Effect of endothelin-1 on the blood pressure response to acute hypoxia and hyperoxia in healthy young men.

Endothelin-1 (ET-1) and its receptors are linked to increases in sensitivity of the chemoreceptors to hypoxic stress and the development of hypertension in preclinical models. We hypothesized ET receptor antagonism would lower resting blood pressure (BP) as well as the acute BP response to chemoreflex stress. Twenty-four men (31 ± 5 years, 26 ± 3 kg/m2) completed two study visits (control, bosentan). On each visit, BP was assessed under three conditions: (1) normoxia (FiO2 0.21), (2) chemoreflex excitation via hypoxia (FiO2 0.05-0.21), (3) chemoreflex inhibition via hyperoxia (FiO2 1.00). Bosentan increased plasma ET-1 (0.94 ± 0.90 to 1.27 ± 0.62 pg/mL, p = 0.004), supporting receptor blockade. Resting diastolic (73 ± 5 to 69 ± 7 mmHg, p = 0.007) and mean (93 ± 7 to 88 ± 7 mmHg, p = 0.005) BP were reduced following bosentan compared to control with no change in systolic BP (p = 0.507). The mean BP response to both acute hypoxia (-0.48 ± 0.38 to -0.25 ± 0.31 mmHg/%, p = 0.004) and hyperoxia (area under the curve -93 ± 108 to -27 ± 66 AU, p = 0.018) were attenuated following bosentan. Acute ET receptor inhibition attenuates the rise in BP during chemoreflex excitation as well as the fall in BP during chemoreflex inhibition in healthy young men. These data support a role for ET-1 in control of resting BP, possibly through a chemoreceptor-mediated mechanism.

Endothelin-1 down-regulates nuclear factor erythroid 2-related factor-2 and contributes to perivascular adipose tissue dysfunction in obesity.

Perivascular adipose tissue (PVAT) negatively regulates vascular muscle contraction. However, in the context of obesity, the PVAT releases vasoconstrictor substances that detrimentally affect vascular function. A pivotal player in this scenario is the peptide endothelin-1 (ET-1), which induces oxidative stress and disrupts vascular function. The present study postulates that obesity augments ET-1 production in the PVAT, decreases the function of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, further increasing reactive oxygen species (ROS) generation, culminating in PVAT dysfunction. Male C57BL/6 mice were fed either a standard or a high-fat diet for 16 weeks. Mice were also treated with saline or a daily dose of 100 mg·kg-1 of the ETA and ETB receptor antagonist Bosentan, for 7 days. Vascular function was evaluated in thoracic aortic rings, with and without PVAT. Mechanistic studies utilized PVAT from all groups and cultured WT-1 mouse brown adipocytes. PVAT from obese mice exhibited increased ET-1 production, increased ECE1 and ETA gene expression, loss of the anticontractile effect, as well as increased ROS production, decreased Nrf2 activity, and downregulated expression of Nrf2-targeted antioxidant genes. PVAT of obese mice also exhibited increased expression of Tyr216-phosphorylated-GSK3ß and KEAP1, but not BACH1 - negative Nrf2 regulators. Bosentan treatment reversed all these effects. Similarly, ET-1 increased ROS generation and decreased Nrf2 activity in brown adipocytes, events mitigated by BQ123 (ETA receptor antagonist). These findings place ET-1 as a major contributor to PVAT dysfunction in obesity and highlight that pharmacological control of ET-1 effects restores PVAT's cardiovascular protective role.

Effects of Bosentan on Hypoxia, Inflammation and Oxidative Stress in Experimental Blunt Thoracic Trauma Model.

