C1q/TNF‐related protein‐9 promotes macrophage polarization and improves cardiac dysfunction after myocardial infarction

Figure 1. Adenoviral CTRP9 delivery ameliorates cardiac function on Day 7 post MI. Ad‐CTRP9 or Ad‐GFP was injected into the jugular vein of rats 5 days before MI. (a–c) Effects of adenoviral CTRP9 supplementation on plasma and tissue CTRP9 protein levels by ELISA and western blot analysis. (d) Survival rate according to survival curve analysis (MI, n = 21, Ad‐GFP, n = 18; Ad‐CTRP9, n = 17). (e,f) Cardiac Masson trichrome staining and infarct area (per group, n = 5). (g–i) Quantitative analysis of the LVEDD, EF, and FS of rat hearts pretreated with Ad‐CTRP9 or Ad‐GFP (per group, successively, n = 7, 6, 6, and 7). *p < 0.05 and **p < 0.01, the connection represents a comparison between two groups. CTRP9: C1q/TNF‐related protein‐9; EF: ejection fraction; ELISA: enzyme‐linked immunosorbent assay; FS, fractional shortening; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; GFP: green fluorescent protein; LVEDD: left ventricular end‐diastolic dimension; MI: myocardial infarction [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2. Ad‐CTRP9 treatment enhances anti‐inflammatory effects and mediates M1 to M2 phenotype polarization in the infarct border zone at Day 3 post‐MI. Rats were treated with Ad‐CTRP9 as shown in the above illustration. (a–f) The effect of Ad‐CTRP9 on M1 (iNOS and CD86) and M2 (Arg1 and CD163) macrophage markers by western blot analysis. (g–i) The protein levels of IL‐1 β, IL‐6, and IL‐10 were measured by tissue ELISA. (j) The matrix metalloproteinase (MMP)‐2 mRNA levels were measured by PCR. (k, l) Representative fluorescent immunostaining images after merging (left, n = 4). The hearts were stained with CD68 (green), CD86/CD163 (red), and DAPI (blue). The double‐positive CD86 and CD68 (CD86+/CD68+) areas of M1 macrophages and CD163+/CD68+ M2 macrophages were quantified (right). Data are presented as the mean ± SEM from three independent experiments. *p < 0.05 and **p < 0.01, the connection represents a comparison between two groups. Arg1: arginase1; CTRP9: C1q/TNF‐related protein‐9; DAPI: 4′,6‐diamidino‐2‐phenylindole; ELISA: enzyme‐linked immunosorbent assay; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; iNOS: inducible nitric oxide synthase; MI: myocardial infarction; mRNA:…

Figure 3. CTRP9 promotes M2 macrophage polarization and decreases inflammatory cytokine production in vitro. Peritoneal macrophages (1 × 106 cells/ml per well) were preincubated with different concentrations of gCTRP9 (1, 3, and 5 μg/ml) for 30 min. After stimulation with 100 ng/ml LPS + 20 ng/ml IFN‐γ or treatment with PBS for 24 hr, the cells were collected. (a–i) Representative mRNA (upper panel) and protein (lower panel) expression of specific M1 and M2 markers were evaluated by qRT‐PCR and western blot analyses. (j) Flow cytometry analysis (left) with quantification (right) of iNOS+/F4/80+ and CD163+/F4/80+ macrophages in total macrophages. (k–m) The supernatant protein levels of IL‐1β, IL‐6, and IL‐10 were measured by ELISA. (n) PCR analysis was performed on mRNA for MMP‐2. At least three independent experiments were performed. Data are presented as the mean ± SEM. *p < 0.05 and **p < 0.01 (vs. control), the connection represents a comparison between two groups. Arg1: arginase1; CTRP9: C1q/TNF‐related protein‐9; ELISA: enzyme‐linked immunosorbent assay; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; IFN‐γ: interferon‐γ; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide;…

Figure 4. CTRP9 deficiency enhances the M1 macrophage phenotype. (a–b) mRNA and protein levels of CTRP9 in 4 kinds of cells. (c) Representative fluorescence microscopy images of GFP fluorescence in macrophages after transfection of shRNA and shCTRP9, and corresponding protein levels determined by western blot analysis. (d–h) Protein levels of M1 and M2 macrophage markers after knockdown of CTRP9 at baseline and in LPS + IFN‐γ‐induced macrophages for 24 hr. Data are presented as the mean ± SEM from four independent experiments. *p < 0.05 and **p < 0.01, the connection represents a comparison between two groups. Arg1: arginase1; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; GFP: green fluorescent protein; IFN‐γ: interferon‐γ; LPS: lipopolysaccharide; mRNA: messenger RNA; SEM: standard error of the mean; shRNA: small hairpin RNA [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5. CTRP9 has a similar function to that of IL‐4 and promotes an anti‐inflammatory macrophage phenotype. (a–d) mRNA levels of specific M1 and M2 markers under treatment with gCTRP9 (3 μg/ml), IL‐4(20 ng/ml), or both by RT‐PCR. (e–g) The supernatant protein levels of IL‐1β, IL‐6, and IL‐10 were measured by ELISA. Data are shown as the mean ± SEM from three independent experiments. *p < 0.05 and **p < 0.01, the connection represents a comparison between two groups. Arg1: arginase1; CTRP9: C1q/TNF‐related protein‐9; ELISA: enzyme‐linked immunosorbent assay; IL: interleukin; mRNA: messenger RNA; RT‐PCR: reverse‐transcription polymerase chain reaction; SEM: standard error of the mean

Figure 6. CTRP9 promotes macrophage polarization partly through AMPK‐mediated activation and largely via an NF‐κB‐dependent pathway. (a–c) The effects of Ad‐GFP and Ad‐CTRP9 transfection on the phosphorylation of AMPK and NF‐κB p65 in the infarct border zone at Day 3 post MI by western blot. (d–f) Effects of treatment of macrophages with gCTRP9 (3 μg/ml) at different time points on AMPK (Thr172) and NF‐κB p65 phosphorylation by western blot analysis. (g–i) Western blot analysis assessed the effects of the AMPK inhibitor compound C on pAMPKα and pNF‐κB p65 protein expression in macrophages responding to gCTRP9 by LPS + IFN‐γ stimulation. Cells were treated with compound C (10 mol/l) for 2 hr and then incubated with CTRP9 (3 µg/ml) for 30 min, followed by LPS + IFN‐γ challenge for 24 hr. (j–n) Effects of two inhibitors (compound C or PDTC; 100 μM) on M1 and M2 marker protein expression in macrophages in response to gCTRP9 via LPS + IFN‐γ stimulation using western blot analysis. Data are shown as the mean ± SEM from three independent experiments; *p < 0.05, **p < 0.01 (vs. control group), &,# p < 0.05, and NS = p > 0.05, the connection represents a comparison between two groups. AMPK:…

Figure 7. CTRP9‐mediated downregulation of TLR4‐MyD88 protein expression is associated with AMPK phosphorylation. (a–c) TLR4 and MyD88 protein expression in the infarct border zone at Day 3 post MI with Ad‐GFP and Ad‐CTRP9 pretreatment. (d–f) Effects of TLR4 and MyD88 on macrophages treated with gCTRP9 (3 μg/ml) at different time points by western blot analysis. (g–j) Protein levels of TLR4 and MyD88 after knockdown of CTRP9 in macrophages for 24 hr. (k–m) Effects of compound C on TLR4 and MyD88 protein expression in macrophages responding to gCTRP9 via LPS + IFN‐γ stimulation using western blot analysis. Data are presented as the mean ± SEM from four independent experiments; *p < 0.05 and **p < 0.01, the connection represents a comparison between two groups. AMPK: adenosine monophosphate kinase; CTRP9: C1q/TNF‐related protein‐9; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; GFP: green fluorescent protein; IFN‐γ: interferon‐γ; LPS: lipopolysaccharide; MI: myocardial infarction; MyD88: myeloid differentiation factor 88; NF‐κB: nuclear factor‐κB; SEM: standard error of the mean; shRNA: small hairpin RNA; TLR4: toll‐like receptor 4

Figure 8. gCTRP9 inhibits TLR4/MD2 complex formation. (a) The competitive binding of bio‐LPS and gCTRP9 to the designed LPS trap was studied by using an ELISA‐based assay. The bio‐LPS (1 μg/ml) and LPS (100‐fold dose) emulously combined with the LPS trap (lanes 1 and 2). The ability of three doses (μg/ml) of CTRP9 to competitively bind bio‐LPS to the LPS trap was tested (lanes 3–5). (b) The construction of TLR4 and MD2 fusion proteins was confirmed by western blot analysis. (c) ELISA‐based analysis of the ability of bio‐LPS to bind to gCTRP9. (d) Verification of the ability of gCTRP9 to effectively bind to the plastic plate in the ELISA system. (e, f) Representative western blot (IB) from a coprecipitation (IP) experiment showing the effects of CTRP9 and anti‐MD2 on MD2/TLR4 complex formation (e) and TLR4/MyD88 complex formation (f). Data are presented as the mean ± SEM from three independent experiments, **p < 0.01; the connection represents a comparison between the two groups. CTRP9: C1q/TNF‐related protein‐9; ELISA: enzyme‐linked immunosorbent assay; LPS: lipopolysaccharide; MyD88: myeloid differentiation factor 88; SEM: standard error of the mean; TLR4: toll‐like receptor 4 [Color…