Suppression of ADAM8 attenuates angiotensin II-induced cardiac fibrosis and endothelial-mesenchymal transition via inhibiting TGF-β1/Smad2/Smad3 pathways

Fig. 1.. Effect of Ang II on cardiac function in vivo. (A) Systolic blood pressure and diastolic blood pressure were recorded via a non-invasive blood pressure monitor. (B) The pulse pressure was analyzed. (C) The immunochemistry assay was applied to detect the ADAM8 expression level in myocardial tissues. (D) The mRNA expression of Adam8 was measured by real-time PCR. (E) ELISA kits were used to examine the content of ADAM8. *P<0.05 and ****P<0.0001. Student’s t-test was used for two-group comparisons.

Fig. 2.. The effect of ADAM8 on Ang II induced cardiac dysfunction in vivo. (A) Representative echocardiographic images from M-mode. (B-E) Quantitative changes of LVESD, LVEDD, LVEF%, and LVFS% in each group. (F, G) The mRNA and protein expression levels of ADAM8 were determined via real-time PCR and western blot, respectively. (H, I) ANP and BNP expression levels were also tested. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Multiple comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test.

Fig. 3.. ADAM8 knockdown attenuates Ang II induced cardiac fibrosis in vivo. (A) Collagen deposition was assessed through Masson’s trichrome staining. (B) The fibrotic area was analyzed. (C-E) Quantitative PCR analysis of mRNA levels of CTGF, collagen I, and collagen III in the hearts. **P<0.01, ***P<0.001 and ****P<0.0001. Multiple comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test.

Fig. 4.. ADAM8 silencing suppresses Ang II-induced EndMT in vivo. (A) Representative western blot bands of α-SMA, FSP1 and VE-cadherin were displayed. (B-D) The quantitative analysis of α-SMA, FSP1 and VE-cadherin was showed. *P<0.05, ***P<0.001 and ****P<0.0001. Multiple comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test.

Fig. 5.. ADAM8 modulates cardiac TGF-β1/Smad2/Smad3 pathways in vivo. (A-C) Western blot analysis for TGF-β1, Smad2, and phosphorylated Smad2 in the heart. (D, E) Representative images and quantitative analysis of Smad3 and phosphorylated Smad3. ***P<0.001 and ****P<0.0001. Multiple comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test.

Fig. 6.. ADAM8 inhibition ameliorated Ang II-induced EndMT in vitro. (A) Primary mouse heart endothelial cells. (B) The purity of endothelial cells was measured via flow cytometry. (C, D) The endothelial cells were infected with lentivirus-shRNA targeting ADAM8. Lentivirus infection efficiency was detected via real-time PCR and western blot. (E-G) The EndMT-related makers, α-SMA, FSP1 and VE-cadherin, were determined by real-time PCR. (H) Endothelial cells were co-immunostained to detect CD31 (green) and FSP-1 (red). **P<0.01, ***P<0.001 and ****P<0.0001. Multiple comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test.