Background and Objectives: In this study, we aimed to investigate the effects of bosentan, an endothelin receptor antagonist, on endothelin-1 (ET-1), hypoxia-inducible factor-1 (HIF-1), nuclear factor-kappa B (NF-κB), and tumor necrosis factor (TNF)-α as inflammation markers, pro-oxidant antioxidant balance (PAB), and total antioxidant capacity (TAC) levels as oxidative stress parameters in lung tissues of rats in an experimental model of pulmonary contusion (PC) induced by blunt thoracic trauma. Materials and Methods: Thirty-seven male Sprague-Dawley rats were divided into five groups. C: The control group (n = 6) consisted of unprocessed and untreated rats. PC3 (n = 8) underwent 3 days of PC. PC-B3 (n = 8) received 100 mg/kg bosentan and was given orally once a day for 3 days. The PC7 group (n = 7) underwent 7 days of PC, and PC-B7 (n = 8) received 100 mg/kg bosentan and was given orally once a day for 7 days. Results: ET-1, NF-κB, TNF-α, HIF-1α, and PAB levels were higher, while TAC activity was lower in all groups compared with the control (p < 0.05). There was no significant difference in ET-1 and TNF-α levels between the PC-B3 and PC-B7 groups and the control group (p < 0.05), while NF-κB, HIF-1α, and PAB levels were still higher in both the PC-B3 and PC-B7 groups than in the control group. Bosentan decreased ET-1, NF-κB, TNF-α, HIF-1α, and PAB and increased TAC levels in comparison to the nontreated groups (p < 0.05). Conclusions: Bosentan decreased the severity of oxidative stress in the lungs and reduced the inflammatory reaction in rats with PC induced by blunt thoracic trauma. This suggests that bosentan may have protective effects on lung injury mechanisms by reducing hypoxia, inflammation, and oxidative stress. If supported by similar studies, bosentan can be used in both pulmonary and emergency clinics to reduce ischemic complications, inflammation, and oxidative stress in some diseases that may be accompanied by ischemia.

The Anti-Atherosclerotic Effects of Endothelin Receptor Antagonist, Bosentan, in Combination with Atorvastatin-An Experimental Study.

Bosentan, an endothelin receptor antagonist (ERA), has potential anti-atherosclerotic properties. We investigated the complementary effects of bosentan and atorvastatin on the progression and composition of the atherosclerotic lesions in diabetic mice. Forty-eight male ApoE-/- mice were fed high-fat diet (HFD) for 14 weeks. At week 8, diabetes was induced with streptozotocin, and mice were randomized into four groups: (1) control/COG: no intervention; (2) ΒOG: bosentan 100 mg/kg/day per os; (3) ATG: atorvastatin 20 mg/kg/day per os; and (4) BO + ATG: combined administration of bosentan and atorvastatin. The intra-plaque contents of collagen, elastin, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNF-a), matrix metalloproteinases (MMP-2, -3, -9), and TIMP-1 were determined. The percentage of lumen stenosis was significantly lower across all treated groups: BOG: 19.5 ± 2.2%, ATG: 12.8 ± 4.8%, and BO + ATG: 9.1 ± 2.7% compared to controls (24.6 ± 4.8%, p < 0.001). The administration of both atorvastatin and bosentan resulted in significantly higher collagen content and thicker fibrous cap versus COG (p < 0.01). All intervention groups showed lower relative intra-plaque concentrations of MCP-1, MMP-3, and MMP-9 and a higher TIMP-1concentration compared to COG (p < 0.001). Importantly, latter parameters presented lower levels when bosentan was combined with atorvastatin compared to COG (p < 0.05). Bosentan treatment in diabetic, atherosclerotic ApoE-/- mice delayed the atherosclerosis progression and enhanced plaques' stability, showing modest but additive effects with atorvastatin, which are promising in atherosclerotic cardiovascular diseases.

Effect of Benidipine Alone and in Combination With Bosentan and Sildenafil in Amelioration of Pulmonary Arterial Hypertension in Experimental Model in Rats.

