Inhibition of Wdr5 Attenuates Ang-II-Induced Fibroblast-to-Myofibroblast Transition in Cardiac Fibrosis by Regulating Mdm2/P53/P21 Pathway

Figure 1. Wdr5 expression was elevated in Ang-II-induced cardiac fibrosis models. (A) Masson’s trichrome staining showed increased fibrosis after 28 days of Ang II infusion (Scale bar 100 μm). (B) is the quantitative statistic of fibrosis areas in (A) (n = 5 in each group). (C) TMT-based quantitative proteomic analysis of ventricle sample showed increased expression of Wdr5. (D) Col1a1, α-SMA, and Wdr5 were significantly elevated in Ang-II-infused animal models in mRNA levels. (E) Fibronectin, col1a1, α-SMA, Wdr5, and H3K4me3 protein levels were significantly increased in Ang-II-infused mice compared with saline group (n = 5 in each group). (F) is the quantitative statistic of (E) graph. (G) IHC results indicated that Wdr5 were upregulated in the ventricle of Ang-II-infused mice (Scale bar 50 μm). (H) is the quantitative analysis of (G). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. saline.

Figure 2. Wdr5 is upregulated in Ang-II-stimulated NRCFs. (A) Col1a1, Col3a1, and α-SMA mRNA levels were upregulated after Ang II stimulation. (B) Col1a1, α-SMA, Wdr5, and H3K4me3 protein levels were upregulated in respond to Ang II. (C) is the quantitative statistic of (B) graph. (D) Immunofluorescence data of H3K4me3 in NRCFs (Scale bar 100 μm). (E) is the quantitative statistic of (D) graph. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. saline.

Figure 3. Pharmacologic inhibition of Wdr5 suppressed the FMT process. (A) Application of MM-401 and OICR-9429 repressed the Ang-II-induced col1a1, col3a1, and α-SMA. (B) Protein levels of col1a1 and α-SMA were downregulated after the application of MM-401 and OICR-9429. (C) is the quantitative analysis of graph (B). (D) Immunofluorescence analysis of α-SMA demonstrated inhibited α-SMA by OICR-9429 (green, α-SMA; blue, DAPI, Scale bar 100 μm). (E) is the quantitative statistics of graph (D) (n = 5 in each group, >3 fields in each sample). (F) Representative images of transwell migration assay and the quantification of migrated NRCFs (n = 5 in each group, >3 fields in each sample). (G) Wound repair assays were performed to assess the migratory capacities of cells that have been treated with DMSO or OICR-9429 (n = 5 in each group, >3 fields in each sample). Scale bar 100 μm. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. saline, # p < 0.05, ## p < 0.01 vs. Ang II group.

Figure 4. Genetic knockdown Wdr5 suppressed Ang-II-induced FMT process. (A) Application of Sh-Wdr5 repressed the Ang-II-induced col1a1, col3a1, and α-SMA in mRNA levels and achieved 60% knockdown of Wdr5 mRNA. (B) Protein levels of col1a1 and α-SMA were downregulated after transfection of Sh-Wdr5 by lentivirus (n = 4 in each group). (C) is the quantitative analysis of graph (B). (D) Immunofluorescence analysis of α-SMA demonstrated inhibited α-SMA by Sh-Wdr5 (green, α-SMA; blue, DAPI, Scale bar 100 μm) and (E) is the quantitative statistics of graph (D) (n = 5 in each group, >3 fields in each sample). (F) Representative images of transwell migration assay and the quantification of migrated NRCFs after transfected with Sh-NC or Sh-Wdr5 (n = 5 in each group, >3 fields in each sample). (G) Wound repair assays were performed to assess the migratory capacities of cells that have been transfected with Sh-NC or Sh-Wdr5 (n = 5 in each group, >3 fields in each sample). Scale bar 100 μm. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. saline, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Ang-II group.

Figure 5. Wdr5 inhibition attenuated fibroblasts proliferation and induced cell senescence. (A) OICR-9429 application for 48 h inhibited the Edu incorporation in fibroblasts compared with a control group (n = 4 in each group, >3 fields for each sample) (B) Sh-Wdr5 infection for 48 h inhibited the Edu incorporation in fibroblasts compared with Sh-NC (n = 4 in each group, >3 fields for each sample). (C,D) CCK8 assay demonstrated that cell viabilities were inhibited after pharmacologic and genetical inhibition of Wdr5 (n = 5 in each group). (E) SA-β Gal staining of OICR9429 or Sh-Wdr5 treated NRCFs. (F) mRNA levels of genes related to senescence-associated secretory phenotype. Scale bar 100 μm. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 between the 2 indicated groups.

Figure 6. Inhibition of Wdr5 reduced the Mdm2/P53/P21 pathway. (A) Application of OICR9429 repressed the Mdm2 and p21 RNA levels. (B) Protein levels of P53 and P21 were upregulated after the application of OICR9429 and overexpressing Mdm2 inhibited the increasement. (C) is the quantitative analysis of graph (B). (D) Application of Sh-Wdr5 repressed the Mdm2 and P21 in mRNA levels. (E) Protein levels of P53 and P21 were upregulated after transfection of Sh-Wdr5 by lentivirus and overexpressing Mdm2 inhibited the increasement. (F) is the quantitative analysis of graph (E). (G) Schematic representation of the Mdm2 gene promoter containing the H3K4me3 enriched sites. TSS represented transcription start site. (H) Cut&Tag-PCR and southern blotting confirmed the enrichment of H3K4me3 on the promoters of Mdm2. (I,J) Cut&Tag-qPCR indicated the decreased enrichment fo H3K4me3 on the promoters of Mdm2 by 3 amplicons. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. saline, # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. Ang-II group.

Figure 7. Pharmacologic inhibition of Wdr5 by OICR-9429 induced selective fibroblast senescence and attenuated the Ang-II-induced fibrosis. (A) Representative fluorescent images of P21, vimentin (a marker of fibroblasts), and DAPI in heart after treatment of OICR9429 and Ang II (Scale bar 50 μm). (B) is the quantitative analysis of graph (A). (C,D) Masson’s trichrome staining showed attenuated fibrosis by treatment of OICR-9429 in the models of Ang II or ISO in different areas. (E,F) OICR9429 did not affect the ventricular systolic function by measuring EF and FS. Data are presented as mean ± SEM. Scale bar 100 μm. ** p < 0.01, #### p < 0.0001 vs. Ang-II group.