Vitamin D inhibits TGFβ₁-mediated myofibroblast contraction.

<p>Representative images of 48 hour timepoint from collagen gel contraction assay are shown in A-D. A) Untreated HCF-av; B) HCF-av treated with TGFβ₁; C) HCF-av treated with 1,25(OH)₂D₃; D) HCF-av treated with TGFβ₁ + 1,25(OH)₂D₃. A time course of gel contraction over 96 hours is shown in (E). Active vitamin D treatment significantly inhibited TGFβ₁-induced gel contraction at all time points post-treatment. All data points represent mean ± SEM. p-values calculated using two way repeated measures analysis of variance with Bonferroni multiple comparison test. *p<0.05, ***p<0.001. F and G) Confocal images of HCF-av at 48 hours following treatment. Stress fibers were stained with phalloidin. HCF-av treated with TGFβ₁(F) demonstrate increased presence of clearly defined stress fibers (white) as compared with cells treated with TGFβ₁ + 1,25(OH)₂D₃ (G). Scale bar: 70μm.</p

Circulating levels of vitamin D are positively associated with myocardial fibrosis in heart failure patients.

<p>Collagen area as a fraction of total myocardial tissue area was significantly higher in patients with vitamin D deficiency as compared with vitamin D insufficient or sufficient patients. There was no difference in collagen area in patients without vitamin D deficiency. All data represent mean ± SEM. p-values were calculated using one way analysis of variance with a Bonferroni multiple comparison test. *p<0.05.</p

Wnt3a inhibits cell proliferation, but increases cell migration and contraction after 72 hour treatment.

<p>(A) Cell proliferation was measured after 72 hours of treatment with Wnt3a or vehicle. Wnt-treated cells grew at 77.4±4.5% the rate of vehicle treated cells (p<0.05). (B) Cells were treated for 72 hours with Wnt3a or vehicle, and then a scratch wound assay was performed to measure cell migration. Wnt-treated cells closed the scratch wound at a significantly faster rate than vehicle-treated cells, as measured 48 hours after injury (78.1±2.1% vs 61.9±3.8%, p<0.05). (C) Cells were treated for 72 hours with Wnt3a or vehicle and then a fibroblast-populated collagen lattice contraction assay was performed. Images of contracted gels taken at 24 hours are shown along with the quantified surface areas of contracted gels. Wnt3a treatment significantly increased the fibroblast-mediated contraction of collagen gels (16.1±0.6% vs 29.4±1.3% of initial surface area, p<0.05). (* denotes p<0.05)</p

Study Cohort Demographic Information.

<p>Data expressed as mean (± SD) or n (%), p-values calculated using one-way analysis of variance for continuous variables and chi-square test for categorical variables.</p><p>Study Cohort Demographic Information.</p

Wnt3a induces canonical Wnt signaling in mouse fibroblasts.

<p>Confocal images of fibroblasts treated for 24 hours with vehicle (top panels) or 250 ng/mL Wnt3a (bottom panels) and immunonstained for β-catenin (green) and nuclei (blue). Wnt3a treatment induced clear nuclear accumulation of β-catenin in murine fibroblasts (arrows). (B) TOPFlash reporter assay demonstrated Wnt3a significantly increased luciferase activity 5.3±1.6 fold after a 24 hour treatment (p<0.05). (C) Wnt3a treatment induced a 255±71 fold increase in the mRNA expression of axin2, a target of classical Wnt signaling (p<0.05). (Scale bar = 23.00 µm in A, * denotes p<0.05)</p

Vitamin D reduces TGFβ₁-mediated phosphorylation of SMAD2.

<p>Representative Western blot images of HCF-av treated for 24 hours (A) and 48 hours (B) with TGFβ₁ in the presence and absence of 1,25(OH)₂D₃, which demonstrate a reduction in pSMAD2 with active vitamin D treatment. Densitometry of Western blot data shows significantly increased phosphorylation of SMAD2 at both 24 hours (C) and 48 hours (D) following treatment with TGFβ₁, which is significantly reduced with co-treatment with 1,25(OH)₂D₃. All data represent mean ± SEM. p-values were calculated using one way analysis of variance with a Bonferroni multiple comparison test. ***p<0.001, ****p<0.0001.</p

Vitamin D treatment inhibits expression of TGFβ₁-mediated α-smooth muscle actin.

<p>A) Representative Western blot of cells 48 hours after treatment. Expression of the discoidin domain receptor 2 (DDR2) is present in human primary adult ventricular cardiac fibroblasts (HCF-av). CYP24 expression was upregulated 48 hours after treatment with 1,25(OH)₂D₃±TGFβ₁. Expression of α-smooth muscle actin (αSMA) was upregulated 48 hours after treatment with TGFβ₁, and significantly reduced with 1,25(OH)₂D₃ co-treatment. B) Densitometry data generated from Western blots of HCF-av cells 48 hours after treatment with TGFβ₁±1,25(OH)₂D₃, and normalized to GAPDH. All data represent mean±SEM. p-values were calculated using one way analysis of variance with a Bonferroni multiple comparison test. *p<0.05, ***</p

Wnt3a-induced change in cell phenotype is dependent on TGF-β expression.

<p>(A) Representative Western blots of vehicle- and Wnt3a-treated fibroblasts showing TGF-β expression, SMAD2 phosphorylation, and smooth muscle α-actin expression at 12, 24, 48, and 72 hours of treatment. (B) Graphical representation of the densitometry results for the blots in A shows, in a sequential manner, that TGF-β expression peaks between 12 and 24 hours, followed by SMAD2 phosphorylation peaking between 24 and 48 hours, which is then followed by smooth muscle α-actin expression peaking after 72 hours of treatment. (C) Western blot of SMAD2 phosphorylation in fibroblasts treated with or without Wnt3a and a TGF-β neutralizing antibody. Densitometry demonstrated the TGF-β neutralizing antibody significantly inhibited Wnt3a-induced SMAD2 phosphorylation (p<0.05). No change was seen in the vehicle-treated cells (p = 0.74). (D) Western blot of smooth muscle α-actin expression in fibroblasts treated with or without Wnt3a and the TGF-β neutralizing antibody. Densitometry confirmed TGF-β neutralization significantly inhibited the Wnt3a-induced smooth muscle α-actin expression (p<0.05). No change was seen in vehicle-treated cells (p = 0.71). (* denotes p<0.05)</p

Wnt3a-induced change in cell phenotype is dependent on β-catenin.

<p>(A) Western blot demonstrated β-catenin siRNA significantly decreased β-catenin expression in vehicle- and Wnt-treated fibroblasts when compared to a scrambled siRNA. (B) Western blot showed knock down of β-catenin expression significantly inhibited the Wnt3a-induced SMAD2 phosporylation (p<0.05). No difference in SMAD2 phosporylation was detected in vehicle treated cells (p = 0.25). (C) Western blot of smooth muscle α-actin expression demonstrated that β-catenin siRNA significantly decreased smooth muscle α-actin expression in Wnt3a-treated fibroblasts (p<0.05). No significant difference was seen in the vehicle-treated cells (p = 0.27). (D) Immunohistochemistry showed Wnt3a promoted smooth muscle α-actin stress fibre formation in control siRNA transfected cells (green, arrows), but β-catenin siRNA completely inhibited the Wnt3a-induced smooth muscle α-actin expression. Cell nuclei are stained blue with DAPI. (Scale bar = 23.00 µm in D, * denotes p<0.05)</p

Vitamin D does not inhibit TGFβ₁-mediated cellular proliferation.

<p>Evaluation of proliferation rates in our treatment groups revealed no significant change in cellular proliferation between cells treated with active vitamin D and TGFβ₁ or TGFβ₁ alone. Proliferation was increased in the presence of TGFβ₁. All data represent mean ± SEM. p-values were calculated using one way analysis of variance with a Bonferroni multiple comparison test.</p