ABSTRACT: Pulmonary arterial hypertension (PAH) is a persistent condition affecting the pulmonary arteries' endothelium. Benidipine, a calcium channel blocker, possesses vasodilatory, anti-inflammatory activity, reduces oxidative stress, and inhibits the activity of Transforming growth factor-ß (TGF-ß) and α-smooth muscle actin (α-SMA). The present study was designed to investigate the effect of benidipine alone and in combination with bosentan and sildenafil on monocrotaline (MCT)-induced pulmonary hypertension in a rat model. PAH was induced by a single-dose administration of MCT in rats. Animals were randomized into different groups and treated with benidipine alone and in combination with bosentan or sildenafil. Various parameters such as hemodynamic parameters, Fulton's index and oxidative stress parameters were performed. Additionally, histopathology of lung and right ventricular of heart tissue, immunohistochemistry, expression of α-SMA, endothelial nitric oxide synthase (eNOS), TGF-ß, and RT-PCR, and an in vitro study using human umbilical vein endothelial cells (HUVECs) was also carried out. Treatment of benidipine and its combination exhibited better prevention in the elevated right ventricular systolic pressure, right ventricular hypertrophy, rise in oxidative stress, and increase in expression of α-SMA and TGF-ß receptor 1 compared with MCT control group rats. In HUVECs, the expression of α-SMA was increased, whereas that of eNOS decreased after TGF-ß exposure and was substantially reversed after pretreatment with benidipine. We concluded that benidipine and its combination with bosentan and sildenafil exhibit beneficial effects in MCT-induced PAH through the eNOS/TGF-ß/α-SMA signaling pathway.

Endothelin-1 Induces Cell Proliferation and Myofibroblast Differentiation through the ETAR/Gαq/ERK Signaling Pathway in Human Cardiac Fibroblasts.

Endothelin-1 (ET-1) has been implicated in the pathogenesis of cardiac fibrosis. Stimulation of endothelin receptors (ETR) with ET-1 leads to fibroblast activation and myofibroblast differentiation, which is mainly characterized by an overexpression of α-smooth muscle actin (α-SMA) and collagens. Although ET-1 is a potent profibrotic mediator, the signal transductions and subtype specificity of ETR contributing to cell proliferation, as well as α-SMA and collagen I synthesis in human cardiac fibroblasts are not well clarified. This study aimed to evaluate the subtype specificity and signal transduction of ETR on fibroblast activation and myofibroblast differentiation. Treatment with ET-1 induced fibroblast proliferation, and synthesis of myofibroblast markers, α-SMA, and collagen I through the ETAR subtype. Inhibition of Gαq protein, not Gαi or Gßγ, inhibited these effects of ET-1, indicating the essential role of Gαq protein-mediated ETAR signaling. In addition, ERK1/2 was required for ETAR/Gαq axis-induced proliferative capacity and overexpression of these myofibroblast markers. Antagonism of ETR with ETR antagonists (ERAs), ambrisentan and bosentan, inhibited ET-1-induced cell proliferation and synthesis of α-SMA and collagen I. Furthermore, ambrisentan and bosentan promoted the reversal of myofibroblasts after day 3 of treatment, with loss of proliferative ability and a reduction in α-SMA synthesis, confirming the restorative effects of ERAs. This novel work reports on the ETAR/Gαq/ERK signaling pathway for ET-1 actions and blockade of ETR signaling with ERAs, representing a promising therapeutic strategy for prevention and restoration of ET-1-induced cardiac fibrosis.

Bosentan attenuates sickle cell disease erythrocyte HbS polymerization and impaired deformability induced by endothelin-1.

