Sovateltide (ILR-1620) Improves Motor Function and Reduces Hyperalgesia in a Rat Model of Spinal Cord Injury.

BACKGROUND: Spinal cord injury (SCI) presents a major global health challenge, with rising incidence rates and substantial disability. Although progress has been made in understanding SCI's pathophysiology and early management, there is still a lack of effective treatments to mitigate long-term consequences. This study investigates the potential of sovateltide, a selective endothelin B receptor agonist, in improving clinical outcomes in an acute SCI rat model. METHODS: Thirty male Sprague-Dawley rats underwent sham surgery (group A) or SCI and treated with vehicle (group B) or sovateltide (group C). Clinical tests, including Basso, Beattie, and Bresnahan scoring, inclined plane, and allodynia testing with von Frey hair, were performed at various time points. Statistical analyses assessed treatment effects. RESULTS: Sovateltide administration significantly improved motor function, reducing neurological deficits and enhancing locomotor recovery compared with vehicle-treated rats, starting from day 7 post injury. Additionally, the allodynic threshold improved, suggesting antinociceptive properties. Notably, the sovateltide group demonstrated sustained recovery, and even reached preinjury performance levels, whereas the vehicle group plateaued. CONCLUSIONS: This study suggests that sovateltide may offer neuroprotective effects, enhancing neurogenesis and angiogenesis. Furthermore, it may possess anti-inflammatory and antinociceptive properties. Future clinical trials are needed to validate these findings, but sovateltide shows promise as a potential therapeutic strategy to improve functional outcomes in SCI. Sovateltide, an endothelin B receptor agonist, exhibits neuroprotective properties, enhancing motor recovery and ameliorating hyperalgesia in a rat SCI model. These findings could pave the way for innovative pharmacological interventions for SCI in clinical settings.

Doxorubicin alters G-protein coupled receptor-mediated vasocontraction in rat coronary arteries.

Doxorubicin (Doxo)-associated cardio-and vasotoxicity has been recognised as a serious complication of cancer chemotherapy. The purpose of this novel paper was to determine the effect of Doxo on G-protein coupled receptor (GPCR)-mediated vasocontraction located on vascular smooth muscle cells. Rat left anterior descending artery segments were incubated for 24 h with 0.5 µM Doxo. The vasocontractile responses by activation of endothelin receptor type A (ETA) and type B (ETB), serotonin receptor 1B (5-HT1B) and thromboxane A2 prostanoid receptor (TP) were investigated by a sensitive myography using specific agonists, while the specificity of the GPCR agonists was verified by applying selective antagonists (i.e. ETA and ETB agonist = 10- 14-10- 7.5 M endothelin-1 (ET-1); ETA antagonist = 10 µM BQ123; ETB agonists = 10- 14-10- 7.5 M sarafotoxin 6c (S6c) and ET-1; ETB antagonist = 0.1 µM BQ788; 5-HT1B agonist = 10- 12-10- 5.5 M 5-carboxamidotryptamine (5-CT); 5-HT1B antagonist = 1 µM GR55562; TP agonist = 10- 12-10- 6.5 M U46619; TP antagonist = 1 µM Seratrodast). Our results show that 0.5 µM Doxo incubation of LAD segments leads to an increased VSMC vasocontraction through the ETB, 5-HT1B and TP GPCRs, with a 2.2-fold increase in ETB-mediated vasocontraction at 10- 10.5 M S6c, a 2.0-fold increase in 5-HT1B-mediated vasocontraction at 10- 5.5 M 5-CT, and a 1.3-fold increase in TP-mediated vasocontraction at 10- 6.5 M U46619. Further studies unravelling the involvement of intracellular GPCR signalling pathways will broaden our understanding of the Doxo-induced vasotoxicity, and thus pave the way to mitigate the adverse effects by potential implementation of adjunct therapy options.

Apelin-13 attenuates injury following ischemic stroke by targeting matrix metalloproteinases (MMP), endothelin- B receptor, occludin/claudin-5 and oxidative stress.

