Fine-mapping of prostate cancer susceptibility loci in a large meta-analysis identifies candidate causal variants

Prostate cancer is a polygenic disease with a large heritable component. A number of common, low-penetrance prostate cancer risk loci have been identified through GWAS. Here we apply the Bayesian multivariate variable selection algorithm JAM to fine-map 84 prostate cancer susceptibility loci, using summary data from a large European ancestry meta-analysis. We observe evidence for multiple independent signals at 12 regions and 99 risk signals overall. Only 15 original GWAS tag SNPs remain among the catalogue of candidate variants identified; the remainder are replaced by more likely candidates. Biological annotation of our credible set of variants indicates significant enrichment within promoter and enhancer elements, and transcription factor-binding sites, including AR, ERG and FOXA1. In 40 regions at least one variant is colocalised with an eQTL in prostate cancer tissue. The refined set of candidate variants substantially increase the proportion of familial relative risk explained by these known susceptibility regions, which highlights the importance of fine-mapping studies and has implications for clinical risk profiling. © 2018 The Author(s).Prostate cancer is a polygenic disease with a large heritable component. A number of common, low-penetrance prostate cancer risk loci have been identified through GWAS. Here we apply the Bayesian multivariate variable selection algorithm JAM to fine-map 84 prostate cancer susceptibility loci, using summary data from a large European ancestry meta-analysis. We observe evidence for multiple independent signals at 12 regions and 99 risk signals overall. Only 15 original GWAS tag SNPs remain among the catalogue of candidate variants identified; the remainder are replaced by more likely candidates. Biological annotation of our credible set of variants indicates significant enrichment within promoter and enhancer elements, and transcription factor-binding sites, including AR, ERG and FOXA1. In 40 regions at least one variant is colocalised with an eQTL in prostate cancer tissue. The refined set of candidate variants substantially increase the proportion of familial relative risk explained by these known susceptibility regions, which highlights the importance of fine-mapping studies and has implications for clinical risk profiling. © 2018 The Author(s).Peer reviewe

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Metabolic hyperemia requires ATP-sensitive K+ channels and H2O2 but not adenosine in isolated mouse hearts

We have previously demonstrated that adenosine-mediated H2O2 production and opening of ATP-sensitive K+ (KATP) channels contributes to coronary reactive hyperemia. The present study aimed to investigate the roles of adenosine, H2O2, and KATP channels in coronary metabolic hyperemia (MH). Experiments were conducted on isolated Langendorffperfused mouse hearts using combined pharmacological approaches with adenosine receptor (AR) knockout mice. MH was induced by electrical pacing at graded frequencies. Coronary flow increased linearly from 14.4 ± 1.2 to 20.6 ± 1.2 ml·min-1·g-1 with an increase in heart rate from 400 to 650 beats/min in wild-type mice. Neither non-selective blockade of ARs by 8-(p-sulfophenyl)theophylline (8-SPT; 50µM) nor selective A2AAR blockade by SCH-58261 (1µM) or deletion affected MH, although resting flow and left ventricular developed pressure were reduced. Combined A2AAR and A2BAR blockade or deletion showed similar effects as 8-SPT. Inhibition of nitric oxide synthesis by N-nitro-L-arginine methyl ester (100µM) or combined 8-SPT administration failed to reduce MH, although resting flows were reduced (by ∼20%). However, glibenclamide (KATP channel blocker, 5µM) decreased not only resting flow (by ∼45%) and left ventricular developed pressure (by ∼36%) but also markedly reduced MH by ∼94%, resulting in cardiac contractile dysfunction. Scavenging of H2O2 by catalase (2,500 U/min) also decreased resting flow (by ∼16%) and MH (by ∼24%) but to a lesser extent than glibenclamide. Our results suggest that while adenosine modulates coronary flow under both resting and ischemic conditions, it is not required for MH. However, H2O2 and KATP channels are important local control mechanisms responsible for both coronary ischemic and metabolic vasodilation.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

CYP-epoxygenases contribute to A2A receptor-mediated aortic relaxation via sarcolemmal KATP channels

Previously, we have shown that A2A adenosine receptor (A2AAR) mediates aortic relaxation via cytochrome P-450 (CYP)-epoxygenases. However, the signaling mechanism is not understood properly. We hypothesized that ATP-sensitive K+ (KATP) channels play an important role in A2AAR-mediated relaxation. Organ bath and Western blot experiments were done using isolated aorta from A2AKO and corresponding wild-type (WT) mice. Aortic rings from WT and A2A knockout (KO) mice were precontracted with submaximal dose of phenylephrine (PE, 10-6 M), and concentration-response curves for pinacidil, cromakalim (nonselective KATP openers), and diazoxide (mitochondrial KATP opener) were obtained. Diazoxide did not have any relaxation effect on PE-precontracted tissues, whereas relaxation to pinacidil (48.09 ± 5.23% in WT vs. 25.41 ± 2.73% in A2AKO; P < 0.05) and cromakalim (51.19 ± 2.05% in WT vs. 38.50 ± 2.26% in A2AKO; P < 0.05) was higher in WT than A2AKO aorta. This suggested the involvement of sarcolemmal rather than mitochondrial KATP channels. Endothelium removal, treatment with SCH 58651 (A2AAR antagonist; 10-6 M), NG-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor) and methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH, CYP-epoxygenases inhibitor; 10-5 M) significantly reduced pinacidil-induced relaxation in WT compared with controls, whereas these treatments did not have any effect in A2AKO aorta. Glibenclamide (KATP channel inhibitor, 10-5 M) blocked 2-p-(2-carboxyethyl)phenethylamino-5′N-ethylcarboxamido adenosine hydrochloride (CGS 21680, A2AAR agonist)-induced relaxation in WT and changed 5′-N-ethylcarboxamide (NECA) (nonselective adenosine analog)-induced response to higher contraction in WT and A2AKO. 5-Hydroxydecanoate (5-HD, mitochondrial KATP channel inhibitor, 10-4 M) had no effect on CGS 21680-mediated response in WT aorta. Our data suggest that A2AAR-mediated vasorelaxation occurs through opening of sarcolemmal KATP channels via CYP-epoxygenases and possibly, nitric oxide, contributing to pinacidil-induced responses. © 2012 the American Physiological Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Contributions of A2A and A2B adenosine receptors in coronary flow responses in relation to the KATP channel using A2B and A2A/2B double-knockout mice

Adenosine plays a role in physiological and pathological conditions, and A2 adenosine receptor (AR) expression is modified in many cardiovascular disorders. In this study, we elucidated the role of the A2BAR and its relationship to the A2AAR in coronary flow (CF) changes using A2B single-knockout (KO) and A2A/2B double-KO (DKO) mice in a Langendorff setup. We used two approaches: 1) selective and nonselective AR agonists and antagonists and 2) A2AKO and A2BKO and A2A/2BDKO mice. BAY 60-6583 (a selective A2B agonist) had no effect on CF in A2BKO mice, whereas it significantly increased CF in wild-type (WT) mice (maximum of 23.3 ± 9 ml·min-1·g-1). 5'-N-ethylcarboxamido adenosine (NECA; a nonselective AR agonist) increased CF in A2BKO mice (maximum of 34.6 ± 4.7 ml·min-1·g-1) to a significantly higher degree compared with WT mice (maximum of 23.1 ± 2.1 ml·min-1·g-1). Also, CGS-21680 (a selective A2A agonist) increased CF in A2BKO mice (maximum of 29 ± 1.9 ml·min-1·g-1) to a significantly higher degree compared with WT mice (maximum of 25.1 ± 2.3 ml·min-1·g-1). SCH-58261 (an A2A-selective antagonist) inhibited the NECA-induced increase in CF to a significantly higher degree in A2BKO mice (19.3 ± 1.6 vs. 0.5 ± 0.4 ml·min-1·g-1) compared with WT mice (19 ± 3.5 vs. 3.6 ± 0.5 ml·min-1·g-1). NECA did not induce any increase in CF in A2A/2BDKO mice, whereas a significant increase was observed in WT mice (maximum of 23.1 ± 2.1 ml·min-1·g-1). Furthermore, the mitochondrial ATP-sensitive K(KATP) channel blocker 5-hydroxydecanoate had no effect on the NECAinduced increase in CF in WT mice, whereas the NECA-induced increase in CF in WT (17.6 ± 2 ml·min-1·g-1), A2AKO (12.5 ± 2.3 ml·min-1·g-1), and A2BKO (16.2 ± 0.8 ml·min-1·g-1) mice was significantly blunted by the KATP channel blocker glibenclamide (to 0.7 ± 0.7, 2.3 ± 1.1, and 0.9 ± 0.4 ml·min-1·g-1, respectively). Also, the CGS-21680-induced (22 ± 2.3 ml·min-1·g-1) and BAY 60-6583-induced (16.4 ± 1.60 ml·min-1·g-1) increase in CF in WT mice was significantly blunted by glibenclamide (to 1.2 ± 0.4 and 1.8 ± 1.2 ml·min-1·g-1, respectively). In conclusion, this is the first evidence supporting the compensatory upregulation of A2AARs in A2BKO mice and demonstrates that both A2AARs and A2BARs induce CF changes through KATP channels. These results identify AR-mediated CF responses that may lead to better therapeutic approaches for the treatment of cardiovascular disorders. © 2011 the American Physiological Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Adenosine increases nasal mucociliary clearance rate in mice through A 2A and A2B adenosine receptors