The effects of endothelin-1 (ET-1) on erythrocytes from sickle cell disease (SCD) patients have been described, but mechanisms of ET-1 regarding primary erythrocyte functions remain unknown. ET-1 is a vasoconstrictor peptide produced by endothelial cells, and the expression of ET-1 is increased in SCD. The present study used ex vivo experiments with sickle cell erythrocytes, ET-1, and bosentan, a dual antagonist of ETA and ETB receptors. We performed a hemoglobin S (HbS) polymerization assay with three concentrations of ET-1 (1, 20, and 50 pg/mL) and bosentan (100 nmol/L). ET-1 increased HbS polymerization at all concentrations, and this effect was suppressed by bosentan. For the deformability assay, red blood cells (RBCs) were incubated on a Sephacryl column with the same concentrations of ET-1 and bosentan. ET-1 decreased deformability, and this effect was reversed by bosentan. To observe erythrocyte adhesion, ET-1 and bosentan were incubated with RBCs in thrombospondin-coated 96-well plate, which demonstrated that ET-1 decreased adhesion but that bosentan enhanced adhesion. We also assessed erythrocyte apoptosis and observed decreased eryptosis induced by ET-1, and these effects were inhibited bosentan. Thus, these findings demonstrated that ET-1 modulates HbS polymerization, erythrocyte deformability, adhesion to thrombospondin, and eryptosis, and these effects were suppressed or enhanced by bosentan.

Endothelin-1 as a novel target for the prevention of metabolic dysfunction with intermittent hypoxia in male participants.

We examined the effect of intermittent hypoxia (IH, a hallmark feature of sleep apnea) on adipose tissue lipolysis and the role of endothelin-1 (ET-1) in this response. We hypothesized that IH can increase ET-1 secretion and plasma free fatty acid (FFA) concentrations. We further hypothesized that inhibition of ET-1 receptor activation with bosentan could prevent any IH-mediated increase in FFA. To test this hypothesis, 16 healthy male participants (32 ± 5 yr, 26 ± 2 kg/m2) were exposed to 30 min of IH in the absence (control) and presence of bosentan (62.5 mg oral twice daily for 3 days prior). Arterial blood samples for ET-1, epinephrine, and FFA concentrations, as well as abdominal subcutaneous adipose tissue biopsies (to assess transcription of cellular receptors/proteins involved in lipolysis), were collected. Additional proof-of-concept studies were conducted in vitro using primary differentiated human white preadipocytes (HWPs). We show that IH increased circulating ET-1, epinephrine, and FFA (P < 0.05). Bosentan treatment reduced plasma epinephrine concentrations and blunted IH-mediated increases in FFA (P < 0.01). In adipose tissue, bosentan had no effect on cellular receptors and proteins involved in lipolysis (P > 0.05). ET-1 treatment did not directly induce lipolysis in differentiated HWP. In conclusion, IH increases plasma ET-1 and FFA concentrations. Inhibition of ET-1 receptors with bosentan attenuates the FFA increase in response to IH. Based on a lack of a direct effect of ET-1 in HWP, we speculate the effect of bosentan on circulating FFA in vivo may be secondary to its ability to reduce sympathoadrenal tone.

DL0805-1, a novel Rho-kinase inhibitor, attenuates lung injury and vasculopathy in a rat model of monocrotaline-induced pulmonary hypertension.

Pulmonary hypertension (PH) is a severe chronic cardiopulmonary dysfunction characterized by impaired of pulmonary circulation. Current therapeutic drugs mainly act as vasodilators, leading to an unsatisfactory prognosis. The Rho/ROCK pathway plays an important role in the cardiovascular system. DL0805-1, a novel Rho kinase inhibitor, synthesized by our institute and showed a protective effect on lung tissues and reduced right ventricular systolic pressure in a hypertensive crisis rat model in our previous study. The present study aims to explore the efficacy of DL0805-1 on PH. The classical PH rat model induced by a single subcutaneous injection of monocrotaline was used to investigate the therapeutic effect of DL0805-1 on PH and the underlying mechanisms. The results showed that the high dose of DL0805-1 had a better effect on the survival rate and controlled right ventricular systolic pressure (RVSP) of PH rats than fasudil. DL0805-1 also exhibited a superior lung protective effect and significantly improved pulmonary vascular function compared with bosentan. Regarding molecular mechanisms, DL0805-1 inhibited the ROCK pathway in both pulmonary arteries and lung tissues. Taken together, DL0805-1 alleviated lung injury and vasculopathy in experimental PH rats. DL0805-1 has the potential to be developed as a candidate drug for the treatment of PH.

Cardioprotective effects of bosentan in 5-fluorouracil-induced cardiotoxicity.

5-fluorouracil (5-FU) is a widely used chemotherapeutic agent but cardiotoxicity challenges its clinical usefulness. Thus, searching for more cardioprotective drugs is highly required to prevent the accompanied cardiac hazards. Up to date, the different mechanisms involved in 5-FU cardiotoxicity are still unclear and there is no evaluation of bosentan's role in controlling these cardiac complications. This forced us to deeply study and evaluate the possible cardiopreserving properties of bosentan and different mechanisms involved in mediating it. 32 Wistar albino rats were included in our experiment and induction of cardiotoxicity was performed via administration of 5-FU (150 mg/kg) on 5th day of the experiment by intraperitoneal (i.p.) injection with or without co-administration of bosentan (50 mg/kg/day) orally for 7days. Our data revealed that 5-FU could induce cardiotoxicity which was detected as significant increases of troponin I, lactate dehydrogenase (LDH), creatine kinase- MB (CK-MB), endothelin receptors, malondialdehyde (MDA), toll like receptor4 (TLR4), myeloid differentiation primary response 88 (MyD88), nuclear factor kappa B (NFκB), and caspase 3 levels. However, there is marked decrease in endothelial nitric oxide synthase (eNOS), reduced glutathione (GSH) and total antioxidant capacity (TAC). In addition, the histopathological examination showed severe toxic features of cardiac injury. Interestingly, co-administration of bosentan could ameliorate 5-FU-induced cardiotoxicity via improving the detected biochemical and histopathological changes besides modulation of TLR4/MyD88/NFκB signaling pathway, eNOS, and endothelin receptors. Bosentan had a significant cardioprotective effect against 5-FU induced cardiac damage. This effect may be attributed to its ability to inhibit endothelin receptors, stimulates eNOS, anti-oxidant, anti-inflammatory, anti-apoptotic properties with modulation of TLR4/MyD88/NFκB signaling pathway.

Primary Human Hepatocyte Spheroids as Tools to Study the Hepatotoxic Potential of Non-Pharmaceutical Chemicals.

Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro.

Bosentan Alters Endo- and Exogenous Bile Salt Disposition in Sandwich-Cultured Human Hepatocytes.

Bosentan, a well-known cholestatic agent, was not identified as cholestatic at concentrations up to 200 µM based on the drug-induced cholestasis (DIC) index value, determined in a sandwich-cultured human hepatocyte (SCHH)-based DIC assay. To obtain further quantitative insights into the effects of bosentan on cellular bile salt handling by human hepatocytes, the present study determined the effect of 2.5-25 µM bosentan on endogenous bile salt levels and on the disposition of 10 µM chenodeoxycholic acid (CDCA) added to the medium in SCHHs. Bosentan reduced intracellular as well as extracellular concentrations of both endogenous glycochenodeoxycholic acid (GCDCA) and glycocholic acid in a concentration-dependent manner. When exposed to 10 µM CDCA, bosentan caused a shift from canalicular efflux to sinusoidal efflux of GCDCA. CDCA levels were not affected. Our mechanistic model confirmed the inhibitory effect of bosentan on canalicular GCDCA clearance. Moreover, our results in SCHHs also indicated reduced GCDCA formation. We confirmed the direct inhibitory effect of bosentan on CDCA conjugation with glycine in incubations with liver S9 fraction. SIGNIFICANCE STATEMENT: Bosentan was evaluated at therapeutically relevant concentrations (2.5-25 µM) in sandwich-cultured human hepatocytes. It altered bile salt disposition and inhibited canalicular secretion of glycochenodeoxycholic acid (GCDCA). Within 24 hours, bosentan caused a shift from canalicular to sinusoidal efflux of GCDCA. These results also indicated reduced GCDCA formation. This study confirmed a direct effect of bosentan on chenodeoxycholic acid conjugation with glycine in liver S9 fraction.

Blocking of interleukin-1 suppresses angiotensin II-induced renal injury.

Clinical hypertension (HT) is associated with renal inflammation and elevated circulating levels of proinflammatory cytokines. Interleukin (IL)-1 receptor antagonist (IL-1Ra) is one of the most important anti-inflammatory cytokines and plays a crucial role in inflammation. Inhibition of IL-1 may contribute to modulation of the Angiotensin II (Ang II)-induced HT response. The present study aimed to elucidate the effects of IL-1Ra and anti-IL-1ß antibody (01BSUR) on Ang II-induced renal injury. To determine the contribution of IL-1Ra to Ang II-induced renal inflammation, male wildtype (WT) and IL-1Ra-deficient (IL-1Ra-/-) mice were infused with Ang II (1000 ng/kg/min) using subcutaneous osmotic pump for 14 days. We checked renal function, histological change, and several mRNA expressions 14 days after infusion. Fourteen days after infusion, systolic blood pressure (197 ± 5 vs 169 ± 9 mmHg, P<0.05) in IL-1Ra-/- mice significantly increased compared with WT mice. Furthermore, on day 14 of Ang II infusion, plasma IL-6 was 5.9-fold higher in IL-1Ra-/- versus WT mice (P<0.001); renal preproendothelin-1 mRNA expression was also significantly higher in IL-1Ra-/- mice (P<0.05). In addition, renal histology revealed greater damage in IL-1Ra-/- mice compared with WT mice 14 days after infusion. Finally, we administrated 01BSUR to both IL-1Ra-/- and WT mice, and 01BSUR treatment decreased Ang II-induced HT and renal damage (glomerular injury and fibrosis of the tubulointerstitial area) in both IL-1Ra-/- and WT mice compared with IgG2a treatment. Inhibition of IL-1 decreased Ang II-induced HT and renal damage in both IL-1Ra-/- and WT mice, suggesting suppression of IL-1 may provide an additional strategy to protect against renal damage in hypertensive patients.

The Influence of Bosentan on MicroRNA-27a/PPARγ/ET-1 Signaling Pathway in Pulmonary Artery Hypertension.

Pulmonary artery hypertension (PAH) is a common and serious disease which is characterized by pulmonary vascular remodeling. Bosentan (BST) is the first approved oral targeted drug of endothelin-1 (ET-1) receptor antagonists for the treatment of PAH. MicroRNA-27a (miR-27a) and peroxisome proliferator-activated receptor γ (PPARγ) were found to be related to the pathogenesis of PAH. To further explore the signal transduction mechanism of BST in the treatment of PAH, we examined the effects of BST on endothelin receptors, miR-27a, and PPARγ. Meanwhile, the influence of miR-27a in the formation and development of PAH was discussed. Our results demonstrated that during the pathophysiology of PAH, miR-27a, PPARγ, and ET-1 were cross-inhibited, which indicated that the miR-27a/PPARγ/ET-1 signaling pathway was dysregulated; in addition, BST could competitively bind to ET-1 receptors and inhibit the miR-27a/PPARγ/ET-1 signaling pathway, thereby delaying the proliferation of PASMCs and affecting the development of PAH. Our results give a new understanding of the pathogenesis and treatment of PAH and provide more reliable evidence for the application of BST in the treatment of PAH in the clinic.

Effect of endothelin on sex-dependent regulation of tone in coronary resistance vessels.

