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1,25 Dihydroxyvitamin D3 Inhibits TGFβ1-Mediated Primary Human Cardiac Myofibroblast Activation.

Meredith A, Boroomand S, Carthy J, Luo Z, McManus B - PLoS ONE (2015)

Bottom Line: Serum 25(OH)D3 levels were assayed using mass spectrometry.Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3.Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Heart Lung Innovation, St. Paul's Hospital, University of British Columbia, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.

ABSTRACT

Aims: Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av), and investigated the relationship between circulating vitamin D (25(OH)D3) and cardiac fibrosis in human myocardial samples.

Methods and results: Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OH)D3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGF)β1 to induce activation, in the presence or absence of active vitamin D (1,25(OH)2D3). Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation.

Conclusions: Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis.

No MeSH data available.


Related in: MedlinePlus

Vitamin D reduces TGFβ1-mediated phosphorylation of SMAD2.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.
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pone.0128655.g004: Vitamin D reduces TGFβ1-mediated phosphorylation of SMAD2.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.

Mentions: TGFβ1 signals via SMAD dependent and independent pathways. We investigated whether vitamin D abrogated the SMAD dependent pathway by evaluating differences in SMAD2 phosphorylation in the presence of active vitamin D by Western blot. Treatment of cells with TGFβ1 significantly increased pSMAD2 levels in HCF-av cells both 24 and 48 hours after treatment (Fig 4) (p<0.0001). Addition of 1,25(OH)2D3 significantly reduced pSMAD2 levels at both timepoints (p<0.001 relative to TGFβ1). Our results indicate that active vitamin D can significantly inhibit TGFβ1-mediated SMAD2 phosphorylation and in this way diminish TGFβ1 downstream signaling events and gene transcription.


1,25 Dihydroxyvitamin D3 Inhibits TGFβ1-Mediated Primary Human Cardiac Myofibroblast Activation.

Meredith A, Boroomand S, Carthy J, Luo Z, McManus B - PLoS ONE (2015)

Vitamin D reduces TGFβ1-mediated phosphorylation of SMAD2.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4462580&req=5

pone.0128655.g004: Vitamin D reduces TGFβ1-mediated phosphorylation of SMAD2.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.
Mentions: TGFβ1 signals via SMAD dependent and independent pathways. We investigated whether vitamin D abrogated the SMAD dependent pathway by evaluating differences in SMAD2 phosphorylation in the presence of active vitamin D by Western blot. Treatment of cells with TGFβ1 significantly increased pSMAD2 levels in HCF-av cells both 24 and 48 hours after treatment (Fig 4) (p<0.0001). Addition of 1,25(OH)2D3 significantly reduced pSMAD2 levels at both timepoints (p<0.001 relative to TGFβ1). Our results indicate that active vitamin D can significantly inhibit TGFβ1-mediated SMAD2 phosphorylation and in this way diminish TGFβ1 downstream signaling events and gene transcription.

Bottom Line: Serum 25(OH)D3 levels were assayed using mass spectrometry.Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3.Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Heart Lung Innovation, St. Paul's Hospital, University of British Columbia, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.

ABSTRACT

Aims: Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av), and investigated the relationship between circulating vitamin D (25(OH)D3) and cardiac fibrosis in human myocardial samples.

Methods and results: Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OH)D3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGF)β1 to induce activation, in the presence or absence of active vitamin D (1,25(OH)2D3). Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation.

Conclusions: Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis.

No MeSH data available.


Related in: MedlinePlus