Oxidative stress, an adverse consequence of brain ischemia-reperfusion injury (IRI), activates matrix metalloproteinase enzymes which cause to destruction of extracellular matrix and tight junction proteins. Oxidative stress during stroke increases serum endothelin-1 and endothelin B receptor (ETBR) expression. Apelin-13, an endogenous peptide, is expressed in numerous tissues that regulate diverse physiological and pathological processes. This study aimed to investigate the effect of intravenous (IV) injection of apelin-13 on cerebral vasogenic edema due to brain IRI. Animals were divided into sham, ischemia, and treat groups. IRI model was induced by middle cerebral artery occlusion (MCAO) for 60 min followed by 23 h reperfusion. Apelin-13 was injected into the tail vein 5 min before reperfusion. Neurological defects were evaluated with longa test. Brain water content and BBB permeability were assessed according to cerebral dry-wet weight and brain Evans blue extraction. Malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were measured using the colorimetric method. Expression of occludin and claudin-5, matrix metalloproteinase- 2 and 9 (MMP-2 & 9) and, ETBR were evaluated using Western blot. Brain IRI was associated with BBB breakdowns and vasogenic edema. Apelin-13 significantly reduced BBB permeability and vasogenic edema. Apelin-13 significantly attenuated IRI-related oxidative stress. Apelin-13 decreased expression of mmp-2, 9 and ETBR, prevented from decrement of occludin and claudin-5 expersion, which protected BBB integrity and reduced vasogenic edema. In conclusion, our results have suggested that an IV injection of apelin-13 could somehow reduce vasogenic edema via targeting oxidative stress and ETBR expression.

Aprocitentan (a Dual Endothelin-Receptor Antagonist) for Treatment-Resistant Hypertension.

ABSTRACT: Treatment-resistant hypertension (TRH) is associated with increased cardiovascular risks and progression of chronic kidney disease. The pathophysiology of TRH is multifactorial, including overactivity of the renin-angiotensin-aldosterone system and sympathetic nervous system, endothelial dysfunction, and volume overload. Endothelin-1 is a vasoconstrictive peptide that causes neurohormonal and sympathetic activation, increased aldosterone synthesis and secretion, endothelial dysfunction, vascular hypertrophy and remodeling, and fibrosis. Endothelin-1 acts through 2 receptors, ETA and ETB. Activation of ETA receptors in vascular smooth muscle cells results in vasoconstriction, whereas ETB receptor activation results in vasoconstriction in the vascular smooth muscle cells and vasodilation through nitric oxide release in endothelial cells. Aprocitentan is novel, oral, dual endothelin-receptor antagonist that has demonstrated a more favorable tolerability and safety profile in early clinical trials compared with other endothelin-receptor antagonists studied. Phase 2 trial data support a significant reduction in blood pressure compared to placebo and similar blood pressure reduction compared to a moderately dosed angiotensin-converting enzyme inhibitor in patients with essential hypertension. An ongoing phase 3 randomized clinical trial is evaluating aprocitentan's efficacy and safety in patients with TRH receiving multiple antihypertensives. Additional research is needed to determine aprocitentan's role in therapy, but this agent may be a suitable treatment option for TRH.

Lignans from Schisandra chinensis ameliorate alcohol and CCl4-induced long-term liver injury and reduce hepatocellular degeneration via blocking ETBR.

