Apilimod alters TGFβ signaling pathway and prevents cardiac fibrotic remodeling

Figures

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  Figure 1. Apilimod prevents cardiac fibrosis development in mice. (A) Cardiac fibrosis was assessed on heart cryosections stained with Sirius red from mice sham-operated or subjected to TAC surgery treated intraperitoneally with Apilimod. Bar is 1 mm. (B) Quantification of cardiac fibrosis from (A). n = 6-8 mice per group. (C-G) Expression levels of myocardial collagen 1 (Col1, C), collagen 3 (Col3, D), fibronectin-1 (E), Ctgf (F) and periostin (G) by qRT-PCR. n = 6-8 mice per group. ANOVA followed by Bonferroni's post-hoc test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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  Figure 2. Apilimod blunts cardiac hypertrophic response in TAC-mice. (A) Heart cryosections of mice treated as indicated were stained with hematoxylin-eosin (H/E) or fluorescent WGA. (B) Quantification of cardiomyocytes cross-sectional area measured from (A). Quantification was performed from 3-4 images across 3-5 mice per group. (C-F) Expression levels of Anp, Bnp, α-skeletal actin and β-Mhc were measured by qRT-PCR in cardiac tissues. n = 4-6 mice per group. ANOVA followed by Bonferroni's post-hoc test, *p < 0.05; ***p < 0.001; ****p < 0.0001.

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  Figure 3. Apilimod inhibits cardiac fibroblast activation. (A) Cardiac fibroblasts were treated with TGFβ in the presence of Apilimod or DMSO, fixed and stained with an anti-collagen 1 antibody (shown in red). Nuclei were stained with DAPI (in blue). Bar is 10 µm. Quantification if shown on the right, n = 41-82 cells across 3 independent experiments. (B-E) Expression levels of collagen 1 (Col1, B), collagen 3 (Col3, C), Tgfβ (D) and Il-6 (E) were assessed by qRT-PCR on treated cardiac fibroblasts. (F-H) Expression level of α-Sma was assessed by qRT-PCR (F), western-blot (G) and immunofluorescence (H) on treated cardiac fibroblasts. (I) Collagen gel contraction assay on TGFβ activated cardiac fibroblasts treated with Apilimod or DMSO for 72 h. n = 4-6 independent experiments. ANOVA followed by Bonferroni's post-hoc test or Student's t-test (unpaired, two-tailed), *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

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  Figure 4. Apilimod reduces cardiac fibroblasts migration and invasion. (A) Migrative properties of treated cardiac fibroblasts were assessed using the wound-healing assay. Nuclei were stained with DAPI (shown in white). Bar is 200 µm. Results are from 3 independent experiments. (B) Cell polarity was scored by the positioning of the Golgi apparatus (stained with GM130, shown in red) towards the migration front. Phalloidin staining of the actin cytoskeleton was used as a counterstain (shown in green). Cell nuclei were stained with DAPI (in blue). Bar is 100 µm. Results are from n = 19-43 cells across 3 independent experiments. (C) Cardiac fibroblasts invasive properties were assessed using a modified Boyden chamber invasion assay. Results are from 3 independent experiments. (D) Expression level of Mmp9 was measured by qRT-PCR in treated cardiac fibroblasts. Results are from 3 independent experiments. ANOVA followed by Bonferroni's post-hoc test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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  Figure 5. PIKfyve inactivation triggers steady-state TGFβ-R2 internalization. (A-B) Isolated primary cardiac fibroblasts (A) or HeLa cells (B) expressing flag-TGFβ-R2 were serum starved and treated with Apilimod or DMSO as indicated. Cells were fixed and stained with an anti-flag antibody. Bar is 10 µm. Results are from n = 9-16 cells across 3 independent experiments. (C-D) Cell surface binding of fluorescent TGFβ (TGFβ-A555, shown in red) on treated- cultured primary cardiac fibroblasts (C) or HeLa cells (D). Nuclei were stained with DAPI (shown in blue). Bar is 10 µm. n = 43-110 cells across 3 independent experiments. Student's t-test (unpaired, two-tailed), ****p < 0.0001. (E) Mouse embryonic fibroblasts (MEF) isolated from PIKfyveβgeo/βgeo mice or wild-type littermates were transfected to express flag-TGFβ-R2, serum-starved and treated with Apilimod as indicated. Cells were fixed and stained with a flag antibody (green) and imaged by confocal microscopy. Bar is 10 µm. Right panel shows the quantification of flag-TGFβ-R2 internalization. n = 4-8 cells across 3 independent experiments. ANOVA followed by Bonferroni's post-hoc test or Student's t-test (unpaired, two-tailed), *p < 0.05;…

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  Figure 6. Apilimod induces TGF-βR2 mislocalization to late-endosomes. (A) Serum-starved HeLa cells expressing flag-TGFβ-R2 were treated as indicated with Apilimod or DMSO, fixed and stained with an anti-flag antibody (green) and an anti-CD63 antibody (red). Bar is 10 µm. Right panel shows the quantification of colocalization. n = 17-37 cells across 3 independent experiments. Student's t-test (unpaired, two-tailed) ***p < 0.001. (B) Non-transfected (NT) or HEK cells expressing flag-TGFβ-R2 were serum-starved and treated with DMSO or Apilimod for the indicated times. Cell lysates were blotted with an anti-flag antibody to assess the amount of remaining flag-TGFβ-R2. RhoGDI was used as a loading control. n = 3 independent experiments.

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  Figure 7. Apilimod prevents TGFβ downstream signaling. (A) Serum-starved HeLa cells expressing flag-TGFβ-R2 were treated with Apilimod or DMSO as indicated, stimulated with TGFβ, fixed and stained with anti-flag (green) and anti-EEA1 (red) antibodies. Bar is 10 µm. (B) Quantification of colocalization between TGFβ-R2 and EEA1. n = 16-37 cells across 3 independent experiments (C) Primary cardiac fibroblasts were serum-starved and pre-treated with DMSO or Apilimod, before stimulation with TGFβ for the indicated time. Cell lysates were blotted for phosphorylated Smad2/3 (p-Smad2/3), total Smad2/3, or RhoGDI as a loading control. Quantification if shown on the right panel. n = 3 independent experiments. ANOVA followed by Bonferroni's post-hoc test or Student's t-test (unpaired, two-tailed), **p < 0.01.

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  Figure 8. Apilimod induces TGFβ receptor internalization in vivo. (A) Schematic of the experimental setting. (B) Cardiac fibroblasts isolated from vehicle or Apilimod-treated mice were plated and processed for cell surface binding of fluorescent TGFβ (TGFβ-A555, shown in red) after the indicated time in culture. Nuclei were stained with DAPI (shown in blue). Bar is 10 µm. Quantification is shown on the right from 34-265 cells from 3 technical replicates from 2-4 mice per group. ANOVA followed by Bonferroni's post-hoc test, **p < 0.01; ****p < 0.0001; ns, non-significant.