DDR2‐mediated autophagy inhibition contributes to angiotensin II‐induced adventitial remodeling

Figures

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  FIGURE 1. Single‐cell analysis of adventitial cells and discoidin domain receptor 2 (DDR2) expression following Ang II stimulation. (A) t‐distributed stochastic neighbour embedding (t‐SNE) plot of adventitial cells, with colours representing different cell clusters. Cluster 1: B cells; Cluster 2: T cells; Cluster 3: Plasma cells; Cluster 4: Endothelial cells; Cluster 5: Fibroblasts of the tunica adventitia; Cluster 6: Lymphocytes; Cluster 7: Macrophages; Cluster 8: Smooth muscle cells; Cluster 9: Trophoblast giant cells. (B) Selected top Gene Ontology (GO) terms of adventitia cells after Ang II treatment. (C) Violin plot of Ddr2 gene expression across cell clusters. (D) Violin plot showing the expression of selected Ddr2 markers following Ang II stimulation, compared with the control group. Statistical significance was assessed using unpaired Student's t‐test. *p < 0.05.

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  FIGURE 2. Discoidin domain receptor 2 (DDR2) deficiency relieved Ang II‐induced AF phenotype switching and autophagy inhibition. AFs were infected with LV‐sh‐DDR2 or scramble for 24 h, followed by treatment with Ang II (100 nmol/L) for 24 h. (A) AF phenotype switching was observed by immunofluorescence staining with α‐SMA (red) and DAPI (blue). Scale bar = 40 µm. (B) The protein expression of α‐SMA and COL1A1 were examined by Western blotting and quantified by densitometry. (C‐D) Representative images and quantification of EdU staining. Scale bar = 200 µm. (E) Representative images of transwell migration assay and quantification of the migrated cells. Scale bar = 200 µm. (F) Representative images of scratch‐wound assay and quantification of the migration area were presented. Scale bar = 500 µm. (G‐H) The expression of DDR2, ATG7, ATG5 and LC3B were examined by Western blotting and quantified by densitometry. Statistical significance was assessed using two‐way ANOVA followed by Bonferroni test. All values are presented as means ± SEM, n = 3–5 per group; *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 3. Discoidin domain receptor 2 (DDR2) overexpression accelerated Ang II‐induced autophagy impairment and AF phenotype switching. AFs were infected with DDR2‐overexpressing lentivirus (LV‐DDR2) or vector for 24 h, followed by treatment with Ang II (100 nmol/L) for 24 h. (A) AF phenotype switching was observed by immunofluorescence staining with α‐SMA (red) and DAPI (blue). Scale bar = 40 µm. (B) The protein expression of α‐SMA and COL1A1 were examined by Western blotting and quantified by densitometry. (C‐D) Representative images and quantification of EdU staining. Scale bar = 200 µm. (E) Representative images of transwell migration assay and quantification of the migrated cells. Scale bar = 200 µm. (F) Representative images of scratch‐wound assay and quantification of the migration area were presented. Scale bar = 500 µm. (G‐H) The expression of DDR2, ATG7, ATG5 and LC3B were examined by Western blotting and quantified by densitometry. Two‐way analysis of variance followed by the Bonferroni's post hoc test was used for statistical analyses. All values are presented as means ± SEM, n = 3–5 per group; *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 4. Rapamycin attenuates Ang II‐induced autophagy inhibition, AF phenotype switching, proliferation and migration. Mouse AFs with or without rapamycin (1 µmol/L) pretreatment for 1 h were treated with either the vehicle or Ang II (100 nmol/L) for 24 h.(A) AF phenotype switching was observed by immunofluorescence staining with α‐SMA (red) and DAPI (blue). Scale bar = 40 µm. (B‐C) The protein expression of α‐SMA and COL1A1 were examined by Western blotting and quantified by densitometry. (D‐E) Representative images and quantification of EdU staining. Scale bar = 200 µm. (F) Representative images of transwell migration assay and quantification of the migrated cells. Scale bar = 200 µm. (G) Representative images of scratch‐wound assay and quantification of the migration area were presented. Scale bar = 400 µm. (H‐K) The expression of ATG7, ATG5, LC3B, p‐mTOR, t‐mTOR, p‐ULK1 and t‐ULK1 were examined by Western blotting and quantified by densitometry. (L‐M) AFs were transfected with mRFP‐GFP‐LC3 adenovirus for 12 h, followed by rapamycin and Ang II exposure and assessment via fluorescence microscope. Yellow dots represent autophagosomes, whereas red dots indicate autolysosomes. Scale bar =…

