Microarray analysis demonstrates upregulation of the endothelin-1 gene with compensatory downregulation of the ETA receptor gene in human portal vein.

High blood pressure in the portal vein, portal hypertension (PH), is the final common pathway in liver cirrhosis regardless of aetiology. Complications from PH are the major cause of morbidity and mortality in these patients. Current drug therapy to reduce portal pressure is mainly limited to ß-adrenergic receptor blockade but about forty percent of patients do not respond. Our aim was to use microarray to measure the expression of ~20,800 genes in portal vein from patients with PH undergoing transplantation for liver cirrhosis (PH, n = 12) versus healthy vessels (control, n = 9) to identify potential drug targets to improve therapy. Expression of 9,964 genes above background was detected in portal vein samples. Comparing PH veins versus control (adjusted p value < 0.05, fold change > 1.5) identified 548 upregulated genes and 1,996 downregulated genes. The 2,544 differentially expressed genes were subjected to pathway analysis. We identified 49 significantly enriched pathways. The endothelin pathway was ranked the tenth most significant, the only vasoconstrictive pathway to be identified. ET-1 gene (EDN1) was significantly upregulated, consistent with elevated levels of ET-1 peptide previously measured in PH and cirrhosis. ETA receptor gene (EDNRA) was significantly downregulated, consistent with an adaptive response to increased peptide levels in the portal vein but there was no change in the ETB gene (EDNRB). The results provide further support for evaluating the efficacy of ETA receptor antagonists as a potential therapy in addition to ß-blockers in patients with PH and cirrhosis.

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.

Co-localization of the sodium-glucose co-transporter-2 channel (SGLT-2) with endothelin ETA and ETB receptors in human cardiorenal tissue.

Endothelin (ET) receptor antagonists are being investigated in combination with sodium-glucose co-transporter-2 inhibitors (SGLT-2i). These drugs primarily inhibit the SGLT-2 transporter that, in humans, is thought to be mainly restricted to the renal proximal convoluted tubule, resulting in increased glucose excretion favouring improved glycaemic control and diuresis. This action reduces fluid retention with ET receptor antagonists. Studies have suggested SGLT-2 may also be expressed in cardiomyocytes of human heart. To understand the potential of combining the two classes of drugs, our aim was to compare the distribution of ET receptor sub-types in human kidney, with SGLT-2. Secondly, using the same experimental conditions, we determined if SGLT-2 expression could be detected in human heart and whether the transporter co-localised with ET receptors. METHODS: Immunocytochemistry localised SGLT-2, ETA and ETB receptors in sections of histologically normal kidney, left ventricle from patients undergoing heart transplantation or controls. Primary antisera were visualised using fluorescent microscopy. Image analysis was used to measure intensity compared with background in adjacent control sections. RESULTS: As expected, SGLT-2 localised to epithelial cells of the proximal convoluted tubules, and co-localised with both ET receptor sub-types. Similarly, ETA receptors predominated in cardiomyocytes; low (compared with kidney but above background) positive staining was also detected for SGLT-2. DISCUSSION: Whether low levels of SGLT-2 have a (patho)physiological role in cardiomyocytes is not known but results suggest the effect of direct blockade of sodium (and glucose) influx via SGLT-2 inhibition in cardiomyocytes should be explored, with potential for additive effects with ETA antagonists.

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.

Expanding the apelin receptor pharmacological toolbox using novel fluorescent ligands.

Introduction: The apelin receptor binds two distinct endogenous peptides, apelin and ELA, which act in an autocrine/paracrine manner to regulate the human cardiovascular system. As a class A GPCR, targeting the apelin receptor is an attractive therapeutic strategy. With improvements in imaging techniques, and the stability and brightness of dyes, fluorescent ligands are becoming increasingly useful in studying protein targets. Here, we describe the design and validation of four novel fluorescent ligands; two based on [Pyr1]apelin-13 (apelin488 and apelin647), and two based on ELA-14 (ELA488 and ELA647). Methods: Fluorescent ligands were pharmacologically assessed using radioligand and functional in vitro assays. Apelin647 was validated in high content imaging and internalisation studies, and in a clinically relevant human embryonic stem cell-derived cardiomyocyte model. Apelin488 and ELA488 were used to visualise apelin receptor binding in human renal tissue. Results: All four fluorescent ligands retained the ability to bind and activate the apelin receptor and, crucially, triggered receptor internalisation. In high content imaging studies, apelin647 bound specifically to CHO-K1 cells stably expressing apelin receptor, providing proof-of-principle for a platform that could screen novel hits targeting this GPCR. The ligand also bound specifically to endogenous apelin receptor in stem cell-derived cardiomyocytes. Apelin488 and ELA488 bound specifically to apelin receptor, localising to blood vessels and tubules of the renal cortex. Discussion: Our data indicate that the described novel fluorescent ligands expand the pharmacological toolbox for studying the apelin receptor across multiple platforms to facilitate drug discovery.

FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.

Inducible apelin receptor knockdown reduces differentiation efficiency and contractility of hESC-derived cardiomyocytes.

AIMS: The apelin receptor, a G protein-coupled receptor, has emerged as a key regulator of cardiovascular development, physiology, and disease. However, there is a lack of suitable human in vitro models to investigate the apelinergic system in cardiovascular cell types. For the first time we have used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and a novel inducible knockdown system to examine the role of the apelin receptor in both cardiomyocyte development and to determine the consequences of loss of apelin receptor function as a model of disease. METHODS AND RESULTS: Expression of the apelin receptor and its ligands in hESCs and hESC-CMs was determined. hESCs carrying a tetracycline-inducible short hairpin RNA targeting the apelin receptor were generated using the sOPTiKD system. Phenotypic assays characterized the consequences of either apelin receptor knockdown before hESC-CM differentiation (early knockdown) or in 3D engineered heart tissues as a disease model (late knockdown). hESC-CMs expressed the apelin signalling system at a similar level to the adult heart. Early apelin receptor knockdown decreased cardiomyocyte differentiation efficiency and prolonged voltage sensing, associated with asynchronous contraction. Late apelin receptor knockdown had detrimental consequences on 3D engineered heart tissue contractile properties, decreasing contractility and increasing stiffness. CONCLUSIONS: We have successfully knocked down the apelin receptor, using an inducible system, to demonstrate a key role in hESC-CM differentiation. Knockdown in 3D engineered heart tissues recapitulated the phenotype of apelin receptor down-regulation in a failing heart, providing a potential platform for modelling heart failure and testing novel therapeutic strategies.

Trans-myocardial Extraction of Endothelin-1 Correlates with Increased Microcirculatory Resistance following Percutaneous Coronary Intervention.

Objective: Coronary microvascular dysfunction (CMD) can complicate successful percutaneous coronary intervention (PCI). The potent endogenous vasoconstrictor peptide Endothelin-1 (ET-1) may be an important mediator. To investigate the mechanism, we sought to define the peri-procedural trans-myocardial gradient (TMG-coronary sinus minus aortic root levels) of ET-1 and its precursor peptide - Big ET-1. We then assessed correlation with pressure-wire indices of CMD: coronary flow reserve (CFR) and index of microvascular resistance (IMR). Methods: Paired blood samples from the guide catheter and coronary sinus were collected before and after pressure-wire-guided PCI from patients with stable angina. Plasma was analysed using a specific enzyme-linked immunosorbent assay for quantification of ET-1 peptides and correlated with pressure-wire data. Non normally distributed continuous variables are presented as median [IQR]. Results: ET-1 and Big ET-1 increased post-PCI in the aorta (ET-1: 0.98 [0.76-1.26] pg/ml to 1.20 [1.03-1.67] pg/ml, P < 0.001 and Big ET-1: 2.74 [1.78-2.50] pg/ml to 3.36 [2.33-3.97] pg/ml, P < 0.001) and coronary sinus (ET-1: 1.00 [0.81-1.28] pg/ml to 1.09 [0.91-1.30] pg/ml, P = 0.03 and Big ET-1: 2.89 [1.95-3.83] pg/ml to 3.56 [2.66-4.83] pg/ml, P = 0.01). TMG of ET-1 shifted negatively compared with baseline following PCI reflecting significantly increased extraction (0.03 [-0.12-0.17] pg/ml pre-PCI versus -0.16 [-0.36-0.07] pg/ml post-PCI, P = 0.01). Increased ET-1 trans-myocardial extraction correlated with higher IMR (Pearson's r = 0.293, P = 0.02) and increased hyperemic transit time (Pearson's r = 0.333, P < 0.01). In subgroup analysis, mean ET-1 trans-myocardial extraction was higher amongst patients with high IMR compared with low IMR (0.73 pg/ml, SD:0.78 versus 0.17 pg/ml, SD:0.42, P = 0.02). There was additionally a numerical trend towards increased ET-1 trans-myocardial extraction in subgroups of patients with low CFR and in patients with Type 4a Myocardial Infarction, albeit not reaching statistical significance. Conclusions: Circulating ET-1 increases post-PCI and upregulated ET-1 trans-myocardial extraction contributes to increased microcirculatory resistance.

