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 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

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

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

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

Versican alters the function of fibroblasts.

<p>(A) Versican increased cell proliferation 1.44 ± 0.07 fold over control cells, p<0.05. (B) Versican expressing fibroblasts showed decreased cell migration, as measure by in vitro scrape wound assay. (C) Cell adhesion was found to be increased in versican-transfected fibroblasts, measured at 30 minutes after seeding. (D) Representative Western blot showing increased integrin β1 expression, as well as phosphorylation of FAK-397, in versican-transfected fibroblasts. (* denotes p<0.05).</p

Versican increases TGF-β signaling in cultured fibroblasts.

<p>(A) Confocal imaging of contracted gels demonstrated the versican-transfected fibroblasts to have increased expression and incorporation of smooth muscle α-actin into their stress fibres (arrows, yellow in overlay). (B) The versican-transfected fibroblasts also displayed increased staining and nuclear localization of phophorylated SMAD2 in contracted collagen gels (arrows, red in overlay). (C) Representative Western blot shows increased phosphorylation of SMAD2 in cultures of versican-transfected fibroblasts (3.13 ±0.61 fold increase, p<0.05). (D) Confocal microscopy confirmed the increased SMAD2 phosphorylation and nuclear accumulation in cultures of versican-transfected fibroblasts (arrows). (Scale bars = 23.00 μm in A, 12.00 μm in B, D)</p

Versican increases fibroblast-mediated contraction of a collagen lattice.

<p>(A) Representative images of contracted collagen gels are shown along with the quantified surface areas of contracted gels. Versican significantly increased the fibroblast-mediated contraction of collagen gels (14.9 ± 0.7% vs 24.8 ± 1.8% of initial gel area, p<0.05). (B) Confocal imaging demonstrated the versican-transfected fibroblasts to be elongated, interconnected, and to have increased stress fibre formation in collagen gels (arrows, red in overlay). Versican was found to localize to the pericellular matrix surrounding elongated cells (arrowheads, green in overlay). (C) A Z-stack image reveals versican (green) forms a pericellular coat around cell protrusions in versican-transfected fibroblasts, suggesting it may be well-situated to influence biological events at the cell membrane. (Scale bars = 23.00 μm in B, 12.00 μm in C, * denotes p<0.05)</p

Versican expression in murine fibroblasts.

<p>(A) Western blot of recombinant versican expression in the cell lysate and conditioned medium of versican-transfected fibroblasts, suggesting the recombinant protein is synthesized and secreted. (B) Cell lysates were digested with chondroitinase ABC prior to Western blotting to confirm the presence of GAG chains on the recombinant versican. In the absence of chondroitinase, versican appeared as a large smear representing molecules of different molecular weights; after chondroitinase treatment, versican appeared as a compact band at the size of the smallest versican molecules from the smear, suggesting the GAG chains were present and had been removed. (C) Immunofluorescence microscopy showed recombinant versican was deposited into the ECM in versican-transfected cells (green, arrows). (Scale bar = 47.00 μm).</p

Versican increases N-cadherin expression.

<p>(A) Light microscopy shows no major change in cell morphology in versican-transfected fibroblasts at both sub-confluent and confluent densities. (B) Representative Western blot showing increased expression of N-cadherin in versican-transfected cells. (C) Confocal microscopy confirmed the increased N-cadherin expression in versican-transfected cells. (Scale bar = 12.00 μm).</p