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©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4
Azhar Maqbool, Emma J Spary, Iain W Manfield, Michaela Ruhmann, Lorena Zuliani-Alvarez, Filomena O Gamboa-Esteves, Karen E Porter, Mark J Drinkhill, Kim S Midwood, Neil A Turner
Azhar Maqbool, Emma J Spary, Karen E Porter, Mark J Drinkhill, Neil A Turner, Division of Cardiovascular and Diabetes Research, Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Leeds LS2 9JT, United Kingdom
Iain W Manfield, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
Michaela Ruhmann, Lorena Zuliani-Alvarez, Kim S Midwood, Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom
Filomena O Gamboa-Esteves, Leeds Institute of Cancer and Pathology, Leeds Teaching Hospitals NHS Trust, Leeds LS9 7TF, United Kingdom
Author contributions: Maqbool A performed the majority of the experiments and manuscript preparation; Spary EJ performed the statistical analysis; Manfield IW was involved in purification of the Tenascin C; Ruhmann M, Zuliani-Alvarez L and Midwood KS were equally involved with the synthesis and purification of the Tenascin C recombinants; Porter KE supplied the human cardiac fibroblasts; Drinkhill MJ generated the mouse infarct model; Gamboa-Esteves FO performed the histology, immunohistochemistry and image capture; Turner NA performed some of the experiments and was involved in experimental design and manuscript preparation.
Supported by The British Heart Foundation, No. CH/92005.
Institutional review board statement: All work on human tissue was carried out with local ethical committee approval (reference number: 01/040 - documents attached) and informed patient consent. All investigations conformed to the principles outlined in the Declaration of Helsinki). All procedures involving animals were carried out in accordance with the Home Office Animals (Scientific Procedures) Act 1986 and the University of Leeds Animal Welfare and Ethical Committee.
Institutional animal care and use committee statement: All procedures involving animals were carried out in accordance with the Home Office Animals (Scientific Procedures) Act 1986 and the University of Leeds Animal Welfare and Ethical Committee.
Conflict-of-interest statement: None.
Data sharing statement: Complete dataset and statistical analyses available from the corresponding author at cvsam@leeds.ac.uk.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Azhar Maqbool, PhD, Lecturer in Cardiovascular Medicine, Division of Cardiovascular and Diabetes Research, Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Clarendon Way, Leeds LS2 9JT, United Kingdom.
cvsam@leeds.ac.uk
Telephone: +44-113-3434171 Fax: +44-113-3434803
Received: March 3, 2016
Peer-review started: March 4, 2016
First decision: March 22, 2016
Revised: April 4, 2016
Accepted: April 14, 2016
Article in press: April 18, 2016
Published online: May 26, 2016
Processing time: 77 Days and 15.3 Hours
AIM: To investigate the effect of Tenascin C (TNC) on the expression of pro-inflammatory cytokines and matrix metalloproteinases in human cardiac myofibroblasts (CMF).
METHODS: CMF were isolated and cultured from patients undergoing coronary artery bypass grafting. Cultured cells were treated with either TNC (0.1 μmol/L, 24 h) or a recombinant protein corresponding to different domains of the TNC protein; fibrinogen-like globe (FBG) and fibronectin type III-like repeats (TNIII 5-7) (both 1 μmol/L, 24 h). The expression of the pro-inflammatory cytokines; interleukin (IL)-6, IL-1β, TNFα and the matrix metalloproteinases; MMPs (MMP1, 2, 3, 9, 10, MT1-MMP) was assessed using real time RT-PCR and western blot analysis.
RESULTS: TNC increased both IL-6 and MMP3 (P < 0.01) mRNA levels in cultured human CMF but had no significant effect on the other markers studied. The increase in IL-6 mRNA expression was mirrored by an increase in protein secretion as assessed by enzyme-linked immunosorbant assay (P < 0.01). Treating CMF with the recombinant protein FBG increased IL-6 mRNA and protein (P < 0.01) whereas the recombinant protein TNIII 5-7 had no effect. Neither FBG nor TNIII 5-7 had any significant effect on MMP3 expression. The expression of toll-like receptor 4 (TLR4) in human CMF was confirmed by real time RT-PCR, western blot and immunohistochemistry. Pre-incubation of cells with TLR4 neutralising antisera attenuated the effect of both TNC and FBG on IL-6 mRNA and protein expression.
CONCLUSION: TNC up-regulates IL-6 expression in human CMF, an effect mediated through the FBG domain of TNC and via the TLR4 receptor.
Core tip: Tenascin C (TNC) is transiently expressed in cardiac tissue following acute myocardial infarction (MI) and MI patients with higher serum TNC levels have worse long term prognosis. This suggests that TNC is important in ventricular remodelling, although a functional role in this process is unclear. We report that TNC stimulates interleukin-6 synthesis in cardiac myofibroblasts, an effect mediated by toll-like receptor 4. As a growing body of evidence suggests that prolongation of the post-infarction inflammatory response results in worse remodelling and dysfunction this important observation may in part explain the mechanism by which TNC induces maladaptive ventricular remodelling following MI.
