via inhibiting NF-κB activation, inducible NOS expression and circulating NO. Reversal of endotoxemic hypotensive by OroA was due to release of endothelial ET-1, upregulated by LPS, from mesenteric arteries, inducing prompt and sustained vasoconstriction via activation of vascular smooth muscle RhoA/ROCK-pathway. In late endotoxemia, OroA-induced vasoconstriction was partly due to decreased circulating NO. Activation of endothelium-dependent constriction in large resistance arteries and suppression of systemic inflammation offer new strategies for acute management of endotoxemic hypotensive shock.

Endothelial dysfunction and vascular disease - a 30th anniversary update.

The endothelium can evoke relaxations of the underlying vascular smooth muscle, by releasing vasodilator substances. The best-characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO) which activates soluble guanylyl cyclase in the vascular smooth muscle cells, with the production of cyclic guanosine monophosphate (cGMP) initiating relaxation. The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDH-mediated responses). As regards the latter, hydrogen peroxide (H2 O2 ) now appears to play a dominant role. Endothelium-dependent relaxations involve both pertussis toxin-sensitive Gi (e.g. responses to α2 -adrenergic agonists, serotonin, and thrombin) and pertussis toxin-insensitive Gq (e.g. adenosine diphosphate and bradykinin) coupling proteins. New stimulators (e.g. insulin, adiponectin) of the release of EDRFs have emerged. In recent years, evidence has also accumulated, confirming that the release of NO by the endothelial cell can chronically be upregulated (e.g. by oestrogens, exercise and dietary factors) and downregulated (e.g. oxidative stress, smoking, pollution and oxidized low-density lipoproteins) and that it is reduced with ageing and in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and EDH, in particular those due to H2 O2 ), endothelial cells also can evoke contraction of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factors. Recent evidence confirms that most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells and that prostacyclin plays a key role in such responses. Endothelium-dependent contractions are exacerbated when the production of nitric oxide is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive and diabetic patients. In addition, recent data confirm that the release of endothelin-1 can contribute to endothelial dysfunction and that the peptide appears to be an important contributor to vascular dysfunction. Finally, it has become clear that nitric oxide itself, under certain conditions (e.g. hypoxia), can cause biased activation of soluble guanylyl cyclase leading to the production of cyclic inosine monophosphate (cIMP) rather than cGMP and hence causes contraction rather than relaxation of the underlying vascular smooth muscle.

ETA and ETB receptors contribute to neuropeptide Y-induced secretion of endothelin-1 in right but not left human ventricular endocardial endothelial cells

Our recent work showed that neuropeptide Y-induced secretion of endothelin-1 (ET-1) in left and right human ventricular endocardial endothelial cells (hLEECs or hREECs respectively) via the activation of neuropeptide Y2 or Y5 receptors depending on the cell type. The aim of this study was to verify whether hLEECs or hREECs secretion of ET-1 induced by NPY is due, in part, to the activation of ETA and/or ETB receptors by the secreted ET-1. Using the technique of indirect immunofluorescence coupled to real 3-D confocal microscopy, as well as ELISA, our results show that in hREECs, the NPY-induced release of ET-1 seems to be due, in part, to the activation of both ETA and ETB receptors. On the other hand, in hLEECs, ETA and ETB receptors do not contribute to the ET-1 released by NPY. Therefore, our results suggest that the NPY-induced release of ET-1 in EECRs is due to NPY receptor activation and the subsequent activation of the ETA and ETB receptors by the released ET-1. However, the release of ET-1 by NPY in hLEECs is mainly due to NPY receptor activation. Furthermore, this secretory process of ET-1 is different between the right and left ventricular cells and highlights the important tuning roles that right and left ventricular EECs possess as well as their contribution to the physiological and pathophysiological states of the underlying heart muscle.

Hypertension in Patients with Chronic Kidney Disease

The prevalence of hypertension in chronic kidney disease (CKD) patients exceeds that of the general population. Uncontrolled hypertension plays a significant role in progression to end stage renal disease and results in increased cardiovascular morbidity and mortality. A complex interplay between various pathophysiologic mechanisms is responsible for the development of hypertension in this patient population. The major factors being extracellular volume overload, increased endothelin-1 release and excess renin-angiotensin-aldosterone system and sympathetic nervous system activity. Dietary and lifestyle modifications have synergistic effects to drug therapy in the control of hypertension. There is no single blood pressure target that is optimal for all CKD patients. It is important to individualize the treatment depending on age, the severity of albuminuria, and comorbidities. Drugs blocking the renin-angiotensin-aldosterone system are the recommended first-line antihypertensive agents for most CKD patients. Intradialytic hypertension may be prevented by individualizing the dialysis prescription and using nondialyzable antihypertensives. New onset of hypertension in the elderly or new onset of difficult to control hypertension in a previously well controlled hypertensive patient should prompt the work up for atherosclerotic renal vascular disease.

