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      Bromodomain and extra‐terminal protein mimic JQ1 decreases inflammation in human vascular endothelial cells: Implications for pulmonary arterial hypertension

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          ABSTRACT

          Background and objective

          Nuclear factor kappa B ( NF‐kB)‐mediated inflammatory gene expression and vascular endothelial cell proliferation/remodelling are implicated in the pathophysiology of the fatal disease, pulmonary arterial hypertension ( PAH). Bromodomain and extra‐terminal ( BET) proteins are essential for the expression of a subset of NF‐kB‐induced inflammatory genes. BET mimics including JQ1+ prevent binding of BETs to acetylated histones and down‐regulate the expression of selected genes.

          Methods

          The effects of JQ1+ on the proliferation of primary human pulmonary microvascular endothelial cells ( HPMECs) from healthy subjects were measured by bromodeoxyuridine ( BrdU) incorporation. Cell cycle progression was assessed by flow cytometry; mRNA and protein levels of cyclin‐dependent kinases ( CDKs), inhibitors and cytokines were determined by reverse transcription‐quantitative PCR ( RT‐qPCR), Western blotting or ELISA. Histone acetyltransferase ( HAT) and deacetylase ( HDAC) activities were determined in nuclear extracts from whole lung of PAH and control patients.

          Results

          JQ1+ significantly inhibited IL6 and IL8 ( IL6 and CXCL8) mRNA and protein in HPMECs compared with its inactive enantiomer JQ1−. JQ1+ decreased NF‐kB p65 recruitment to native IL6 and IL8 promoters. JQ1+ showed a concentration‐dependent decrease in HPMEC proliferation compared with JQ1−‐treated cells. JQ1+ induced G1 cell cycle arrest by increasing the expression of the CDK inhibitors (CDKN) 1A (p21 cip) and CDKN2D ( p19 INK4D ) and decreasing that of CDK2, CDK4 and CDK6. JQ1+ also inhibited serum‐stimulated migration of HPMECs. Finally, HAT activity was significantly increased in the lung of PAH patients.

          Conclusion

          Inhibition of BETs in primary HPMECs decreases inflammation and remodelling. BET proteins could be a target for future therapies for PAH.

          Abstract

          Endothelial cell inflammation and proliferation are important in the pathogenesis of pulmonary arterial hypertension ( PAH). Bromodomain and extra‐terminal ( BET) mimics reduced inflammation and cell proliferation in primary human pulmonary vascular endothelial cells. The enhanced histone acetyltransferase ( HAT) activity in PAH suggests that BET mimics may be effective in PAH although experiments in disease cells/tissues are required.

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          Most cited references21

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          Guidelines for the diagnosis and treatment of pulmonary hypertension.

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            Decreased histone deacetylase activity in chronic obstructive pulmonary disease.

            Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation that is greater in patients with advanced disease. We asked whether there is a link between the severity of disease and the reduction in histone deacetylase (HDAC) activity in the peripheral lung tissue of patients with COPD of varying severity. HDAC is a key molecule in the repression of production of proinflammatory cytokines in alveolar macrophages. HDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of specimens of surgically resected lung tissue from nonsmokers without COPD, patients with COPD of varying severity, and patients with pneumonia or cystic fibrosis. Alveolar macrophages from nonsmokers, smokers, and patients with COPD and bronchial-biopsy specimens from nonsmokers, healthy smokers, patients with COPD, and those with mild asthma were also examined. Total RNA extracted from lung tissue and macrophages was used for quantitative reverse-transcriptase-polymerase-chain-reaction assay of HDAC1 through HDAC8 and interleukin-8. Expression of HDAC2 protein was quantified with the use of Western blotting. Histone-4 acetylation at the interleukin-8 promoter was evaluated with the use of a chromatin immunoprecipitation assay. Specimens of lung tissue obtained from patients with increasing clinical stages of COPD had graded reductions in HDAC activity and increases in interleukin-8 messenger RNA (mRNA) and histone-4 acetylation at the interleukin-8 promoter. The mRNA expression of HDAC2, HDAC5, and HDAC8 and expression of the HDAC2 protein were also lower in patients with increasing severity of disease. HDAC activity was decreased in patients with COPD, as compared with normal subjects, in both the macrophages and biopsy specimens, with no changes in HAT activity, whereas HAT activity was increased in biopsy specimens obtained from patients with asthma. Neither HAT activity nor HDAC activity was changed in lung tissue from patients with cystic fibrosis or pneumonia. Patients with COPD have a progressive reduction in total HDAC activity that reflects the severity of the disease. Copyright 2005 Massachusetts Medical Society.
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              RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain.

              Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.
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                Author and article information

                Contributors
                s.mumby@imperial.ac.uk
                Journal
                Respirology
                Respirology
                10.1111/(ISSN)1440-1843
                RESP
                Respirology (Carlton, Vic.)
                John Wiley & Sons, Ltd (Chichester, UK )
                1323-7799
                1440-1843
                18 August 2016
                January 2017
                : 22
                : 1 ( doiID: 10.1111/resp.2017.22.issue-1 )
                : 157-164
                Affiliations
                [ 1 ] Vascular BiologyImperial College London LondonUK
                [ 2 ] Airway Disease Section, National Heart and Lung InstituteImperial College London LondonUK
                [ 3 ] Faculty of MedicineSouth Paris University ClamartFrance
                [ 4 ] Pulmonary Hypertension: Pathophysiology and Therapeutic InnovationINSERM Research Unit 999 ClamartFrance
                [ 5 ] Pulmonary Resuscitation Respiratory and Service, National Reference Centre for Pulmonary Hypertension Severe, Assistance Publique Hôpitaux de Paris Hôpital Antoine Béclère ParisFrance
                Author notes
                [*] [* ]Correspondence: Sharon Mumby, Airways Disease Section, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK. Email: s.mumby@ 123456imperial.ac.uk
                Article
                RESP12872
                10.1111/resp.12872
                5215513
                27539364
                ad597009-9256-4dec-80d9-a7a48119f3e6
                © 2016 The Authors. Respirology published by John Wiley & Sons Australia, Ltd on behalf of Asian Pacific Society of Respirology

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 24 September 2015
                : 17 April 2016
                : 31 May 2016
                Page count
                Figures: 0, Tables: 0, Pages: 8, Words: 2748
                Funding
                Funded by: British Heart Foundation
                Award ID: PG/14/27/30679
                Funded by: Royal Brompton Hospital Charitable Fund
                Funded by: NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton
                Funded by: Harefield NHS Foundation Trust
                Funded by: Wellcome Trust Programme
                Award ID: 093080/Z/10/Z
                Funded by: Imperial College London
                Funded by: National Funding Agency for Research
                Award ID: ANR‐JSV1‐001
                Funded by: European Respiratory Society Long‐Term Fellowship
                Categories
                Original Article
                ORIGINAL ARTICLES
                Pulmonary Vascular Disease
                Custom metadata
                2.0
                resp12872
                resp12872-hdr-0001
                January 2017
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.0.0 mode:remove_FC converted:05.01.2017

                Respiratory medicine
                bromodomain and extra‐terminal proteins,human pulmonary microvascular endothelial cells,inflammation,proliferation,pulmonary hypertension

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