Macrophage hypoxia signaling regulates cardiac fibrosis via Oncostatin M

The fibrogenic response in tissue-resident fibroblasts is determined by the balance between activation and repression signals from the tissue microenvironment. While the molecular pathways by which transforming growth factor-1 (TGF-β1) activates pro-fibrogenic mechanisms have been extensively studied and are recognized critical during fibrosis development, the factors regulating TGF-β1 signaling are poorly understood. Here we show that macrophage hypoxia signaling suppresses excessive fibrosis in a heart via oncostatin-m (OSM) secretion. During cardiac remodeling, Ly6Chi monocytes/macrophages accumulate in hypoxic areas through a hypoxia-inducible factor (HIF)-1α dependent manner and suppresses cardiac fibroblast activation. As an underlying molecular mechanism, we identify OSM, part of the interleukin 6 cytokine family, as a HIF-1α target gene, which directly inhibits the TGF-β1 mediated activation of cardiac fibroblasts through extracellular signal-regulated kinase 1/2-dependent phosphorylation of the SMAD linker region. These results demonstrate that macrophage hypoxia signaling regulates fibroblast activation through OSM secretion in vivo.

Data represent the transthoracic echocardiography data in unoperated control (cont) (n = 11) and mHIF-1 CKO (n = 9). Ejection fraction, left ventricular end-diastolic dimension (LVDd), left ventricular dimeter at end systole (LVDs), thickness of interventricular septum (IVSth) and thickness of posterior LV wall (PWth) are measured. The Mann-Whitney U test was used to compare differences between cont and mHIF-1 CKO mice. Data show the mean and the standard deviation (error bar). n.s., not statistically significant. The average number of CD31 positive cells within 5 fields were counted 14 days after TAC operation. Control (cont) (n = 5) and mHIF-1 CKO (n = 5). Mouse CD31 antibody (DIA-310; diluted 1:200, Dianova, Hamburg, Germany) was used for the CD31 stainig in mouse heart tissues. The Mann-Whitney U test was used to compare differences between cont and mHIF-1 CKO. Data show the mean and the standard deviation (error bar). n.s., not statistically significant.
The average number of TUNEL positive cells within 5 fields were counted 3 days after TAC operation. Control (cont) (n = 5) and mHIF-1 CKO (n = 5). Cardio TACS in situ apoptosis detection kit (R & D systems) was used for the TUNEL staining. The Mann-Whitney U test was used to compare differences between cont and mHIF-1 CKO. Data show the mean and the standard deviation (error bar). n.s., not statistically significant. Data represent the transthoracic echocardiography data 28 days after TAC operation in control (cont) (n = 13) and mHIF-1 CKO mice (n = 16). Left ventricular end-diastolic dimension (LVDd), left ventricular dimeter at end systole (LVDs), thickness of interventricular septum (IVSth) and thickness of posterior LV wall (PWth) are measured. The Mann-Whitney U test was used to compare differences between cont and mHIF-1 CKO mice. Data show the mean and the standard deviation (error bar). n.s., not statistically significant. *, p < 0.05.
Clodoronate liposomes (200 l) and control liposomes (200 l) were injected intravenously 2 days after TAC operation. Masson's trichrome staining was performed using cardiac tissues of mHIF-1 CKO mice 14 days after TAC operation. Fibrotic area was calculated compared to the total surface are (SA). The Mann-Whitney U test was used to compare differences between control liposomes (n = 6) and clodronate liposomes (n = 9) groups. Data show the mean and the standard deviation (error bar). n.s., not statistically significant.
Data represent the transthoracic echocardiography data 42 days after TAC operation in control (cont) (n = 8) and mHIF-1 mice (n = 10). Ejection fraction, left ventricular end-diastolic dimension (LVDd), left ventricular dimeter at end systole (LVDs), thickness of interventricular septum (IVSth) and thickness of posterior LV wall (PWth) are measured. The Mann-Whitney U test was used to compare differences between cont and mHIF-1 CKO mice. Data show the mean and the standard deviation (error bar). *, p < 0.05.
The transcript levels of Osm in bone marrow-derived macrophages (BMDMs) were assessed by quantitative PCR. Bone marrow cells were collected from tibia of adult male mice at the age of 6-8 weeks. Monocyte-colony stimulating factor (M-CSF) was added at day 0 (40 ng per ml), day 3 (40 ng per ml) and day 6 (20 ng per ml). M-CSF was purchased from R & D Systems (Minneapolis, MN, USA). We exposed BMDMs to hypoxia (1% O2) or normoxia and collected total RNA at 12 h time point. Data show the mean and the SD of technical triplicates from a representative experiment. Two-tailed t-test with Welch's correction was used for the statistical analysis (t = 4.256, df = 2.131). *, p < 0.05.
The effect of each hypoxic inducible secretory factor in fibroblasts activation was examined (20 ng per ml for HB-EGF, TIMP1, CXCL1, MANF, JAG1 and 10 ng per ml for OSM). Thirty minutes after pretreatment with hypoxia inducible secretory factors, C3H/10T1/2 cells were stimulated with TGF-1 (2.5 ng per ml, 12 h) and the relative expression of SMA mRNA was calculated. Myocardial infarction model was performed as follows. Mice were intubated and ventilated. After exposing the heart at the fourth left intercostal space, the left coronary artery was permanently ligated with an 8-0 nylon.Fibrotic area was calculated compared to the total surface area (SA) using cardiac tissues 14 days after myocardial infarction operated mice. The Mann-Whitney U test was used to compare differences between cont (n = 8) and mHIF-1 CKO (n = 7). Data show the mean and the standard deviation (error bar). *, p < 0.05.
After pretreatment with the OSM (10 ng per ml) or IL6 (20 ng per ml), primary cariac fibroblasts were stimulated with TGF-1 (2.5 ng per ml, 12 h) and the relative expression level of SMA mRNA was calculated. Data show the mean and the SD of technical triplicates from a representative experiment. The one-way ANOVA and Dunnett's multiple comparisons test was used for the statistical analysis (F (2, 6) = 197.2). *, p < 0.05.
Co-transfection of HIF-1 and aryl hydrocarbon receptor nuclear translocator (ARNT) significantly activated the promoter activity of pGL3mOSM(-1023 to +129)/HRE. Data show the mean and the SD of technical triplicates from a representative experiment. The two-way ANOVA and Sidak's multiple comparison test was performed for the statistical analysis (F (1, 8) = 49.37). n.s., not statistically significant. *, p < 0.05 vs pcDNA3. Construction of reporter plasmids. phHIF-1 and phARNT were generated as described 1 . Murine liver genomic DNA was isolated using Wizard Genomic DNA Purification Kit (Promega) and used for construction of Osm reporter plasmids. A fragment of the promoter region of Osm (-1023 to +129bp relative to TSS (transcription start site)) was amplified using a pair of primers (forward;5'-GATCGCAGATCTCGAACTGGGTCCTGGTACTCTGGC-3', reverse;5'-CTAGCCCGGGCTCGATGCTGTGGCTTCCAAGCATGGC-3'). PCR product was subcloned into the Xho1 site of the promoter in pGL3-Basic vector (Promega) using In-Fusion HD Cloning Kit (Z9633N, TaKaRa Clontech, Japan), yielding pGL3mOSM(-1023 to +129). A fragment containing the HRE region of Osm (-4151 to -3772 bp relative to TSS) was amplified using a pair of primers (forward;5'-GGATCCAGCCTCAGCAGAGCCAGTCCGA -3', reverse;5'-GGATCCGGTTGGCTATGGGGACAGGAGTG -3') and inserted at BamH1 site of the enhancer in pGL3mOSM(-1023 to +129), yielding pGL3mOSM(-1023 to +129)/HRE. The luciferase assays were performed by transfecting reporter constructs into COS7 cells using Fugene HD (Promega, Madison, WI, USA) according to manufacturer's instrument. The CMV-beta-galactosidase plasmid was co-transfected as an internal control for transfection efficiency. The cells were harvested using passive lysis buffer (Promega) at 48 h after transfection and assayed for the luciferase activity by means of the Dual-Glo Luciferase assay system (Promega, Madison, WI, USA) and a luminometer (

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After pretreatment with OSM (10 ng per ml, 30 min) in the presence of U0126 (20 M, 60 min) or DMSO, primary cardiac fibroblasts were stimulated with TGF-1 (2.5 ng per ml, 12 h) and the relative expression level of SMA mRNA was calculated. Data show the mean and the SD of technical triplicates from a representative experiment. Two-tailed t-test with Welch's correction was used for the statistical analysis (t = 6.142, df = 3.924