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  FIGURE 5. CQ aggravated Ang II‐induced autophagy inhibition and AF phenotype switching. Mouse AFs with or without CQ (20 µmol/L) pretreatment for 1 h were treated with either the vehicle or Ang II (100 nmol/L) for 24 h. (A) AF phenotype switching was observed by immunofluorescence staining with α‐SMA (red) and DAPI (blue). Scale bar = 40 µm. (B) The protein expression of α‐SMA and COL1A1 were examined by Western blotting and quantified by densitometry. (C‐D) Representative images and quantification of EdU staining. Scale bar = 200 µm. (E) Representative images of transwell migration assay and quantification of the migrated cells. Scale bar = 200 µm. (F) Representative images of scratch‐wound assay and quantification of the migration area were presented. Scale bar = 500 µm. (G‐H) The expression of SQSTM1 and LC3B were examined by Western blotting and quantified by densitometry. Two‐way analysis of variance followed by the Bonferroni's post hoc test was used for statistical analyses. Non‐parametric Kruskal‐Wallis tests with Dunn‐Benjamini‐Hochberg post hoc corrections were applied for multi‐group comparisons of protein levels. All values are presented as means ± SEM, n = 3–5 per group; *p <…

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  FIGURE 6. PI3K/AKT/mTOR signalling is required for discoidin domain receptor 2 (DDR2)‐mediated autophagy inhibition. AFs were infected with LV‐sh‐DDR2 or scramble for 24 h, followed by treatment with Ang II (100 nmol/L) for 24 h.(A) Representative Western blot of PI3K level following immunoprecipitation with anti‐DDR2 antibody in untreated AFs. (B) Quantification of the activity of PI3K by PI3K activity assay kit. (C‐D) The expression of p‐AKT and t‐AKT were examined by Western blotting and quantified by densitometry. (E‐G) The expression of p‐mTOR, t‐mTOR, p‐p70S6K, t‐p70S6K, p‐4EBP‐1, t‐4EBP‐1, p‐ULK1 and t‐ULK1 were examined by Western blotting and quantified by densitometry. Non‐parametric Kruskal–Wallis tests with Dunn's post hoc comparisons adjusted by the Benjamini‐Hochberg method were applied for multi‐group analyses. All values are presented as means ± SEM, n = 3–5 per group; *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 7. PI3K/AKT signal inhibition attenuated Ang II‐induced adventitial remodelling. Mouse AFs with or without LY294002 (20 εmM) pretreatment for 1 h were treated with either the vehicle or Ang II (100 nM) for 24 h. (A) AF phenotype switching was observed by immunofluorescence staining with α‐SMA (red) and DAPI (blue). Scale bar = 40 µm. (B‐C) The protein expression of α‐SMA and COL1A1 were examined by Western blotting and quantified by densitometry. (D) Representative images and quantification of EdU staining. Scale bar = 200 µm. (E‐F) Representative images of transwell migration assay and quantification of the migrated cells. Scale bar = 200 µm. (G‐H) Representative images of the in vitro scratch‐wound assay and quantification of the migration area were presented. Scale bar = 500 µm. (I‐L) The expression of p‐AKT, t‐AKT, p‐mTOR, t‐mTOR, p‐ULK1, t‐ULK1 and LC3B were examined by Western blotting and quantified by densitometry. Statistical significance was assessed using two‐way ANOVA followed by Bonferroni test. Non‐parametric Kruskal–Wallis tests with Dunn–Benjamini–Hochberg post hoc corrections were applied for multi‐group comparisons of protein levels. All values are presented as…

