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

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

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

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 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

Versican V1 overexpression induces a myofibroblast-like phenotype in cultured fibroblasts

Background: Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts. Methods and Results: The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle a-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2. Conclusions: Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts