Limits...
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: 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.Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts.

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 treatment inhibits expression of TGFβ1-mediated α-smooth muscle actin.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<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4462580&req=5

pone.0128655.g001: Vitamin D treatment inhibits expression of TGFβ1-mediated α-smooth muscle actin.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<0.001.

Mentions: We confirmed the primary cells we used in our in vitro experiments were fibroblasts by Western blot for discoidin domain receptor 2 (DDR2). Western blot for VDR was used to confirm primary cardiac fibroblast expression of the vitamin D receptor. To confirm the validity of our model system, we examined whether our HCF-av cells expressed a functional VDR. CYP24 is a well known VDR target gene, which is upregulated in the presence of the VDR ligand 1,25(OH)2D3. Treatment of HCF-av cells with 1,25(OH)2D3 resulted in upregulation of CYP24, consistent with previous biomedical literature on vitamin D signaling (Fig 1A).


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 treatment inhibits expression of TGFβ1-mediated α-smooth muscle actin.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<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128655.g001: Vitamin D treatment inhibits expression of TGFβ1-mediated α-smooth muscle actin.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<0.001.
Mentions: We confirmed the primary cells we used in our in vitro experiments were fibroblasts by Western blot for discoidin domain receptor 2 (DDR2). Western blot for VDR was used to confirm primary cardiac fibroblast expression of the vitamin D receptor. To confirm the validity of our model system, we examined whether our HCF-av cells expressed a functional VDR. CYP24 is a well known VDR target gene, which is upregulated in the presence of the VDR ligand 1,25(OH)2D3. Treatment of HCF-av cells with 1,25(OH)2D3 resulted in upregulation of CYP24, consistent with previous biomedical literature on vitamin D signaling (Fig 1A).

Bottom Line: 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.Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts.

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