Endothelin ETA receptors predominate in chronic thromboembolic pulmonary hypertension.

AIMS: Endothelin-1 levels are raised in chronic thromboembolic pulmonary hypertension. Our aim in this study was to identify the presence of endothelin receptors in patients with CTEPH by analysing tissue removed at pulmonary endarterectomy. MAIN METHODS: Pulmonary endarterectomy tissue cross-sections were analysed using autoradiography with [(125)I]-ET-1 using ligands selective for ETA or ETB to determine sub-type distribution. The precise cellular localisation of ETA and ETB receptors was determined using selective antisera to both sub-types and compared with haematoxylin and eosin, Elastic Van Gieson and smooth muscle actin labelled sections. KEY FINDINGS: Two patterns of ET-1 binding were found. In sections with frequent recanalised channels, ET-1 bound to the smooth muscle cells surrounding the channels. In sections where there was less organised thrombus with no obvious re-canalisation, minimal ET-1 binding was observed. Some contractile type smooth muscle cells not associated with recanalised channels and diffusely spread throughout the PEA material were associated with ET receptor antibody binding on immunohistochemistry. There was a greater expression of the ETA receptor type in the specimens. SIGNIFICANCE: The presence of ET-1 receptors in the chronic thrombus in proximal CTEPH suggests ET-1 could act not only on the distal vasculopathy in the unobstructed vessels but may also stimulate smooth muscle cell proliferation within chronic clot. The abundance of ET receptors within the tissue provides evidence that the ET pathway is involved in the pathology of chronic thrombus reorganisation leading to CTEPH providing a rationale for the repurposing of ET receptor antagonists in the treatment of this condition.We acknowledge the support of the referring UK centres for PH; the Pulmonary Hypertension Association-UK, Wellcome Trust award WT107715/Z/15/Z, Programmes in Translational Medicines and Therapeutics (085686) and in Metabolic and Cardiovascular Disease (096822/Z/11/Z), the British Heart Foundation PG/09/050/27734, MRC and the NIHR Cambridge Biomedical Research Centre. We also acknowledge the support of the Cambridge NIHR BRC Cell Phenotyping Hub and the Papworth Hospital Research Tissue Bank.This is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.lfs.2016.02.03

Expression of the apelin receptor, a novel potential therapeutic target, and its endogenous ligands in diverse stem cell populations in human glioblastoma

Glioblastoma multiforme (GBM) is one of the most common and lethal forms of brain cancer, carrying a very poor prognosis (median survival of ~15 months post-diagnosis). Treatment typically involves invasive surgical resection of the tumour mass, followed by radiotherapy and adjuvant chemotherapy using the alkylating agent temozolomide, but over half of patients do not respond to this drug and considerable resistance is observed. Tumour heterogeneity is the main cause of therapeutic failure, where diverse progenitor glioblastoma stem cell (GSC) lineages in the microenvironment drive tumour recurrence and therapeutic resistance. The apelin receptor is a class A GPCR that binds two endogenous peptide ligands, apelin and ELA, and plays a role in the proliferation and survival of cancer cells. Here, we used quantitative whole slide immunofluorescent imaging of human GBM samples to characterise expression of the apelin receptor and both its ligands in the distinct GSC lineages, namely neural-progenitor-like cells (NPCs), oligodendrocyte-progenitor-like cells (OPCs), and mesenchymal-like cells (MES), as well as reactive astrocytic cells. The data confirm the presence of the apelin receptor as a tractable drug target that is common across the key cell populations driving tumour growth and maintenance, offering a potential novel therapeutic approach for patients with GBM

The biased apelin receptor agonist, MM07, reverses Sugen/hypoxia-induced pulmonary arterial hypertension as effectively as the endothelin antagonist macitentan

Introduction: Pulmonary arterial hypertension (PAH) is characterised by endothelial dysfunction and pathological vascular remodelling, resulting in the occlusion of pulmonary arteries and arterioles, right ventricular hypertrophy, and eventually fatal heart failure. Targeting the apelin receptor with the novel, G protein-biased peptide agonist, MM07, is hypothesised to reverse the developed symptoms of elevated right ventricular systolic pressure and right ventricular hypertrophy. Here, the effects of MM07 were compared with the clinical standard-of-care endothelin receptor antagonist macitentan.Methods: Male Sprague-Dawley rats were randomised and treated with either normoxia/saline, or Sugen/hypoxia (SuHx) to induce an established model of PAH, before subsequent treatment with either saline, macitentan (30 mg/kg), or MM07 (10 mg/kg). Rats were then anaesthetised and catheterised for haemodynamic measurements, and tissues collected for histopathological assessment.Results: The SuHx/saline group presented with significant increases in right ventricular hypertrophy, right ventricular systolic pressure, and muscularization of pulmonary arteries compared to normoxic/saline controls. Critically, MM07 was as at least as effective as macitentan in significantly reversing detrimental structural and haemodynamic changes after 4 weeks of treatment.Discussion: These results support the development of G protein-biased apelin receptor agonists with improved pharmacokinetic profiles for use in human disease

A novel cyclic biased agonist of the apelin receptor, MM07, is disease modifying in the rat monocrotaline model of pulmonary arterial hypertension.

BACKGROUND AND PURPOSE: Apelin is an endogenous vasodilatory and inotropic peptide that is down-regulated in human pulmonary arterial hypertension, although the density of the apelin receptor is not significantly attenuated. We hypothesised that a G protein-biased apelin analogue MM07, which is more stable than the endogenous apelin peptide, may be beneficial in this condition with the advantage of reduced β-arrestin-mediated receptor internalisation with chronic use. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats received either monocrotaline to induce pulmonary arterial hypertension or saline and then daily i.p. injections of either MM07 or saline for 21 days. The extent of disease was assessed by right ventricular catheterisation, cardiac MRI, and histological analysis of the pulmonary vasculature. The effect of MM07 on signalling, proliferation, and apoptosis of human pulmonary artery endothelial cells was investigated. KEY RESULTS: MM07 significantly reduced the elevation of right ventricular systolic pressure and hypertrophy induced by monocrotaline. Monocrotaline-induced changes in cardiac structure and function, including right ventricular end-systolic and end-diastolic volumes, ejection fraction, and left ventricular end-diastolic volume, were attenuated by MM07. MM07 also significantly reduced monocrotaline-induced muscularisation of small pulmonary blood vessels. MM07 stimulated endothelial NOS phosphorylation and expression, promoted proliferation, and attenuated apoptosis of human pulmonary arterial endothelial cells in vitro. CONCLUSION AND IMPLICATIONS: Our findings suggest that chronic treatment with MM07 is beneficial in this animal model of pulmonary arterial hypertension by addressing disease aetiology. These data support the development of G protein-biased apelin receptor agonists with improved pharmacokinetic profiles for use in human disease.the Medical Research Council MC_PC_14116 [to APD] Wellcome Trust [107715/Z/15/Z to APD], Programme in Metabolic and Cardiovascular Disease [096822/Z/11/Z to PY; 203814/Z/16/A to TLW], Parke Davis Fellowship [to PY], British Heart Foundation [FS/14/59/31282 to CR] and in part by the National Institute for Health Research Cambridge Biomedical Research Centre

Differential expression in humans of the viral entry receptor ACE2 compared with the short deltaACE2 isoform lacking SARS-CoV-2 binding sites.

ACE2 is a membrane protein that regulates the cardiovascular system. Additionally, ACE2 acts as a receptor for host cell infection by human coronaviruses, including SARS-CoV-2 that emerged as the cause of the on-going COVID-19 pandemic and has brought unprecedented burden to economy and health. ACE2 binds the spike protein of SARS-CoV-2 with high affinity and shows little variation in amino acid sequence meaning natural resistance is rare. The discovery of a novel short ACE2 isoform (deltaACE2) provides evidence for inter-individual differences in SARS-CoV-2 susceptibility and severity, and likelihood of developing subsequent 'Long COVID'. Critically, deltaACE2 loses SARS-CoV-2 spike protein binding sites in the extracellular domain, and is predicted to confer reduced susceptibility to viral infection. We aimed to assess the differential expression of full-length ACE2 versus deltaACE2 in a panel of human tissues (kidney, heart, lung, and liver) that are implicated in COVID-19, and confirm ACE2 protein in these tissues. Using dual antibody staining, we show that deltaACE2 localises, and is enriched, in lung airway epithelia and bile duct epithelia in the liver. Finally, we also confirm that a fluorescently tagged SARS-CoV-2 spike protein monomer shows low binding at lung and bile duct epithelia where dACE2 is enriched.This research was funded in whole, or in part by: Wellcome Trust (WT107715/Z/15/Z, A.P.D., and J.J.M.); Wellcome Trust Programme in Metabolic and Cardiovascular Disease (203814/Z/16/A, T.L.W., D.N.), Wellcome Trust Major Award (208363/Z/17/Z) for Imaging Core (G.S.); British Heart Foundation (FS/17/61/33473 A.P.D., R.G.C.M; TG/18/4/33770, A.P.D., J.J.M.; FS/18/46/33663, S.S.). Cambridge Biomedical Research Centre Biomedical Resources Grant (University of Cambridge, Cardiovascular Theme, RG64226). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care

The G Protein Biased Small Molecule Apelin Agonist CMF-019 is Disease Modifying in Endothelial Cell Apoptosis In Vitro and Induces Vasodilatation Without Desensitisation In Vivo

Signaling through the apelin receptor is beneficial for a number of diseases including pulmonary arterial hypertension. The endogenous small peptides, apelin and elabela/toddler, are downregulated in pulmonary arterial hypertension but are not suitable for exogenous administration owing to a lack of bioavailability, proteolytic instability and susceptibility to renal clearance. CMF-019, a small molecule apelin agonist that displays strong bias towards G protein signaling over β-arrestin (∼400 fold), may be more suitable. This study demonstrates that in addition to being a positive inotrope, CMF-019 caused dose-dependent vasodilatation in vivo (50 nmol 4.16 ± 1.18 mmHg, **p < 0.01; 500 nmol 6.62 ± 1.85 mmHg, **p < 0.01), without receptor desensitization. Furthermore, CMF-019 rescues human pulmonary artery endothelial cells from apoptosis induced by tumor necrosis factor α and cycloheximide (5.66 ± 0.97%, **p < 0.01) by approximately 50% of that observable with rhVEGF (11.59 ± 1.85%, **p < 0.01), suggesting it has disease-modifying potential in vitro. CMF-019 displays remarkable bias at the apelin receptor for a small molecule and importantly recapitulates all aspects of the cardiovascular responses to the endogenous ligand, [Pyr1]apelin-13, in vivo. Additionally, it is able to protect human pulmonary artery endothelial cells from apoptosis, suggesting that the beneficial effects observed with apelin agonists extend beyond hemodynamic alleviation and address disease etiology itself. These findings support CMF-019 as a G protein biased small molecule apelin agonist in vitro and in vivo that could form the basis for the design of novel therapeutic agents in chronic diseases, such as, pulmonary arterial hypertension

Human embryonic stem cell-derived cardiomyocyte platform screens inhibitors of SARS-CoV-2 infection.

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated

The G Protein Biased Small Molecule Apelin Agonist CMF-019 is Disease Modifying in Endothelial Cell Apoptosis In Vitro and Induces Vasodilatation Without Desensitisation In Vivo

Signaling through the apelin receptor is beneficial for a number of diseases including pulmonary arterial hypertension. The endogenous small peptides, apelin and elabela/toddler, are downregulated in pulmonary arterial hypertension but are not suitable for exogenous administration owing to a lack of bioavailability, proteolytic instability and susceptibility to renal clearance. CMF-019, a small molecule apelin agonist that displays strong bias towards G protein signaling over β-arrestin (∼400 fold), may be more suitable. This study demonstrates that in addition to being a positive inotrope, CMF-019 caused dose-dependent vasodilatation in vivo (50 nmol 4.16 ± 1.18 mmHg, **p < 0.01; 500 nmol 6.62 ± 1.85 mmHg, **p < 0.01), without receptor desensitization. Furthermore, CMF-019 rescues human pulmonary artery endothelial cells from apoptosis induced by tumor necrosis factor α and cycloheximide (5.66 ± 0.97%, **p < 0.01) by approximately 50% of that observable with rhVEGF (11.59 ± 1.85%, **p < 0.01), suggesting it has disease-modifying potential in vitro. CMF-019 displays remarkable bias at the apelin receptor for a small molecule and importantly recapitulates all aspects of the cardiovascular responses to the endogenous ligand, [Pyr1]apelin-13, in vivo. Additionally, it is able to protect human pulmonary artery endothelial cells from apoptosis, suggesting that the beneficial effects observed with apelin agonists extend beyond hemodynamic alleviation and address disease etiology itself. These findings support CMF-019 as a G protein biased small molecule apelin agonist in vitro and in vivo that could form the basis for the design of novel therapeutic agents in chronic diseases, such as, pulmonary arterial hypertension

Development and validation of an LC-MS/MS method for detection and quantification of in vivo derived metabolites of [Pyr 1 ]apelin-13 in humans

Abstract: [Pyr1]apelin-13 is the predominant apelin peptide isoform in the human cardiovascular system and plasma. To date, few studies have investigated [Pyr1]apelin-13 metabolism in vivo in rats with no studies examining its stability in humans. We therefore aimed to develop an LC-MS/MS method for detection and quantification of intact [Pyr1]apelin-13 and have used this method to identify the metabolites generated in vivo in humans. [Pyr1]apelin-13 (135 nmol/min) was infused into six healthy human volunteers for 120 minutes and blood collected at time 0 and 120 minutes after infusion. Plasma was extracted in the presence of guanidine hydrochloride and analysed by LC-MS/MS. Here we report a highly sensitive, robust and reproducible method for quantification of intact [Pyr1]apelin-13 and its metabolites in human plasma. Using this method, we showed that the circulating concentration of intact peptide was 58.3 ± 10.5 ng/ml after 120 minutes infusion. We demonstrated for the first time that in humans, [Pyr1]apelin-13 was cleaved from both termini but the C-terminal was more susceptible to cleavage. Consequently, of the metabolites identified, [Pyr1]apelin-13(1–12), [Pyr1]apelin-13(1–10) and [Pyr1]apelin-13(1–6) were the most abundant. These data suggest that apelin peptides designed for use as cardiovascular therapeutics, should include modifications that minimise C-terminal cleavage

Development and validation of an LC-MS/MS method for detection and quantification of in vivo derived metabolites of [Pyr 1 ]apelin-13 in humans

Abstract: [Pyr1]apelin-13 is the predominant apelin peptide isoform in the human cardiovascular system and plasma. To date, few studies have investigated [Pyr1]apelin-13 metabolism in vivo in rats with no studies examining its stability in humans. We therefore aimed to develop an LC-MS/MS method for detection and quantification of intact [Pyr1]apelin-13 and have used this method to identify the metabolites generated in vivo in humans. [Pyr1]apelin-13 (135 nmol/min) was infused into six healthy human volunteers for 120 minutes and blood collected at time 0 and 120 minutes after infusion. Plasma was extracted in the presence of guanidine hydrochloride and analysed by LC-MS/MS. Here we report a highly sensitive, robust and reproducible method for quantification of intact [Pyr1]apelin-13 and its metabolites in human plasma. Using this method, we showed that the circulating concentration of intact peptide was 58.3 ± 10.5 ng/ml after 120 minutes infusion. We demonstrated for the first time that in humans, [Pyr1]apelin-13 was cleaved from both termini but the C-terminal was more susceptible to cleavage. Consequently, of the metabolites identified, [Pyr1]apelin-13(1–12), [Pyr1]apelin-13(1–10) and [Pyr1]apelin-13(1–6) were the most abundant. These data suggest that apelin peptides designed for use as cardiovascular therapeutics, should include modifications that minimise C-terminal cleavage