Objectives/Hypothesis: Mucociliary clearance (MCC) is an important mechanism of host defense in the upper and lower respiratory tract. Impaired MCC plays a critical role in the development and perpetuation of chronic rhinosinusitis (CRS). The aim of this investigation was to determine the influence of adenosine on nasal MCC, and to determine the receptors mediating this physiology in vivo. Study Design: Prospective study using an animal model. Methods: Nasal MCC was measured by whole-nose scintigraphic acquisition in vivo. The effects of both endogenous and exogenous adenosine were investigated in wild-type and adenosine receptor knockout (A 2A-/-, A 2B-/-, A 2A-/-A 2B-/-, and A 1-/- A 3-/-) mice. Results: Exogenous adenosine aerosol robustly enhanced nasal MCC. The augmentation of MCC by adenosine was abolished in mice lacking both A2A and A2B receptors, but remained robust in mice lacking either A2A or A2B. Likewise, basal nasal MCC was reduced in mice lacking both the A2A and A2B receptors, but was statistically identical among wild-type mice and mice lacking either A2A or A2B. Conclusions: These findings indicate that activation of both Gs-coupled adenosine receptors can accelerate nasal MCC. Targeting these receptors may represent a novel therapeutic approach for enhancing MCC in CRS. Laryngoscope, 2012 Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Adenosine A2A receptor and vascular response: role of soluble epoxide hydrolase, adenosine A1 receptor and angiotensin-II

Previously, we have reported that the coronary reactive hyperemic response was reduced in adenosine A2A receptor-null (A2AAR−/−) mice, and it was reversed by the soluble epoxide hydrolase (sEH) inhibitor. However, it is unknown in aortic vascular response, therefore, we hypothesized that A2AAR-gene deletion in mice (A2AAR−/−) affects adenosine-induced vascular response by increase in sEH and adenosine A1 receptor (A1AR) activities. A2AAR−/− mice showed an increase in sEH, AI AR and CYP450-4A protein expression but decrease in CYP450-2C compared to C57Bl/6 mice. NECA (adenosine-analog) and CCPA (adenosine A1 receptor-agonist)-induced dose-dependent vascular response was tested with t-AUCB (sEH-inhibitor) and angiotensin-II (Ang-II) in A2AAR−/− vs. C57Bl/6 mice. In A2AAR−/−, NECA and CCPA-induced increase in dose-dependent vasoconstriction compared to C57Bl/6 mice. However, NECA and CCPA-induced dose-dependent vascular contraction in A2AAR−/− was reduced by t-AUCB with NECA. Similarly, dose-dependent vascular contraction in A2AAR−/− was reduced by t-AUCB with CCPA. In addition, Ang-II enhanced NECA and CCPA-induced dose-dependent vascular contraction in A2AAR−/− with NECA. Similarly, the dose-dependent vascular contraction in A2AAR−/− was also enhanced by Ang-II with CCPA. Further, t-AUCB reduced Ang-II-enhanced NECA and CCPA-induced dose-dependent vascular contraction in A2AAR−/− mice. Our data suggest that the dose-dependent vascular contraction in A2AAR−/− mice depends on increase in sEH, A1AR and CYP4A protein expression.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Involvement of NADPH oxidase in A<inf>2A</inf> adenosine receptor-mediated increase in coronary flow in isolated mouse hearts

Adenosine increases coronary flow mainly through the activation of A2A and A2B adenosine receptors (ARs). However, the mechanisms for the regulation of coronary flow are not fully understood. We previously demonstrated that adenosine-induced increase in coronary flow is in part through NADPH oxidase (Nox) activation, which is independent of activation of either A1 or A3ARs. In this study, we hypothesize that adenosine-mediated increase in coronary flow through Nox activation depends on A2A but not A2BARs. Functional studies were conducted using isolated Langendorff-perfused mouse hearts. Hydrogen peroxide (H2O2) production was measured in isolated coronary arteries from WT, A2AAR knockout (KO), and A2BAR KO mice using dichlorofluorescein immunofluorescence. Adenosine-induced concentration-dependent increase in coronary flow was attenuated by the specific Nox2 inhibitor gp91 ds-tat or reactive oxygen species (ROS) scavenger EUK134 in both WT and A2B but not A2AAR KO isolated hearts. Similarly, the A2AAR selective agonist CGS-21680-induced increase in coronary flow was significantly blunted by Nox2 inhibition in both WT and A2BAR KO, while the A2BAR selective agonist BAY 60-6583-induced increase in coronary flow was not affected by Nox2 inhibition in WT. In intact isolated coronary arteries, adenosine-induced (10 μM) increase in H2O2 formation in both WT and A2BAR KO mice was attenuated by Nox2 inhibition, whereas adenosine failed to increase H2O2 production in A2AAR KO mice. In conclusion, adenosine-induced increase in coronary flow is partially mediated by Nox2-derived H2O2, which critically depends upon the presence of A2AAR.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Interactions between A2A adenosine receptors, hydrogen peroxide, and KATP channels in coronary reactive hyperemia