BACKGROUND/AIMS: Sex dependent differences in coronary artery vasoregulation may be due to variations in responses to endogenous vasoactive compounds including endothelin (ET-1) and nitric oxide (NO). METHODS: Septal coronary arteries (<200 µm) from healthy, sexually mature male, female and ovariectomized (i.e. surgical menopause) Sprague-Dawley rats were used. Myogenic tone, measured by pressure myography, was initially determined for all vessel segments studied before and after exposure to the nonselective ETA/ETB receptor blocker, bosentan (1 µM). Vasoconstrictor responses (vascular endothelium intact) to cumulative ET-1 (10-12 - 10-9 M) were assessed in a separate set of septal coronary vessels. Additional studies, examined the vasoconstrictor effects of ET-1 after NO blockade with L-NAME (200 µM). RESULTS: Myogenic tone was 26 ± 7% in male, 20 ± 7% in female (p = 0.04 versus male) and 24 ± 3% in ovariectomized (p = NS versus male/female) vessels. Antagonism of ET-1 receptors produced a greater reduction in myogenic tone in male, compared to female rats over a similar range of intraluminal pressure (20-80 mmHg). Robust constrictor responses to cumulative concentrations of ET-1 were observed in all vessels; however, male rats exhibited greater sensitivity to vasoconstrictor effects of ET-1. After exposure to L-NAME vessel responses to ET-1 were normalized in male and female (not studied in ovariectomized) groups. CONCLUSIONS: These findings confirm marked sex differences for myogenic tone and vessel constrictor responses to ET-1 in coronary resistance vessels. Results also suggest greater sensitivity to vasoconstrictor effects of ET-1 in male coronary resistance vessels.

Additive protective effects of sacubitril/valsartan and bosentan on vascular remodelling in experimental pulmonary hypertension.

AIMS: Although right ventricular (RV) function is an important determinant of morbidity and mortality in patients with pulmonary arterial hypertension (PAH), there is no treatment targeting directly the RV. We evaluate the efficacy of sacubitril/valsartan (LCZ 696) as add-on therapy to bosentan in rats with severe pulmonary hypertension (PH). METHODS AND RESULTS: Combination therapy of LCZ 696 and bosentan has additive vascular protective effects against the pulmonary vascular remodelling and PH in two preclinical models of severe PH. Compared with monotherapy, co-treatment of LCZ 696 (30 or 68 mg/kg/day for 2 weeks, per os) and bosentan (100 mg/kg/day for 2 weeks, per os) started 7 days after monocrotaline (MCT) injection substantially reduces pulmonary pressures, vascular remodelling, and RV hypertrophy and fibrosis in rats. Consistent with these observations, co-treatment of rats with established PH induced by sugen/hypoxia (SuHx) with LCZ 696 (30 mg/kg/day for 3 weeks, per os) and bosentan (100 mg/kg/day for 3 weeks, per os) started 5 weeks after Sugen injection partially attenuate total pulmonary vascular resistance and cardiovascular structures. We also obtained evidence showing that LCZ 696 has anti-proliferative effect on cultured human pulmonary artery smooth muscle cells derived from patients with idiopathic PAH, an effect that is more pronounced in presence of bosentan. Finally, we found that the plasma levels of atrial natriuretic peptide (ANP) and cyclic guanosine monophosphate (cGMP) are higher in rats co-treated with LCZ 696 (30 mg/kg/day) and bosentan (100 mg/kg/day) than in MCT and SuHx rats treated with vehicle. CONCLUSION: Dual therapy with LCZ 696 plus bosentan proved significantly superior beneficial effect to LCZ 696 or bosentan alone on vascular remodelling and severity of experimental PH.

Switching of cell fate through the regulation of cell growth during drug-induced intrahepatic cholestasis.

Understanding the fundamental mechanisms that govern the fate of cells during drug-induced intrahepatic cholestasis provides strategies for the establishment of evaluation methods for drug screening. In the present study, the aggregates of a differentiated human hepatic cell line, HepaRG, were incubated in medium with Y27632 or bosentan to clarify the changes in the behavior of bile canaliculi (BC) with the growth of cells during drug-induced intrahepatic cholestasis. With elapsed exposure time, the aggregates in the culture with bosentan caused the dilation of BC, and the hepatocytes ultimately exhibited apoptotic death after the disruption of BC. Y27632 caused the disruption of BC in the aggregates after dilation. However, there was no change in the number of cells within the aggregates in the culture with Y27632, in spite of its cytotoxicity. After 144 h from the start of Y27632 exposure, the aggregates showed the rearrangement of BC. To inhibit cell division, the aggregates exposed to Y27632, which exhibited disruption of BC, were treated with mitomycin C for 2 h and continuously exposed to Y27632. The inhibition of cell division could not induce the rearrangement of BC within these aggregates, which was similar to the phenomenon observed in the aggregates exposed to bosentan. These findings indicate that growth is an important factor that influences the switching of cell fate toward survival or death in drug-induced intrahepatic cholestasis process. Thus, the autoregulation of growth is a major contributor to the rearrangement of BC within aggregates.

Blockade of endothelin receptor A enhances the therapeutic efficacy of gemcitabine in pancreatic cancer cells.

Pancreatic adenocarcinoma is currently one of the leading causes of cancer-related death worldwide. The high rate of mortality in pancreatic cancer patients is due to the inability to detect early-stage disease and the disease being highly refractory to therapy. Gemcitabine has been the standard chemotherapy for advanced pancreatic cancer patients for the last two decades. However, gemcitabine resistance develops within a few weeks of treatment, and the associated mechanism remains poorly understood. Therefore, a novel therapeutic strategy is needed to overcome the limited clinical efficacy of gemcitabine in pancreatic adenocarcinoma. In this study, we demonstrated that ET-1/ETAR axis gene expression was upregulated in pancreatic cancer cells after treatment with gemcitabine. Additionally, ETAR expression was significantly higher in tumor tissues than in normal tissues, and patients with high ETAR expression had a notably worse overall survival rate than those with low ETAR expression. Furthermore, our results revealed that bosentan, an ETAR antagonist, enhanced the growth-inhibiting and proapoptotic effects of gemcitabine on pancreatic cancer cells. Thus, our findings indicate that blockade of the ET-1/ETAR axis signaling pathway promotes the antiproliferative effect of gemcitabine on pancreatic cancer. Therefore, combination of ETAR blockade and gemcitabine serves as an effective therapeutic approach to achieve clinical benefits in pancreatic adenocarcinoma patients.

Endothelin-1 Mediates the Systemic and Renal Hemodynamic Effects of GPR81 Activation.

GPR81 (G-protein-coupled receptor 81) is highly expressed in adipocytes, and activation by the endogenous ligand lactate inhibits lipolysis. GPR81 is also expressed in the heart, liver, and kidney, but roles in nonadipose tissues are poorly defined. GPR81 agonists, developed to improve blood lipid profile, might also provide insights into GPR81 physiology. Here, we assessed the blood pressure and renal hemodynamic responses to the GPR81 agonist, AZ'5538. In male wild-type mice, intravenous AZ'5538 infusion caused a rapid and sustained increase in systolic and diastolic blood pressure. Renal artery blood flow, intrarenal tissue perfusion, and glomerular filtration rate were all significantly reduced. AZ'5538 had no effect on blood pressure or renal hemodynamics in Gpr81-/- mice. Gpr81 mRNA was expressed in renal artery vascular smooth muscle, in the afferent arteriole, in glomerular and medullary perivascular cells, and in pericyte-like cells isolated from kidney. Intravenous AZ'5538 increased plasma ET-1 (endothelin 1), and pretreatment with BQ123 (endothelin-A receptor antagonist) prevented the pressor effects of GPR81 activation, whereas BQ788 (endothelin-B receptor antagonist) did not. Renal ischemia-reperfusion injury, which increases renal extracellular lactate, increased the renal expression of genes encoding ET-1, KIM-1 (Kidney Injury Molecule 1), collagen type 1-α1, TNF-α (tumor necrosis factor-α), and F4/80 in wild-type mice but not in Gpr81-/- mice. In summary, activation of GPR81 in vascular smooth muscle and perivascular cells regulates renal hemodynamics, mediated by release of the potent vasoconstrictor ET-1. This suggests that lactate may be a paracrine regulator of renal blood flow, particularly relevant when extracellular lactate is high as occurs during ischemic renal disease.