Simultaneous induction of differentiation and senescence in cardiac fibroblasts by TGF-β1: effects of senotherapeutics drugs

Figures

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  FIGURE 1. TGF-β1 induces the differentiation of cardiac fibroblasts (CF) into cardiac myofibroblasts (CMF). CFs were treated with TGF-β1 (10 ng/ml) or vehicle (1X PBS) for 7 days. Immunofluorescence for α-SMA (green) and nuclei with DAPI (blue) was performed; representative images at 10× (scale bar 100 µm) are shown to evaluate α-SMA assembly into stress fibers (A), percentage of cells with α-SMA assembly (B), cells with increased cellular area (C) (N = 5). To measure soluble collagen (D), after 7 days of TGF-β1 treatment, cells were further incubated for 48 h in fresh medium with ascorbic acid (100 µM), and soluble collagen was quantified by Sirius Red staining and spectrophotometry, expressed as µg collagen/µg protein (N = 7). Control vs TGF-β1 were compared using unpaired Mann -Whitney; ** p < 0.01.

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  FIGURE 2. TGF-β1 alters cell cycle proteins without inducing DNA damage. CFs were treated with TGF-β1 (10 ng/mL) for 7 days or vehicle (1X PBS). Immunofluorescence (A,B) and Western blot (C) were performed for γ-H2AX (green; nuclei counterstained with DAPI, blue). Representative images taken with epifluorescence microscopy at ×20 magnification, indicated at the bottom right corner, where the scale bar denotes 100 μm. p15–p16 (D) p21 (E) and Rb phosphorylation (F,G) were analyzed by Western blot, with β-Tubulin as a loading control. In (G) the ALK5 inhibitor SB431542 (10 μM, 7 days) was included. N = 5 for graphs (A–F) N = 3 for (G). Control vs. TGF-β1 comparisons were performed by unpaired Mann–Whitney; p-Rb with SB431542 was analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. ***p < 0.001, **p < 0.01. D7 (doxorubicin 100 nM, 7 days) was included as a positive control.

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  FIGURE 3. TGF-β1 reduces proliferation and increases SA-β-gal staining. CFs were treated with TGF-β1 (10 ng/ml) or vehicle (1X PBS) for 7 days. After treatment, immunofluorescence for Ki-67 (green; (A, B)) was performed. Nuclei were stained with DAPI (blue) and the cytoskeleton (F-actin) with rhodamine (red). Representative images were acquired using epifluorescence microscopy at 20× magnification, indicated at the bottom right, with a scale bar of 100 µm. Additionally, SA-β-gal staining was performed at pH 6 (C, D). Representative images were acquired by brightfield microscopy at 10× magnification, indicated at the bottom right, with a scale bar of 100 µm. C7 = Control 7 days; T7 = TGF-β1 7 days. N = 5. In both graphs, Control vs TGF-β1 groups were compared by unpaired non-parametric Mann -Whitney test. **p < 0.01.

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  FIGURE 4. SASP profile of senescent myofibroblasts induced by TGF-β1. CFs were treated with TGF-β1 (10 ng/mL) or vehicle (1X PBS) for 7 days. After this period, cells were incubated for 48 h in DMEM-F12 medium, and secreted cytokines were measured by Luminex: IL-1β (A) IL-6 (B) IL-10 (C) IL-5 (D) MCP-1 (E) and TNF-α [(F) pg/mL], expressed as pg/µg protein of cell lysate except for TNF-α; N = 8. Except for TNF-α, Control vs. TGF-β1 was compared by Mann–Whitney; *p < 0.05, ns: not significant. VCAM-1 (G) was analyzed by Western blot (β-Tubulin as loading control; N = 5); Control vs. TGF-β1, Mann–Whitney, **p < 0.01. LD: limit of detection.

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  FIGURE 5. TGF-β1 increases Bax levels. CFs were treated with TGF-β1 (10 ng/mL) for 7 days or vehicle (1X PBS). Bax (A) BCL-2 (B) and BCL-xL (C) levels were analyzed by Western blot, with β-Tubulin as a loading control. Ratios of BCL-xL/Bax (D) and BCL-2/Bax (E) were also calculated to assess the apoptotic state of the cells. Control vs. TGF-β1 comparisons were performed using unpaired Mann–Whitney; *p < 0.05, ***p < 0.001.

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  FIGURE 6. Senolytic drugs reduce viability and SA-β-gal staining of senescent CMF. After treatment with TGF-β1 (10 ng/mL) for 7 days, medium was replaced and cells were exposed to increasing doses of Navitoclax (A) Dasatinib (B) Quercetin (C) or a fixed dose of Dasatinib + Quercetin (D). Cell viability was assessed by MTT after 72 h of drug treatment. Additionally, SA-β-gal staining (E,F) was performed in senescent CMF treated with Navitoclax (1 µM) or the combination of Dasatinib (150 nM) + Quercetin (100 µM). For figures (A, N = 8) and (B–C, N = 6), Vehicle (V, 1% DMSO) was compared against drug-treated groups using one-way ANOVA followed by Dunnett´s multiple comparisons pos-hoc test; (D, N = 6) was analyzed by unpaired Mann–Whitney; (E, N = 6) was analyzed by one-way ANOVA followed by Šídák’s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  FIGURE 7. TGF-β1 induces the differentiation of cardiac fibroblasts (CFs) into senescent cardiac myofibroblasts (CMFs). Senescent CMFs produce more collagen, display a low pro-inflammatory but high pro-apoptotic profile, and are selectively vulnerable to Navitoclax and Dasatinib + Quercetin, which reduce both cell viability and senescence markers.