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 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

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

The projected increase in the prevalence of COPD in Canada in men (A) and women (B) across disease severities.

<p>The projected increase in the prevalence of COPD in Canada in men (A) and women (B) across disease severities.</p

The estimated monetary benefit of each hypothetical intervention assuming different scenarios for effectiveness of these interventions in reducing smoking rates (intervention I), progression of disease (intervention II), and exacerbation rates (intervention II).

<p>The estimated monetary benefit of each hypothetical intervention assuming different scenarios for effectiveness of these interventions in reducing smoking rates (intervention I), progression of disease (intervention II), and exacerbation rates (intervention II).</p

Key input parameters in the model.

a<p>rates were calculated based on relative risk of mortality per smoking status <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746-Gamble1" target="_blank">[25]</a>, 2002 Canadian life tables, and 2010 mortality estimates, Statistics Canada (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746.s002" target="_blank">Appendix S2</a>).</p>b<p>were estimated based on the symptom rates among smokers, previous smokers, and never smokers reported by Mannino et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746-Mannino2" target="_blank">[36]</a> for men and women and also proportion of patients without COPD reported by Buist et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746-Buist1" target="_blank">[9]</a>.</p>c<p>Estimated based on the reported rates for men and women in Buist et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746-Buist1" target="_blank">[9]</a> (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746.s002" target="_blank">Appendix S2</a>).</p>d<p>Estimated based on COPD specific mortality rate of 30.4 per 10,000 (Camp et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746-Camp1" target="_blank">[12]</a>) and probabilities of major exacerbations.</p>e<p>Estimated based on progression rates in Hoogendoorn et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746-Hoogendoorn4" target="_blank">[31]</a> (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746.s002" target="_blank">Appendix S2</a>).</p>f<p>these weights are EQ-5D Scores.</p>g<p>Costs in in Mannino et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746-Mannino2" target="_blank">[36]</a> were multiplied by 1.155 to reflect the changes in Canadian Consumer Price Index (CPI) between 2002 and 2011.</p>h<p>Estimated based on proportion of major/minor exacerbations used in Spencer et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046746#pone.0046746-Spencer1" target="_blank">[13]</a>.</p

Outcomes of hypothetical interventions.

<p>I. Decreasing smoking start rate (by testing for COPD predisposition in early smokers).</p><p>II. Decreasing progression rates (by access to new pharmacogenomic agents).</p><p>III. Decreasing exacerbations (by prediction of exacerbators).</p

The results of one-way sensitivity analyses on the total cost related to COPD in Canada over the next 25 years.

<p>The results of one-way sensitivity analyses on the total cost related to COPD in Canada over the next 25 years.</p