Apelin is expressed throughout the human kidney, is elevated in chronic kidney disease & associates independently with decline in kidney function.

AIMS: Chronic kidney disease (CKD) is common and cardiovascular disease (CVD) is its commonest complication. The apelin system is a potential therapeutic target for CVD but data relating to apelin in CKD are limited. We examined expression of the apelin system in human kidney, and investigated apelin and Elabela/Toddler (ELA), the endogenous ligands for the apelin receptor, in patients with CKD. METHODS: Using autoradiography, immunohistochemistry and enzyme-linked immunosorbent assay, we assessed expression of apelin, ELA and the apelin receptor in healthy human kidney, and measured plasma apelin and ELA in 155 subjects (128 patients with CKD, 27 matched controls) followed up for 5 years. Cardiovascular assessments included blood pressure, arterial stiffness (pulse wave velocity) and brachial artery flow-mediated dilation. Surrogate markers of endothelial function (plasma asymmetric dimethylarginine and endothelin-1) and inflammation (C-reactive protein and interleukin-6) were measured. RESULTS: The apelin system was expressed in healthy human kidney, throughout the nephron. Plasma apelin concentrations were 60% higher in women than men (6.48 [3.62-9.89] vs. 3.95 [2.02-5.85] pg/mL; P < .0001), and increased as glomerular filtration rate declined (R = -0.41, P < .0001), and albuminuria rose (R = 0.52, P < .0001). Plasma apelin and ELA were associated with vascular dysfunction. Plasma apelin associated independently with a 50% decline in glomerular filtration rate at 5 years. CONCLUSION: We show for the first time that the apelin system is expressed in healthy human kidney. Plasma apelin is elevated in CKD and may be a potential biomarker of risk of decline in kidney function. Clinical studies exploring the therapeutic potential of apelin agonism in CKD are warranted.

Endothelin-1 is increased in the plasma of patients hospitalised with Covid-19.

Virus induced endothelial dysregulation is a well-recognised feature of severe Covid-19 infection. Endothelin-1 (ET-1) is the most highly expressed peptide in endothelial cells and a potent vasoconstrictor, thus representing a potential therapeutic target. ET-1 plasma levels were measured in a cohort of 194 Covid-19 patients stratified according to the clinical severity of their illness. Hospitalised patients, including those who died and those developing acute myocardial or kidney injury, had significantly elevated ET-1 plasma levels during the acute phase of infection. The results support the hypothesis that endothelin receptor antagonists may provide clinical benefit for certain Covid-19 patients.

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.

In vitro metabolism of synthetic Elabela/Toddler (ELA-32) peptide in human plasma and kidney homogenates analyzed with mass spectrometry and validation of endogenous peptide quantification in tissues by ELISA.

BACKGROUND: Elabela/Toddler (ELA) is a novel endogenous ligand of the apelin receptor, whose signalling has emerged as a therapeutic target, for example, in cardiovascular disease and cancer. Shorter forms of ELA-32 have been predicted, including ELA-21 and ELA-11, but metabolism and stability of ELA-32 in humans is poorly understood. We, therefore, developed an LC-MS/MS assay to identify ELA-32 metabolites in human plasma and tissues. METHOD: Human kidney homogenates or plasma were incubated at 37 °C with ELA-32 and aliquots withdrawn over 2-4 h into guanidine hydrochloride. Proteins were precipitated and supernatant solid-phase extracted. Peptides were extracted from coronary artery, brain and kidney by immunoprecipitation or solid-phase extraction following acidification. All samples were reduced and alkylated before analysis on an Orbitrap mass spectrometer in high and nano flow mode. RESULTS: The half-life of ELA-32 in plasma and kidney were 47.2 ±â€¯5.7 min and 44.2 ±â€¯3 s, respectively. Using PEAKS Studio and manual data analysis, the most important fragments of ELA-32 with potential biological activity identified were ELA-11, ELA-16, ELA-19 and ELA-20. The corresponding fragments resulting from the loss of C-terminal amino acids were also identified. Endogenous levels of these peptides could not be measured, as ELA peptides are prone to oxidation and poor chromatographic peaks. CONCLUSIONS: The relatively long ELA plasma half-life observed and identification of a potentially more stable fragment, ELA-16, may suggest that ELA could be a better tool compound and novel template for the development of new drugs acting at the apelin receptor.

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.

Plasma levels of apelin are reduced in patients with liver fibrosis and cirrhosis but are not correlated with circulating levels of bone morphogenetic protein 9 and 10.

BACKGROUND: The peptide apelin is expressed in human healthy livers and is implicated in the development of hepatic fibrosis and cirrhosis. Mutations in the bone morphogenetic protein receptor type II (BMPR-II) result in reduced plasma levels of apelin in patients with heritable pulmonary arterial hypertension. Ligands for BMPR-II include bone morphogenetic protein 9 (BMP9), highly expressed in liver, and BMP10, expressed in heart and to a lesser extent liver. However, it is not known whether reductions in BMP9 and/or BMP10, with associated reduction in BMPR-II signalling, correlate with altered levels of apelin in patients with liver fibrosis and cirrhosis. METHODS: Plasma from patients with liver fibrosis (n = 14), cirrhosis (n = 56), and healthy controls (n = 25) was solid-phase extracted using a method optimised for recovery of apelin, which was measured by ELISA. RESULTS: Plasma apelin was significantly reduced in liver fibrosis (8.3 ± 1.2 pg/ml) and cirrhosis (6.5 ± 0.6 pg/ml) patients compared with controls (15.4 ± 2.0 pg/ml). There was no obvious relationship between apelin and BMP 9 or BMP10 previously measured in these patients. Within the cirrhotic group, there was no significant correlation between apelin levels and disease severity scores, age, sex, or treatment with ß-blockers. CONCLUSIONS: Apelin was significantly reduced in plasma of patients with both early (fibrosis) and late-stage (cirrhosis) liver disease. Fibrosis is more easily reversible and may represent a potential target for new therapeutic interventions. However, it remains unclear whether apelin signalling is detrimental in liver disease or is beneficial and therefore, whether an apelin antagonist or agonist have clinical use.

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 ß-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.

Apelin peptides linked to anti-serum albumin domain antibodies retain affinity in vitro and are efficacious receptor agonists in vivo.

The apelin receptor is a potential target in the treatment of heart failure and pulmonary arterial hypertension where levels of endogenous apelin peptides are reduced but significant receptor levels remain. Our aim was to characterise the pharmacology of a modified peptide agonist, MM202, designed to have high affinity for the apelin receptor and resistance to peptidase degradation and linked to an anti-serum albumin domain antibody (AlbudAb) to extend half-life in the blood. In competition, binding experiments in human heart MM202-AlbudAb (pKi  = 9.39 ± 0.09) bound with similar high affinity as the endogenous peptides [Pyr1 ]apelin-13 (pKi  = 8.83 ± 0.06) and apelin-17 (pKi  = 9.57 ± 0.08). [Pyr1 ]apelin-13 was tenfold more potent in the cAMP (pD2  = 9.52 ± 0.05) compared to the ß-arrestin (pD2  = 8.53 ± 0.03) assay, whereas apelin-17 (pD2  = 10.31 ± 0.28; pD2  = 10.15 ± 0.13, respectively) and MM202-AlbudAb (pD2  = 9.15 ± 0.12; pD2  = 9.26 ± 0.03, respectively) were equipotent in both assays, with MM202-AlbudAb tenfold less potent than apelin-17. MM202-AlbudAb bound to immobilised human serum albumin with high affinity (pKD  = 9.02). In anaesthetised, male Sprague Dawley rats, MM202-AlbudAb (5 nmol, n = 15) significantly reduced left ventricular systolic pressure by 6.61 ± 1.46 mm Hg and systolic arterial pressure by 14.12 ± 3.35 mm Hg and significantly increased cardiac contractility by 533 ± 170 mm Hg/s, cardiac output by 1277 ± 190 RVU/min, stroke volume by 3.09 ± 0.47 RVU and heart rate by 4.64 ± 2.24 bpm. This study demonstrates that conjugating an apelin mimetic peptide to the AlbudAb structure retains receptor and in vivo activity and may be a new strategy for development of apelin peptides as therapeutic agents.

Development and validation of an LC-MS/MS method for detection and quantification of in vivo derived metabolites of [Pyr1]apelin-13 in humans.

[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.

Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] peptides that improve diet induced obesity are G protein biased ligands at the apelin receptor.

BACKGROUND: Apelin signalling pathways have important cardiovascular and metabolic functions. Recently, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)], were reported to function independent of the apelin receptor in vivo to produce beneficial metabolic effects without modulating blood pressure. We aimed to show that these peptides bound to the apelin receptor and to further characterise their pharmacology in vitro at the human apelin receptor. METHODS: [Pyr1]apelin-13 saturation binding experiments and competition binding experiments were performed in rat and human heart homogenates using [125I]apelin-13 (0.1 nM), and/or increasing concentrations of apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] (50pM-100µM). Apelin-36 and its analogues apelin-36-[F36A], apelin-36-[L28A], apelin-36-[L28C(30kDa-PEG)], apelin-36-[A28 A13] and [40kDa-PEG]-apelin-36 were tested in forskolin-induced cAMP inhibition and ß-arrestin assays in CHO-K1 cells heterologously expressing the human apelin receptor. Bias signaling was quantified using the operational model for bias. RESULTS: In both species, [Pyr1]apelin-13 had comparable subnanomolar affinity and the apelin receptor density was similar. Apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] competed for binding of [125I]apelin-13 with nanomolar affinities. Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to apelin-36 at recruiting ß-arrestin. Bias analysis suggested that these peptides were G protein biased. Additionally, [40kDa-PEG]-apelin-36 and apelin-36-[F36A] retained nanomolar potencies in both cAMP and ß-arrestin assays whilst apelin-36-[A13 A28] exhibited a similar profile to apelin-36-[L28C(30kDa-PEG)] in the ß-arrestin assay but was more potent in the cAMP assay. CONCLUSIONS: Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the apelin receptor, suggesting that the apelin receptor is an important therapeutic target in metabolic diseases.

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 ß-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.

Detection of Atherosclerotic Inflammation by 68Ga-DOTATATE PET Compared to [18F]FDG PET Imaging.

BACKGROUND: Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography ([18F]FDG PET), [18F]FDG lacks cell specificity, and coronary imaging is unreliable because of myocardial spillover. OBJECTIVES: This study tested the efficacy of gallium-68-labeled DOTATATE (68Ga-DOTATATE), a somatostatin receptor subtype-2 (SST2)-binding PET tracer, for imaging atherosclerotic inflammation. METHODS: We confirmed 68Ga-DOTATATE binding in macrophages and excised carotid plaques. 68Ga-DOTATATE PET imaging was compared to [18F]FDG PET imaging in 42 patients with atherosclerosis. RESULTS: Target SSTR2 gene expression occurred exclusively in "proinflammatory" M1 macrophages, specific 68Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo 68Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval [CI]: 0.28 to 0.99; p = 0.02). 68Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBRmax) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range [IQR]: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003). 68Ga-DOTATATE mTBRmax predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve [ROC AUC]: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and [18F]FDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001). [18F]FDG mTBRmax differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial [18F]FDG spillover rendered coronary [18F]FDG scans uninterpretable in 27 patients (64%). Coronary 68Ga-DOTATATE PET scans were readable in all patients. CONCLUSIONS: We validated 68Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that 68Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than [18F]FDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography [VISION]; NCT02021188).