ETHNOPHARMACOLOGICAL RELEVANCE: Chemical hepatotoxicity, especially alcoholic liver injury (ALI), commonly occurs in young and middle-aged people who drink heavily. ALI is extremely harmful and can induce severe disease states, such as hepatitis, liver fibrosis, cirrhosis, or liver cancer, which are similar to CCl4-induced liver disease states in animals. In recent studies, the pathological changes of hepatocytes and the hepatic stellate cell have shown a significant connection between endoplasmic reticulum (ER) stress and the development of liver pathology in patients. However, the detailed pathological mechanism needs to be further studied. Schisandra chinensis, (S. chinensis), a fruit-bearing vine used in Traditional Chinese Medicine (TCM), has been used to treat chronic or acute diseases, including liver disease. S. chinensis-derived lignans (SCDLs) in particular have been shown to alleviate liver pathological changes. AIM OF THE STUDY: This study sought to elucidate the mechanisms underlying SCDL-mediated hepatoprotection. MATERIALS AND METHODS: We first used in silico target prediction and computational simulation methods to identify putative lignan-binding targets relative to the hepatoprotective effect. A gene microarray analysis was performed to identify differently expressed genes that might have significance in the disease pathological process. We then used histological analyses in a mice hepatotoxicity model to test the effectiveness of SCDLs in vivo, and a hepatocellular toxicity model to analyze the candidate-compound-mediated hepatoprotection and expression states of the key targets in vitro. RESULTS: The in silico analysis results indicated that endothelin receptor B (ETBR/EDNRB) is likely a significant node during the liver pathological change process and a promising key target for the SCDL compound schisantherin D on the hepatoprotective effect; experimental studies showed that schisantherin D alleviated the EtOH- and ET-1-induced HL-7702 cell (belongs to liver parenchymal cell lines) injury ratio, decreased the expression of ETBR, and inhibited ECMs and ET-1 secretion in LX-2 cells (one form of hepatic stellate cells). SCDLs ameliorated EtOH- and CCl4-induced fibrosis formation in mice liver tissue. Liver tissue western blots of SCDL-treated mice showed downregulated α-SMA, ETBR, PLCß, CHOP, Bax, and the apoptotic factors of cleaved-caspase 12, cleaved-caspase 9, and cleaved-caspase 3 hinted at an anti-apoptosis and hepatoprotective effect. The SCDL treatment also elevated serum glutathione (GSH) and reduced the serum-transforming growth factor-ß1 (TGF-ß1) level. CONCLUSION: The findings indicated that SCDLs prevent hepatotoxicity via their anti-fibrotic, anti-oxidant, and anti-apoptosis properties. ETBR may be the key factor in promoting chemical hepatotoxicity.

Endothelin A and B Receptors: Potential Targets for Microcirculatory-Mitochondrial Therapy in Experimental Sepsis.

The hypoxia-sensitive endothelin (ET) system plays an important role in circulatory regulation through vasoconstrictor ETA and ETB2 and vasodilator ETB1 receptors. Sepsis progression is associated with microcirculatory and mitochondrial disturbances along with tissue hypoxia. Our aim was to investigate the consequences of treatments with the ETA receptor (ETA-R) antagonist, ETB1 receptor (ETB1-R) agonist, or their combination on oxygen dynamics, mesenteric microcirculation, and mitochondrial respiration in a rodent model of sepsis. Sprague Dawley rats were subjected to fecal peritonitis (0.6 g kg i.p.) or a sham operation. Septic animals were treated with saline or the ETA-R antagonist ETR-p1/fl peptide (100 nmol kg i.v.), the ETB1-R agonist IRL-1620 (0.55 nmol kg i.v.), or a combination therapy 22 h after induction. Invasive hemodynamic monitoring and blood gas analysis were performed during a 90-min observation, plasma ET-1 levels were determined, and intestinal capillary perfusion (CPR) was detected by intravital videomicroscopy. Mitochondrial Complex I (CI)- and CII-linked oxidative phosphorylation (OXPHOS) was evaluated by high-resolution respirometry in liver biopsies. Septic animals were hypotensive with elevated plasma ET-1. The ileal CPR, oxygen extraction (ExO2), and CI-CII-linked OXPHOS capacities decreased. ETR-p1/fl treatment increased ExO2 (by >45%), CPR, and CII-linked OXPHOS capacity. The administration of IRL-1620 countervailed the sepsis-induced hypotension (by >30%), normalized ExO2, and increased CPR. The combined ETA-R antagonist-ETB1-R agonist therapy reduced the plasma ET-1 level, significantly improved the intestinal microcirculation (by >41%), and reversed mitochondrial dysfunction. The additive effects of a combined ETA-R-ETB1-R-targeted therapy may offer a tool for a novel microcirculatory and mitochondrial resuscitation strategy in experimental sepsis.

Selective ETA vs. dual ETA/B receptor blockade for the prevention of sunitinib-induced hypertension and albuminuria in WKY rats.

AIMS: Although effective in preventing tumour growth, angiogenesis inhibitors cause off-target effects including cardiovascular toxicity and renal injury, most likely via endothelin (ET)-1 up-regulation. ET-1 via stimulation of the ETA receptor has pro-hypertensive actions whereas stimulation of the ETB receptor can elicit both pro- or anti-hypertensive effects. In this study, our aim was to determine the efficacy of selective ETA vs. dual ETA/B receptor blockade for the prevention of angiogenesis inhibitor-induced hypertension and albuminuria. METHODS AND RESULTS: Male Wistar Kyoto (WKY) rats were treated with vehicle, sunitinib (angiogenesis inhibitor; 14 mg/kg/day) alone or in combination with macitentan (ETA/B receptor antagonist; 30 mg/kg/day) or sitaxentan (selective ETA receptor antagonist; 30 or 100 mg/kg/day) for 8 days. Compared with vehicle, sunitinib treatment caused a rapid and sustained increase in mean arterial pressure of ∼25 mmHg. Co-treatment with macitentan or sitaxentan abolished the pressor response to sunitinib. Sunitinib did not induce endothelial dysfunction. However, it was associated with increased aortic, mesenteric, and renal oxidative stress, an effect that was absent in mesenteric arteries of the macitentan and sitaxentan co-treated groups. Albuminuria was greater in the sunitinib- than vehicle-treated group. Co-treatment with sitaxentan, but not macitentan, prevented this increase in albuminuria. Sunitinib treatment increased circulating and urinary prostacyclin levels and had no effect on thromboxane levels. These increases in prostacyclin were blunted by co-treatment with sitaxentan. CONCLUSIONS: Our results demonstrate that both selective ETA and dual ETA/B receptor antagonism prevents sunitinib-induced hypertension, whereas sunitinib-induced albuminuria was only prevented by selective ETA receptor antagonism. In addition, our results uncover a role for prostacyclin in the development of these effects. In conclusion, selective ETA receptor antagonism is sufficient for the prevention of sunitinib-induced hypertension and renal injury.

Mitochondrial-Derived Peptide MOTS-c Attenuates Vascular Calcification and Secondary Myocardial Remodeling via Adenosine Monophosphate-Activated Protein Kinase Signaling Pathway.

INTRODUCTION: Vascular calcification (VC) is a complex, regulated process involved in many disease entities. So far, there are no treatments to reverse it. Exploring novel strategies to prevent VC is important and necessary for VC-related disease intervention. OBJECTIVE: In this study, we evaluated whether MOTS-c, a novel mitochondria-related 16-aa peptide, can reduce vitamin D3 and nicotine-induced VC in rats. METHODS: Vitamin D3 plus nicotine-treated rats were injected with MOTS-c at a dose of 5 mg/kg once a day for 4 weeks. Blood pressure, heart rate, and body weight were measured, and echocardiography was performed. The expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and the angiotensin II type 1 (AT-1) and endothelin B (ET-B) receptors was determined by Western blot analysis. RESULTS: Our results showed that MOTS-c treatment significantly attenuated VC. Furthermore, we found that the level of phosphorylated AMPK was increased and the expression levels of the AT-1 and ET-B receptors were decreased after MOTS-c treatment. CONCLUSIONS: Our findings provide evidence that MOTS-c may act as an inhibitor of VC by activating the AMPK signaling pathway and suppressing the expression of the AT-1 and ET-B receptors.

Endothelin-1 increases CHSY-1 expression in aortic endothelial cells via transactivation of transforming growth factor ß type I receptor induced by type B receptor endothelin-1.

OBJECTIVES: TGF-ß through hyperelongation of glycosaminoglycan (GAG) chains leads to binding of low-density lipoproteins to the proteoglycans. The vasoactive peptide, endothelin-1 (ET-1), plays a key role in the development of atherosclerosis. This study addressed the question whether ET-1 by activating the Rho kinase and cytoskeletal rearrangement can transactivate the TGF-ß receptor leading to phosphorylation of the transcription factor Smad2 and increased expression of the GAG chain synthesizing enzyme such as chondroitin synthase-1 (CHSY-1) in bovine aortic endothelial cells (BAECs). METHODS: In this study, intermediates in ET-1-induced Smad2C phosphorylation and the protein level of CHSY-1 were identified and quantified by Western blotting. KEY FINDINGS: Endothelin-1 caused time-dependent phosphorylation of Smad2C which was inhibited in the presence of the endothelin B receptor antagonist, BQ788. The response to ET-1 was inhibited by the Rho/ROCK kinase antagonist, Y27632 and by cytochalasin D, an inhibitor of actin polymerization but the ET-1-mediated pSmad2C was not inhibited by the matrix metalloproteinase (MMP) inhibitor, GM6001. ET-1 increased CHSY-1 protein level, which was inhibited in the presence of BQ788, cytochalasin D and Y27632. CONCLUSIONS: Endothelin-1 signalling via the ETB receptor utilizes cytoskeletal rearrangement and Rho kinase but not MMPs leading to TßRI transactivation signalling and phosphorylation of Smad2C and through this pathway increased the level of CHSY-1.

Ovarian hormones modulate endothelin-1 receptor responses in young women.

OBJECTIVE: We recently demonstrated ETBR mediate vasodilation in young but not postmenopausal women; it is unclear if this is related to age or a decline in ovarian hormones. The purpose of this study was to test the hypothesis that ETBR responses are modulated by ovarian hormones. METHODS: We measured cutaneous vasodilatory responses in 12 young women (22 ± 1 years, 23 ± 1 kg/m2 ) during the ML (days 20-25) and EF (days 2-5) phases of the menstrual cycle. Cutaneous microdialysis perfusions of lactated Ringer (control), ETBR antagonist (BQ-788, 300 nmol/L), and ETAR antagonist (BQ-123, 500 nmol/L) were performed, followed by local heating to 42°C. RESULTS: Serum estradiol (ML: 118 ± 16 vs EF: 44 ± 9 pg/mL, P < 0.05) and progesterone (ML: 8.3 ± 1.0 vs EF: 0.7 ± 0.2 ng/mL, P < 0.05) were higher during ML vs EF phase. ETBR blockade decreased vasodilation during ML (control: 91 ± 2 vs BQ-788: 83 ± 2%CVCmax, P < 0.05) but not EF (control: 89 ± 2 vs BQ-788: 89 ± 1%CVCmax). ETAR blockade also decreased vasodilation during ML (control: 91 ± 2 vs BQ-123: 87 ± 2%CVCmax, P < 0.05) but not EF (control: 89 ± 2 vs BQ-123: 92 ± 2%CVCmax). CONCLUSIONS: These data suggest that fluctuations in ovarian hormones modulate ETBR and ETAR responses in young women.

Bucindolol attenuates the vascular remodeling of pulmonary arteries by modulating the expression of the endothelin-1 A receptor in rats with pulmonary arterial hypertension.

The aim of this study was to investigate the role of the ß-adrenergic blocker bucindolol on endothelial dysfunction and pulmonary vascular remodeling in rats with pulmonary arterial hypertension (PAH). Male Wistar rats were divided into four groups: control, monocrotaline (MCT), control?+?bucindolol and monocrotaline?+?bucindolol (MCT?+?BCD). PAH was induced by an injection of monocrotaline (60?mg/kg i.p.). After two weeks, the animals were treated for seven days with bucindolol (2?mg/kg/day i.p.) or vehicle. Echocardiography was performed upon treatment completion to analyze pulmonary vascular resistance (PVR) and right ventricle (RV) myocardial performance index. Lungs were collected for oxidative stress and western blot analysis, and the pulmonary artery was analyzed for histological and immunohistochemical parameters. The MCT?+?BCD group showed a decrease (32%) in the protein expression of endothelin-1 type A receptor (ETAR) and in the ratio of ETA/endothelin-1 type B receptor (ETBR) (62%) as compared to the MCT group. Bucindolol treatment did not alter oxidative stress, as determined by lipid peroxidation analysis and antioxidant enzyme activities and expression, endothelial nitric oxide synthase immunocontent and decreased nitrotyrosine levels. Moreover, bucindolol improved vascular remodeling of the pulmonary artery in the MCT?+?BCD group by decreasing (21%) PVR and increasing RV workload in relation to MCT.

Homocysteine up-regulates ETB receptors via suppression of autophagy in vascular smooth muscle cells.

The change of autophagy is implicated in cardiovascular diseases (CVDs). Homocysteine (Hcy) up-regulates endothelin type B (ETB) receptors in vascular smooth muscle cells (VSMCs). However, it is unclear whether autophagy is involved in Hcy-induced-up-regulation of ETB receptors in VSMCs. The present study was designed to examine the hypothesis that Hcy up-regulates ETB receptors by inhibiting autophagy in VSMCs. Hcy treated the rat superior mesenteric artery (SMA) without endothelium in the presence and absence of AICAR, rapamycin or MHY1485 for 24 h. The contractile responses to sarafotoxin 6c (S6c) (an ETB receptor agonist) were studied using a sensitive myograph. Levels of protein expression were determined using Western blot analysis. Punctate staining of LC3B was exanimated by immunofluorescence using confocal microscopy. The results showed that Hcy inhibited AMPK, and activated mTOR, followed by impairing autophagy, and increased the levels of ETB receptor protein expression and the ETB receptor-mediated contractile responses to S6c in SMA without endothelium. However, these effects were reversed by AICAR or rapamycin. Additionally, MHY1485 up-regulated the AICAR-inhibited ETB receptor-mediated contractile response and the levels of ETB receptor protein expression in presence of Hcy. In conclusion, this suggested that Hcy up-regulated ETB receptors by inhibiting autophagy in VSMCs via AMPK/mTOR signaling pathway.

Tail vein injection of mmLDL upregulates mouse mesenteric artery ETB receptors via activation of the ERK1/2 pathway.

Minimally modified low density lipoprotein (mmLDL) is a risk factor for cardiovascular disease. This study investigated the effect of mmLDL on mouse mesenteric artery endothelin type B (ETB) receptors and its molecular mechanism. Mice were injected with normal saline (NS group), mmLDL in the tail vein (mmLDL group), or with both mmLDL and an intraperitoneal injection of the ERK1/2 pathway-specific inhibitor U0126 (mmLDL+U0126 group). The dose-response curve of mesenteric artery contraction induced by sarafotoxin 6c (S6c), the ETB receptor agonist, was measured using a sensitive myograph system. ELISAs, RT-PCR and Western blot were used to determine the serum concentrations of mouse oxidized low density lipoprotein (oxLDL), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) as well as the expression of ETB receptors, ICAM-1, VCAM-1 and phosphorylated-extracellular signal-regulated kinase 1/2 (p-ERK1/2). The S6c-induced contraction dose-response curve was significantly enhanced by mmLDL treatment and showed a significantly higher Emax value than in the NS group (P<0.001), and the ETB receptor mRNA and protein expression in the vascular wall was significantly higher than in the NS group. The serum concentration and expression of ICAM-1 and VCAM-1 were also increased by mmLDL treatment, but intraperitoneal injection of U0126 inhibited these changes as well as the increase in p-ERK1/2 protein in the vessel wall caused by mmLDL. ICAM-1 and VCAM-1 serum concentrations were positively correlated with the S6c-induced maximum contraction of blood vessels. Increased in vivo levels of mmLDL increased the serum concentrations and expression of ICAM-1 and VCAM-1 by activating the ERK1/2 pathway, resulting in the expression of ETB receptors and the enhancement of contractile function in vascular smooth muscle. Understanding the effect of mmLDL on ETB receptors and its mechanism can provide ideas for cardiovascular disease prevention and treatment.

Targeting endothelin-1 receptor/ß-arrestin1 network for the treatment of ovarian cancer.

INTRODUCTION: Endothelin-1 receptor (ET-1R)/ß-arrestin1 (ß-arr1) signaling is dysregulated in ovarian cancer. This signaling circuit enables cancer cells to engage several signaling and transcriptional networks that are pervasively intertwined, and represent a potential therapeutic target for developing novel agents for ovarian cancer treatment. Areas covered: In this article, we discuss the role of the signaling network between ET-1R and key pathways mediated by the scaffold protein ß-arr1, as part of signaling complex, or as a transcription co-activator, promoting precise control of transcription of different genes, including ET-1. Therefore ET-1R/ß-arr1 is an actionable node involved in the activation of a persistent feedback loop that contributes to bypass signaling. Targeting ET-1R empowering this circuit can represent a necessary measure to reach clinical efficacy. Preclinical studies demonstrate that blocking ET-1R by FDA approved dual ETAR/ETBR antagonist prevents ß-arr1 network formation, offering a novel therapeutic strategy in ovarian cancer patients. Expert opinion: The information provided in this review about the ET-1R/ß-arr1 hub represents an invaluable tool for both identifying the interconnected pathways involved in ovarian cancer and targeting them more effectively. The new perspective arising from ET-1R therapeutics will likely prompt a valuable frame for the design of new promising combinatorial therapy, blocking compensatory networks.

Renin-angiotensin system blockade alone or combined with ETA receptor blockade: effects on the course of chronic kidney disease in 5/6 nephrectomized Ren-2 transgenic hypertensive rats.

BACKGROUND: Early addition of endothelin (ET) type A (ETA) receptor blockade to complex renin-angiotensin system (RAS) blockade has previously been shown to provide better renoprotection against progression of chronic kidney disease (CKD) in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX). In this study, we examined if additional protection is provided when ETA blockade is applied in rats with already developed CKD. METHODS: For complex RAS inhibition, an angiotensin-converting enzyme inhibitor along with angiotensin II type 1 receptor blocker was used. Alternatively, ETA receptor blocker was added to the RAS blockade. The treatments were initiated 6 weeks after 5/6 NX and the follow-up period was 50 weeks. RESULTS: When applied in established CKD, addition of ETA receptor blockade to the complex RAS blockade brought no further improvement of the survival rate (30% in both groups); surprisingly, aggravated albuminuria (588 ± 47 vs. 245 ± 38 mg/24 h, p < 0.05) did not reduce renal glomerular injury index (1.25 ± 0.29 vs. 1.44 ± 0.26), did not prevent the decrease in creatinine clearance (203 ± 21 vs. 253 ± 17 µl/min/100 g body weight), and did not attenuate cardiac hypertrophy to a greater extent than observed in 5/6 NX TGR treated with complex RAS blockade alone. CONCLUSIONS: When applied in the advanced phase of CKD, addition of ETA receptor blockade to the complex RAS blockade brings no further beneficial renoprotective effects on the CKD progression in 5/6 NX TGR, in addition to those seen with RAS blockade alone.

Dual endothelin receptor antagonists contrast the effects induced by endothelin-1 on cultured human microvascular endothelial cells.

OBJECTIVES: To evaluate the ability of dual endothelin (ET) receptor antagonists (ETA/ETB -ETA/BRAs) to contrast the ET-1-induced effects on cultured human microvascular endothelial cells (HMVECs). METHODS: Some cultured HMVECs were untreated, or treated with ET-1 (100nM) or transforming growth factor ß1 (TGFß1, 10ng/mL) alone for 6 days, in order to induce the endothelial-to-mesenchymal transition (EndoMT). Other cultured HMVECs were pre-treated for 1hr with ETA/BRAs bosentan (10µM) or macitentan (1µM, 10µM) before the stimulation with ET-1 for 6 days. At the end of treatments, a mechanical injury was induced to cultured HMVECs (by scratching the cell monolayer with a sterile tip), and then the cell ability to re-fill the damaged area was determined after 24hrs. EndoMT phenotype markers and monocyte chemoattractant protein-1 (MCP-1) were evaluated by qRT-PCR and Western blotting. Statistical analysis was performed using Mann-Whitney-U non-parametric test. RESULTS: Both ET-1 and TGFß1 induced EndoMT and the MCP-1 over-expression in cultured HMVECs, as well as reduced the process of endothelial cell damage repair. Pre-treatment with ETA/BRAs let cultured HMVECs to significantly restore the in vitro damage of the cell monolayer and antagonised the EndoMT process as well as the MCP-1 over-expression (range p<0.05 - p<0.001). Conversely, untreated or TGFß1-treated HMVECs were found unaffected by the ETA/BRAs treatments. CONCLUSIONS: The treatment with dual ETA/BRAs seems to partially restore the altered cell function induced by ET-1 in cultured endothelial cells, and might justify their therapeutic efficiency in clinical conditions characterised by increased concentrations of ET-1.

Positive feedback role of TRPC3 in TNF-α-mediated vasogenic edema formation induced by status epilepticus independent of ETB receptor activation.

Brain-blood barrier (BBB) disruption results in vasogenic edema, which is involved in the pathogenesis of epilepsy. Following status epilepticus (SE), up-regulated transient receptor potential canonical channel-3 (TRPC3), a Ca2+-permeable cation channels in endothelial cells, is relevant to vasogenic edema formation in the rat piriform cortex. In addition, pyrazole-3 (Pyr-3, a TRPC3 inhibitor) attenuated SE-induced vasogenic edema. However, the upstream regulators of TRPC3 expression in vasogenic edema formation have been unclear. In the present study, soluble tumor necrosis factor p55 receptor (sTNFp55R, a TNF-α inhibitor), SN50 (a nuclear factor-κB (NFκB) inhibitor), BQ-788 (an endothelin B (ETB) receptor inhibitor) and Pyr-3 effectively prevented vasogenic edema following SE. sTNFp55R and SN50 (but not BQ-788) attenuated SE-induced up-regulation of endothelial TRPC3 expression. Pyr-3 ameliorated SE-induced NFκB p65-Thr435 phosphorylation and ETB receptor expression. In addition, Pyr-3 mitigated NFκB p65-Thr435 phosphorylation induced by recombinant TNF-α. These findings indicate that TNF-α-mediated NFκB p65-Thr435 phosphorylation may up-regulate TRPC3 expression, which participates in vasogenic edema formation via increasing endothelial nitric oxide synthase expression following SE, independent of ETB receptor activation. Therefore, we suggest that TRPC3 may be involved in a positive feedback loop of NFκB/ETB receptor signaling pathway.

Activation of purinergic receptors (P2) in the renal medulla promotes endothelin-dependent natriuresis in male rats.

Renal endothelin-1 (ET-1) and purinergic signaling systems regulate Na(+) reabsorption in the renal medulla. A link between the renal ET-1 and purinergic systems was demonstrated in vitro, however, the in vivo interaction between these systems has not been defined. To test whether renal medullary activation of purinergic (P2) receptors promotes ET-dependent natriuresis, we determined the effect of increased medullary NaCl loading on Na(+) excretion and inner medullary ET-1 mRNA expression in anesthetized adult male Sprague-Dawley rats in the presence and absence of purinergic receptor antagonism. Isosmotic saline (NaCl; 284 mosmol/kgH2O) was infused into the medullary interstitium (500 µl/h) during a 30-min baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH2O) for two further 30-min urine collection periods. Na(+) excretion was significantly increased during intramedullary infusion of hyperosmotic saline. Compared with isosmotic saline, hyperosmotic saline infused into the renal medulla caused significant increases in inner medullary ET-1 mRNA expression. Renal intramedullary infusion of the P2 receptor antagonist suramin inhibited the increase in Na(+) excretion and inner medullary ET-1 mRNA expression induced by NaCl loading in the renal medulla. Activation of medullary P2Y2/4 receptors by infusion of UTP increased urinary Na(+) excretion. Combined ETA and ETB receptor blockade abolished the natriuretic response to intramedullary infusion of UTP. These data demonstrate that activation of medullary P2 receptors promotes ET-dependent natriuresis in male rats, suggesting that the renal ET-1 and purinergic signaling systems interact to efficiently facilitate excretion of a NaCl load.

The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries.

Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ETA receptor mRNA and protein levels as well as contractile responses mediated by ETA receptors. The immunoreactivity of increased ETA receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ETB receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries.

Discovery of Dual ETA/ETB Receptor Antagonists from Traditional Chinese Herbs through in Silico and in Vitro Screening.

Endothelin-1 receptors (ETAR and ETBR) act as a pivotal regulator in the biological effects of ET-1 and represent a potential drug target for the treatment of multiple cardiovascular diseases. The purpose of the study is to discover dual ETA/ETB receptor antagonists from traditional Chinese herbs. Ligand- and structure-based virtual screening was performed to screen an in-house database of traditional Chinese herbs, followed by a series of in vitro bioassay evaluation. Aristolochic acid A (AAA) was first confirmed to be a dual ETA/ETB receptor antagonist based intracellular calcium influx assay and impedance-based assay. Dose-response curves showed that AAA can block both ETAR and ETBR with IC50 of 7.91 and 7.40 µM, respectively. Target specificity and cytotoxicity bioassay proved that AAA is a selective dual ETA/ETB receptor antagonist and has no significant cytotoxicity on HEK293/ETAR and HEK293/ETBR cells within 24 h. It is a feasible and effective approach to discover bioactive compounds from traditional Chinese herbs using in silico screening combined with in vitro bioassay evaluation. The structural characteristic of AAA for its activity was especially interpreted, which could provide valuable reference for the further structural modification of AAA.