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  FIGURE 8. Discoidin domain receptor 2 (DDR2)‐mediated autophagy inhibition promotes ROS accumulation in Ang II stimulated AF. Mouse AFs with or without NAC (100 µmol/L) pretreatment for 1 h were treated with either the vehicle or Ang II (100 nmol/L) for 24 h. (A) Representative images of mitochondrial superoxide stained by MitoSOX. Scale bar = 40 µm. (B) AF phenotype switching was observed by immunofluorescence staining with α‐SMA (red) and DAPI (blue). Scale bar = 40 µm. (C) The protein expression of α‐SMA and COL1A1 were examined by Western blotting and quantified by densitometry. (D‐E) Representative images and quantification of EdU staining. Scale bar = 200 µm. (F) Representative images of transwell migration assay and quantification of the migrated cells. Scale bar = 200 µm. (G) Representative images of the in vitro scratch‐wound assay and quantification of the migration area were presented. Scale bar = 500 µm. Statistical significance was assessed using two‐way ANOVA followed by Bonferroni test. All values are presented as means ± SEM, n = 3–5 per group; **p < 0.01, ***p < 0.001.

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  FIGURE 9. Rapamycin partially attenuated Ang II‐induced adventitial remodelling and AF phenotype switching in C57BL/6J mice. Saline or Ang II was administrated to mice for 4 weeks with or without rapamycin administration (4 mg/kg per day). (A) Representative haematoxylin and eosin (HE), Masson's trichrome and sirius red staining of the aortic cross‐sections. Scale bar = 100 µm. (B) Quantification of the adventitial thickness. (C) Quantification of the fibrosis ratio. (D) Representative collagen I staining of the aortic cross‐sections. Scale bar = 100 µm. (E‐F) The adventitial fibrosis indicated by the expression level of α‐SMA and COL1A1 were examined by Western blotting and quantified by densitometry; (G‐J) The expression of p‐mTOR, t‐mTOR, p‐AKT, t‐AKT, p‐ULK1, t‐ULK1, ATG7, ATG5 and LC3B were examined by Western blotting and quantified by densitometry. (K‐L) Electron micrograph of autophagosome (marked with arrow) from the adventitia of the respective group and the quantification of autophagic vacuole. Scale bar = 2 µm (upper) and 1 µm (down). Two‐way analysis of variance followed by the Bonferroni's post hoc test was used for statistical analyses. Non‐parametric Kruskal–Wallis tests…

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  FIGURE 10. Deletion of Ddr2 alleviates adventitial remodelling and autophagy restriction in mice with Ang II infusion. Saline or Ang II was administrated to mice for 4 weeks in Ddr2‐cKO and Ddr2fl/fl mice. (A) Representative haematoxylin and eosin (HE), Masson's trichrome and sirius red staining of the aortic cross‐sections. Scale bar = 100 µm. (B) Quantification of the adventitial thickness. (C) Quantification of the fibrosis ratio. (D) Representative collagen I staining of the aortic cross‐sections. Scale bar = 100 µm. (E‐F) The adventitial fibrosis indicated by the expression level of α‐SMA and collagen I were examined by Western blotting and quantified by densitometry; (G‐H) The protein expression of DDR2 was examined by Western blotting and quantified by densitometry; (I‐L) The expression of p‐mTOR, t‐mTOR, p‐AKT, t‐AKT, p‐ULK1, t‐ULK1, ATG7, ATG5 and LC3B were examined by Western blotting and quantified by densitometry. (M‐N) Electron micrograph of autophagosome (marked with arrow) from the adventitia of the respective group and the quantification of autophagic vacuole. Scale bar = 2 µm (upper) and 1 µm (down). Two‐way analysis of variance followed by the Bonferroni's post hoc test…

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  FIGURE 11. Discoidin domain receptor 2 (DDR2) promotes PI3K/Akt/mTOR mediated autophagy inhibition, which contributes to Ang II‐induced AF phenotypic switch.