Myocardial metabolites such as adenosine mediate reactive hyperemia, in part, by activating ATP-dependent K+ (KATP) channels in coronary smooth muscle. In this study, we investigated the role of adenosine A2A and A2B receptors and their signaling mechanisms in reactive hyperemia. We hypothesized that coronary reactive hyperemia involves A2A receptors, hydrogen peroxide (H2O2), and KATP channels. We used A2A and A2B knockout (KO) and A2A/2B double KO (DKO) mouse hearts for Langendorff experiments. Flow debt for a 15-s occlusion was repaid 128 ± 8% in hearts from wild-type (WT) mice; this was reduced in hearts from A2A KO and A2A/2B DKO mice (98 ± 9 and 105 ± 6%; P < 0.05), but not A2B KO mice (123 ± 13%). Patch-clamp experiments demonstrated that adenosine activated glibenclamide-sensitive KATP current in smooth muscle cells from WT and A2B KO mice (90 ± 23% of WT) but not A2A KO or A2A/A2B DKO mice (30 ± 4 and 35 ± 8% of WT; P < 0.05). Additionally, H2O2 activated KATP current in smooth muscle cells (358 ± 99%; P < 0.05). Catalase, an enzyme that breaks down H2O2, attenuated adenosine-induced coronary vasodilation, reducing the percent increase in flow from 284 ± 53 to 89 ± 13% (P < 0.05). Catalase reduced the repayment of flow debt in hearts from WT mice (84 ± 9%; P < 0.05) but had no effect on the already diminished repayment in hearts from A2A KO mice (98 ± 7%). Our findings suggest that adenosine A2A receptors are coupled to smooth muscle KATP channels in reactive hyperemia via the production of H2O2 as a signaling intermediate. © 2013 the American Physiological Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Role of ω-hydroxylase in adenosine-mediated aortic response through MAP kinase using a 2A-receptor knockout mice

Previously, we have shown that A 2A adenosine receptor (A 2AAR) knockout mice (KO) have increased contraction to adenosine. The signaling mechanism(s) for A 2AAR is still not fully understood. In this study, we hypothesize that, in the absence of A 2AAR, ω-hydroxylase (Cyp4a) induces vasoconstriction through mitogen-activated protein kinase (MAPK) via upregulation of adenosine A1 receptor (A 1AR) and protein kinase C (PKC). Organ bath and Western blot experiments were done using isolated aorta from A 2AKO and corresponding wild-type (WT) mice. Isolated aortic rings from WT and A2AKO mice were precontracted with submaximal dose of phenylephrine (10 -6 M), and concentration responses for selective A 1AR, A 2AAR agonists, angiotensin II and cytochrome P-450-epoxygenase, 20-hydroxyeicosatrienoic acid (20- HETE) PKC, PKC-α, and ERK1/2 inhibitors were obtained. 2-p-(2- Carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680, A 2AAR agonist) induced concentration-dependent relaxation in WT, which was blocked by methylsulfonyl-propargyloxyphenylhexanamide (cytochrome P-450-epoxygenase inhibitor; 10 -5 M) and also with removal of endothelium. A 1 agonist, 2-chloro-N 6- cyclopentyladenosine (CCPA) produced higher contraction in A 2AKO aorta than WT (49.2 ± 8.5 vs. 27 ± 5.9% at 10 -6 M, P < 0.05). 20-HETE produced higher contraction in A 2AKO than WT (50.6 ± 8.8 vs. 21.1 ± 3.3% at 10 -7 M, P < 0.05). Contraction to CCPA in WT and A 2AKO aorta was inhibited by PD-98059 (p42/p44 MAPK inhibitor; 10 -6 M), chelerythrine chloride (nonselective PKC blocker; 10 -6 M), Gö-6976 (selective PKC-α inhibitor; 10 -7 M), and HET0016 (20-HETE inhibitor; 10 -5 M). Also, contraction to 20- HETE in WT and A 2AKO aorta was inhibited by PD-98059 and Gö-6976. Western blot analysis indicated the upregulation of A 1AR, Cyp4a, PKC-α, and phosphorylated-ERK1/2 in A 2AKO compared with WT (P < 0.05), while expression of Cyp2c29 was significantly higher in WT. CCPA (10 -6 M) increased the protein expression of PKC-α and phosphorylated-ERK1/2, while HET0016 significantly reduced the CCPA-induced increase in expression of these proteins. These data suggest that, in the absence of A 2AAR, Cyp4a induces vasoconstriction through MAPK via upregulation of A 1AR and PKC-α. © 2012 the American Physiological Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe