ADAMTS8 Promotes Cardiac Fibrosis Partly Through Activating EGFR Dependent Pathway

Figure 1. ADAMTS8 expression was increased in DCM patients with severe cardiac fibrosis. (A,B) Representative Masson staining (scale bar = 100 μm) and quantification of fibrosis. (C,D) Representative western blotting and quantification of ADAMTS8, α-SMA, and Collagen1 expression in the ventricular septum from hearts obtained from patients with DCM and normal hearts. (E) Immunofluorescence staining of ADAMTS8 and Vimentin in the ventricular septum from hearts from patients with DCM and normal hearts (scale bar = 40 μm). Data were presented as mean ± SEM. ***P < 0.001 vs. Normal group.

Figure 2. Increased ADAMTS8 expression was associated with cardiac fibrosis in the MI rat model. (A) Representative Masson staining images from the heart of the MI rat model (scale bar = 100 μm). (B) Changes of ADAMTS8 protein expressions in the border zone and the remote area at the indicated time points post-MI. (C) Western blotting and quantification of the expressions of α-SMA and Collagen I in infarcted and border zones of rat hearts. (D) Representative images of double immunofluorescence staining of ADAMTS8 (green) and vimentin (red) (scale bar = 200 μm). (E,F) The ADAMTS8 protein level in cardiac myocytes and cardiac fibroblasts under hypoxia conditions for 12, 24, and 48 h assessed via western blotting. Data were presented as mean ± SEM. *P < 0.05 vs. 0 day (Sham). **P < 0.01 vs. 0 day (Sham) or ctrl group.

Figure 3. ADAMTS8 expression was increased in the TAC-induced cardiac fibrosis rat model. (A,B) Representative Sirius Red and Masson staining images from a sham heart and a TAC-induced heart (scale bar = 200 μm). (C) Immunofluorescence staining of ADAMTS8 (green) and vimentin (red) in normal and TAC-induced rats (scale bar = 100 μm). (D) ADAMTS8, α-SMA, and Collagen1 expressions in the sham group and TAC model group via western blotting and quantified according to the immunoblots. (E,F) The ADAMTS8 protein level in cardiac myocytes and cardiac fibroblasts treated with PBS or Ang-II. Data were presented as mean ± SEM. **P < 0.01, ***P < 0.001 vs. sham.

Figure 4. ADAMTS8 promoted myofibroblast formation in vitro. (A) Knockdown efficiency of three different interference sequences of ADAMTS8 detected by qPCR and western blotting. (B) The overexpression efficiency of ADAMTS8 verified by fluorescence and Western blotting. (C,D) α-SMA, Collagen I, and CTGF levels in cultured cardiac fibroblasts infected with ADAMTS8-overexpressing adenovirus or si-ADAMTS8 detected via western blotting. (E) Immunofluorescence for the expression of α-SMA (red) and nuclei (DAPI: blue) (scale bar = 20 μm). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and NS indicates no significance vs. Ctrl or between the two indicated groups.

Figure 5. ADAMTS8 mediated the interaction between cardiomyocytes and cardiac fibroblasts. (A) Wound repair assays to assess the migration ability of cardiac fibroblasts co-cultured with cardiomyocytes transfected with ADAMTS8 overexpressing adenovirus (Ad-ADAMTS8-Co) or siRNA (si-ADAMTS8-Co). (B) Transwell migration assay and quantification of migrated cardiac fibroblasts (scale bar = 50 μm). (C) Quantification by CCK-8 assay (n = 4 in each group). (D,E) p27kip1 and PCNA levels in cardiac fibroblasts co-cultured with cardiomyocytes transfected with ADAMTS8 overexpressing adenovirus (Ad-ADAMTS8-Co) or siRNA (si-ADAMTS8-Co) detected via western blotting. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 between the two indicated groups.

Figure 6. ADAMTS8-mediated mitochondrial dysfunction in cardiac fibroblasts and activated ROS-sensitive pathways to regulate the fibrotic response. (A,C) Representative Western blot of DRP-1 (p-drp1) phosphorylated at Ser637 and total drp1 (t-drp1) in cultured cardiac fibroblasts infected with si-ADAMTS8 or ADAMTS8-overexpressing adenovirus. (B) Real-time polymerase chain reaction (RT-PCR) analyses of Mfn1 and Mfn2 mRNA in cultured cardiac fibroblasts infected with si-ADAMTS8. Average expression values were normalized to GAPDH mRNA. (D) RT-PCR analyses of NOX4 mRNA in cultured cardiac fibroblasts infected with ADAMTS8-overexpressing adenovirus average expression values were normalized to GAPDH mRNA. (E) Representative images of dihydroethidium (DHE) staining of CFs infected with ADAMTS8-overexpressing adenovirus. (F,G) Activation of MAPK and AKT in cultured cardiac fibroblasts overexpressing ADAMTS8 or downregulating ADAMTS8 detected via western blotting. (H) Cultured cardiac fibroblasts infected with ADAMTS8 overexpressing adenovirus treated with the MAPK pathway inhibitor SB203580 or AKT inhibitor LY294002 (25 uM) after quiescence, followed by analysis of the cell lysates with indicated…

Figure 7. ADAMTS8 activated YAP, and inhibiting the EGFR expression blocked the ADAMTS8-induced YAP activation in cultured cardiac fibroblasts. (A,B) The phosphorylation levels of EGFR and its downstream MAPK and AKT were all attenuated in the gefitinib group. (C) EGFR inhibitor abated ADAMTS8-induced cardiac fibroblast activation. (D) Cardiac fibroblasts co-cultured with cardiomyocytes transfected with ADAMTS8 overexpressing adenovirus (Ad-ADAMTS8-Co) were treated with or without gefitinib, followed by analysis of the cell lysates with indicated antibodies. (E) The protein levels of collagen1 and α-SMA were all partly declined in the verteporfin group. (F) Cardiac fibroblasts co-cultured with cardiomyocytes infected with ADAMTS8 overexpressing adenovirus were treated with the MAPK pathway inhibitor SB203580 or AKT inhibitor LY294002 after quiescence, followed by analysis of the cell lysates with indicated antibodies.

Figure 8. ADAMTS8 overexpression promoted cardiac fibrosis and impaired cardiac function in vivo. (A–C) Representative pictures from Sirius red and Masson staining and quantitative analysis of interstitial fibrotic area in the infarct border zone of Ad-v Sham, Ad-ADAMTS8 Sham, Ad-v MI, and Ad-ADAMTS8 MI rats (scale bar = 50 μm). (D) Representative M-mode images of Ad-v Sham, Ad-ADAMTS8 Sham, Ad-v MI, and Ad-ADAMTS8 MI groups; EF was quantified via echocardiography. (E) Collage1 and α-SMA expression levels were quantified by western blotting. (F) Activation of the EGFR, MAPK, and AKT signaling pathways of a rat model of MI following adenovirus injection was detected via western blotting in vivo. Data were presented as mean ± SEM. **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, and NS indicates no significance between the two indicated groups.

Figure 9. Mebendazole down-regulated ADAMTS8 and ameliorated cardiac fibrosis and heart failure. (A) Representative Western blots and quantification of ADAMTS8 in cardiac fibroblasts treated with mebendazole (5 mol/L) for 48 h (n = 3 each). (B) Representative Western blots and quantification of ADAMTS8, PCNA, p27kip1, α-SMA, and Collage1 expression in CFs treated with or without Mebendazole (n = 3 each). (C) Representative Western blotting of ADAMTS8, α-SMA, and Collagen1 protein levels in the LVs of rats followed by treatment with vehicle or mebendazole for 14 days after MI or TAC (n = 5 each). (D) Representative pictures from Sirius red and Masson staining in the infarct border zone of rats followed by treatment with vehicle or mebendazole for 21 days after MI(scale bar = 100 μm). (E,F) Representative M-mode echocardiograms obtained on 28th day post-MI. Cardiac function was measured by detecting EF%, FS%, LVDD, and LVSD post-MI. Data were presented as mean ± SEM. ***P < 0.001, ##P < 0.01 and ###P < 0.001 between the two indicated groups.

Figure 10. Illustrating ADAMTS8-mediated activation of EGFR signaling pathway by hypoxia and Ang-II stimulation.