Novel 24-h ovine model of brain death to study the profile of the endothelin axis during cardiopulmonary injury.

BackgroundUpregulation of the endothelin axis has been observed in pulmonary tissue after brain death, contributing to primary graft dysfunction and ischaemia reperfusion injury. The current study aimed to develop a novel, 24-h, clinically relevant, ovine model of brain death to investigate the profile of the endothelin axis during brain death-associated cardiopulmonary injury. We hypothesised that brain death in sheep would also result in demonstrable injury to other transplantable organs.MethodsTwelve merino cross ewes were randomised into two groups. Following induction of general anaesthesia and placement of invasive monitoring, brain death was induced in six animals by inflation of an extradural catheter. All animals were supported in an intensive care unit environment for 24 h. Animal management reflected current human donor management, including administration of vasopressors, inotropes and hormone resuscitation therapy. Activation of the endothelin axis and transplantable organ injury were assessed using ELISA, immunohistochemistry and standard biochemical markers.ResultsAll animals were successfully supported for 24 h. ELISA suggested early endothelin-1 and big endothelin-1 release, peaking 1 and 6 h after BD, respectively, but there was no difference at 24 h. Immunohistochemistry confirmed the presence of the endothelin axis in pulmonary tissue. Brain dead animals demonstrated tachycardia and hypertension, followed by haemodynamic collapse, typified by a reduction in systemic vascular resistance to 46 ± 1 % of baseline. Mean pulmonary artery pressure rose to 186 ± 20 % of baseline at induction and remained elevated throughout the protocol, reaching 25 ± 2.2 mmHg at 24 h. Right ventricular stroke work increased 25.9 % above baseline by 24 h. Systemic markers of cardiac and hepatocellular injury were significantly elevated, with no evidence of renal dysfunction.ConclusionsThis novel, clinically relevant, ovine model of brain death demonstrated that increased pulmonary artery pressures are observed after brain death. This may contribute to right ventricular dysfunction and pulmonary injury. The development of this model will allow for further investigation of therapeutic strategies to minimise the deleterious effects of brain death on potentially transplantable organs.Electronic supplementary materialThe online version of this article (doi:10.1186/s40635-015-0067-9) contains supplementary material, which is available to authorized users.

A novel role of endothelium in activation of latent pro-membrane type 1 MMP and pro-MMP-2 in rat aorta.

Aortic stiffness is an independent risk factor for progression of cardiovascular diseases. Degradation of elastic fibres in aorta due to angiotensin II (ANGII)-stimulated overactivation of latent membrane type 1 matrix metalloproteinase (MT1MMP) and matrix metalloproteinase-2 (MMP2) is regarded to represent an important cause of aortic stiffness. Therefore, clarification of the causal mechanisms triggering the overactivation of these MMPs is of utmost importance. This study addresses the endothelium as a novel key activator of latent pro-MT1MMP and pro-MMP2 in rat aorta. Using a co-culture model of rat aortic endothelial cells (ECs) and smooth muscle cells (SMCs), we found that ANGII stimulation resulted in activation of latent pro-MT1MMP and pro-MMP2 in SMCs exclusively when co-cultured with ECs (assessed with western blot and gelatin zymography, respectively). EC-specific AT1 receptor stimulation triggered endothelin-1 release and paracrine action on SMCs. Endothelin-1 increased expression and activity of pro-protein convertase furin in SMCs via endothelin receptor type A (assessed with qPCR and furin activity assay, respectively). Consequently, furin acted in two ways. First, it increased the activation of latent pro-MT1MMP and, second, it activated pro-αvβ3 integrin. Both pathways led to overactivation of latent pro-MMP2. In vitro findings in the co-culture model were fully consistent with the ex vivo findings obtained in isolated rat aorta. We propose that the endothelium under ANGII stimulation acts as a novel and key activator of latent pro-MT1MMP and pro-MMP2 in SMCs of rat aorta. Therefore, endothelium may critically contribute to pathophysiology of aortic stiffness.