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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: 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 inhibits TGFβ1-mediated myofibroblast contraction.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.
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pone.0128655.g002: Vitamin D inhibits TGFβ1-mediated myofibroblast contraction.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.

Mentions: TGFβ1 is a potent pro-fibrotic mediator known to induce myofibroblastic activation in cardiac fibroblasts. It strongly upregulates the expression of the myofibroblast marker αSMA, and drives a secretory and contractile phenotype in these cells. As such, TGFβ1 is a critical mediator of aberrant extracellular matrix deposition and remodeling in fibrotic diseases. We evaluated the expression of αSMA following treatment of HCF-av with TGFβ1 in the presence and absence of 1,25(OH)2D3 to determine whether treatment with active vitamin D could abrogate TGFβ1-mediated myofibroblast activation. TGFβ1 treatment resulted in significant upregulation of αSMA expression (p<0.05) at 48 hours following treatment. Co-treatment of cells with 1,25(OH)2D3 reduced αSMA levels to baseline (p<0.001) (Fig 1B). Myofibroblastic activation is characterized by stress fiber formation. Stress fibers incorporate αSMA, allowing myofibroblasts to generate increased contractile force on the matrix surrounding them, and their formation is induced by TGFβ1. Confocal imaging of f-actin stained cells following treatment with TGFβ1 ± 1,25(OH)2D3 demonstrated a reduction in stress fiber staining intensity in the presence of active vitamin D3 (Fig 2G).


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 inhibits TGFβ1-mediated myofibroblast contraction.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.
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pone.0128655.g002: Vitamin D inhibits TGFβ1-mediated myofibroblast contraction.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.
Mentions: TGFβ1 is a potent pro-fibrotic mediator known to induce myofibroblastic activation in cardiac fibroblasts. It strongly upregulates the expression of the myofibroblast marker αSMA, and drives a secretory and contractile phenotype in these cells. As such, TGFβ1 is a critical mediator of aberrant extracellular matrix deposition and remodeling in fibrotic diseases. We evaluated the expression of αSMA following treatment of HCF-av with TGFβ1 in the presence and absence of 1,25(OH)2D3 to determine whether treatment with active vitamin D could abrogate TGFβ1-mediated myofibroblast activation. TGFβ1 treatment resulted in significant upregulation of αSMA expression (p<0.05) at 48 hours following treatment. Co-treatment of cells with 1,25(OH)2D3 reduced αSMA levels to baseline (p<0.001) (Fig 1B). Myofibroblastic activation is characterized by stress fiber formation. Stress fibers incorporate αSMA, allowing myofibroblasts to generate increased contractile force on the matrix surrounding them, and their formation is induced by TGFβ1. Confocal imaging of f-actin stained cells following treatment with TGFβ1 ± 1,25(OH)2D3 demonstrated a reduction in stress fiber staining intensity in the presence of active vitamin D3 (Fig 2G).

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