Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein induced type I collagen processing in conditioned media of human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 24 h, and conditioned media were collected and concentrated for Western blotting. Stain-free gel bands from corresponding cell lysate samples were used as the loading control. Representative Western blots of ( A ) type I procollagen and pC-collagen (tropocollagen with PICP), detected using anti-C-telo antibody (n = 16), ( B ) type I procollagen, pC-collagen (tropocollagen with PICP), and PICP, detected using anti-PICP antibody (n = 8), and ( C ) type I procollagen, detected using anti-PINP antibody (n = 8). Schematic molecular structures and approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Cell Culture, Recombinant, Western Blot, Staining

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: Silencing ADAMTS-2 inhibited WISP-1 protein-induced type I collagen processing in conditioned media of human cardiac fibroblasts (HCFs). HCFs were either transfected with control SiRNA (1.228 μM), ADAMTS SiRNAs (614 nM/target gene), or left untransfected prior to seeding on a 12-well plate. After culture in supplemented fibroblast growth medium for 24 h, HCFs were starved in serum-free medium (SFM) for 48 h. The medium was then replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) and HCFs cultured for 15 h for qPCR analysis, and 24 h or 96 h for Western blotting analysis. ( A ) Quantification of ADAMTS-2 mRNA expression using qPCR analysis. Data were normalised to 36B4 housekeeping gene and expressed as the relative fold change to the untransfected HCFs (Control). ( B ) Quantification of ADAMTS-2 protein expression (168 h post-transfection) using Western blotting analysis. Data were normalised to stain-free gel bands and expressed as the relative fold change to the untransfected HCFs (Control). ( C ) Representative Western blots of type I procollagen and pC-collagen (tropocollagen with PICP) detected using anti-C-telo antibody. Stain-free gel bands from corresponding cell lysate samples were used as loading control. Quantification of pC-collagen I protein expression (96 h post-transfection) was expressed as the relative fold change to the WISP-1 protein treatment group. Data shown as mean ± SEM (n = 4–6). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Transfection, Recombinant, Cell Culture, Western Blot, Expressing, Staining

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein promoted Akt phosphorylation via integrin β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin αVβ5-blocking antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Cell Culture, Recombinant, Lysis, Western Blot, Expressing, MANN-WHITNEY, Incubation, Blocking Assay

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein promoted human cardiac fibroblasts (HCFs) activation. HCFs were cultured on soft substrate plates (8 kPa) in supplemented fibroblast growth medium for 24 h. HCFs were starved in serum-free medium (SFM) for 48 h, then the medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) and cultured for 24 h. ( A ) HCFs were fixed for immunocytochemical staining with anti-α-SMA antibody. α-SMA positive cells are stained green, and nuclei are stained blue with DAPI (4′,6-diamidino-2-phenylindole). Some positive cells are indicated by white arrows. Scale bar represents 50 μm. Quantification of positive α-SMA staining was expressed as the relative fold change to the control of the percentage of positive α-SMA staining cells to total cells on soft substrate. Data shown as mean ± SEM (n = 8). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Quantification of α-SMA protein expression and ( C ) quantification of PCNA protein expression using Western blotting analysis. Data were normalised to stain-free gel bands and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( D ) Quantification of accumulated migration distance per cell over the duration of consecutive images (21 h 30 min). Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Student’s t test. * indicates p < 0.05.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Activation Assay, Cell Culture, Recombinant, Staining, MANN-WHITNEY, Expressing, Western Blot, Migration

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 deficiency attenuated angiotensin II (AngII)-induced coronary artery perivascular fibrosis. Cardiac fibrosis was induced by subcutaneous AngII infusion (1000 ng/kg/min) for 28 days via osmotic pumps in WISP-1 +/+ and WISP-1 −/− mice. Representative images showing type I collagen (dark brown) staining using anti-C-telo antibody in left ventricular tissues with and without AngII infusion. Nuclei are stained blue with haematoxylin. Non-immune IgG was used as the negative control. Quantification of positive type I collagen staining was expressed as the percentage of positive collagen I staining area to total tissue area. Data shown as mean ± SEM (n = 5–8). Red arrows indicate some positive staining (dark brown). Scale bar represents 100 μm. Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Staining, Negative Control

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: A schematic summary of the findings of this study. WISP-1 promotes cardiac fibroblasts’ phenotypic switch from quiescent fibroblasts to myofibroblasts (activated fibroblasts), promoting collagen processing and accumulation. WISP-1 activates Akt signalling via integrin β1/FAK/ILK in cardiac fibroblasts. Deletion of WISP-1 attenuates angiotensin II (AngII)-induced cardiac fibrotic remodelling in vivo. Figure key is illustrated on the top left-hand side of the figure. Purple ↓ denotes promotion; black ↑ denotes increase; ┤ denotes inhibition.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: In Vivo, Inhibition