title: Myocardial fibrosis induced by exposure to subclinical lipopolysaccharide is associated with decreased miR-29c and enhanced NOX2 expression in mice.
abstract: BACKGROUND
  Exposure to subclinical levels of lipopolysaccharide (LPS) occurs commonly and is seemingly well tolerated. However, recurrent LPS exposure induces cardiac fibrosis over 2 to 3 months in a murine model, not mediated by the renin-angiotensin system. Subclinical LPS induces cardiac fibrosis by unique mechanisms.
  
  
  METHODS
  In C57/Bl6 mice, LPS (10 mg/kg) or saline (control) were injected intraperitoneally once a week for 1-4 weeks. Mice showed no signs of distress, change in activity, appetite, or weight loss. Mice were euthanized after 3 days, 1, 2, or 4 weeks to measure cardiac expression of fibrosis-related genes and potential mediators (measured by QRT-PCR), including micro-RNA (miR) and NADPH oxidase (NOX). Collagen fraction area of the left ventricle was measured with picrosirius red staining. Cardiac fibroblasts isolated from adult mouse hearts were incubated with 0, 0.1, 1.0 or 10 ng/ml LPS for 48 hours.
  
  
  RESULTS
  Cardiac miR expression profiling demonstrated decreased miR-29c after 3 and 7 days following LPS, which were confirmed by QRT-PCR. The earliest changes in fibrosis-related genes and mediators that occurred 3 days after LPS were increased cardiac expression of TIMP-1 and NOX-2 (but not of NOX-4). This persisted at 1 and 2 weeks, with additional increases in collagen Iα1, collagen IIIα1, MMP2, MMP9, TIMP1, TIMP2, and periostin. There was no change in TGF-β or connective tissue growth factor. Collagen fraction area of the left ventricle increased after 2 and 4 weeks of LPS. LPS decreased miR-29c and increased NOX-2 in isolated cardiac fibroblasts.
  
  
  CONCLUSIONS
  Recurrent exposure to subclinical LPS induces cardiac fibrosis after 2-4 weeks. Early changes 3 days after LPS were decreased miR-29c and increased NOX2 and TIMP1, which persisted at 1 and 2 weeks, along with widespread activation of fibrosis-related genes. Decreased miR-29c and increased NOX2, which induce cardiac fibrosis in other conditions, may uniquely mediate LPS-induced cardiac fibrosis.
authors.affiliation: Cardiology Section, Department of Medicine and Research Service, Veterans Administration San Diego Healthcare System, San Diego, California, United States of America; University of California San Diego, San Diego, California, United States of America.
authors.forename: Wilbur Y W
authors.initials: WY
authors.lastname: Lew
authors.affiliation: Cardiology Section, Department of Medicine and Research Service, Veterans Administration San Diego Healthcare System, San Diego, California, United States of America; University of California San Diego, San Diego, California, United States of America.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Bayna
authors.affiliation: Cardiology Section, Department of Medicine and Research Service, Veterans Administration San Diego Healthcare System, San Diego, California, United States of America; University of California San Diego, San Diego, California, United States of America.
authors.forename: Erminia
authors.initials: E
authors.lastname: Dalle Molle
authors.affiliation: Cardiology Section, Department of Medicine and Research Service, Veterans Administration San Diego Healthcare System, San Diego, California, United States of America; University of California San Diego, San Diego, California, United States of America.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Contu
authors.affiliation: Humanitas Research Hospital, University of Milan, Rozzano, Milan, Italy.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Condorelli
authors.affiliation: Cardiology Section, Department of Medicine and Research Service, Veterans Administration San Diego Healthcare System, San Diego, California, United States of America; University of California San Diego, San Diego, California, United States of America.
authors.forename: Tong
authors.initials: T
authors.lastname: Tang
mesh_terms: D000818:Animals; D009202:Cardiomyopathies / Q000139:chemically induced*; D015815:Cell Adhesion Molecules / Q000096:biosynthesis; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis; D004719:Endomyocardial Fibrosis / Q000139:chemically induced*; D017379:Hypertrophy, Left Ventricular / Q000139:chemically induced*; D008070:Lipopolysaccharides; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000096:biosynthesis*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis
chemical_list: D015815:Cell Adhesion Molecules; D024042:Collagen Type I; D008070:Lipopolysaccharides; C547014:MIRN29 microRNA, mouse; D008562:Membrane Glycoproteins; D035683:MicroRNAs; C082838:Postn protein, mouse; C560825:Timp1 protein, mouse; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D020778:Matrix Metalloproteinase 2; C522362:Mmp2 protein, mouse; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse