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Ablation of the androgen receptor from vascular smooth muscle cells demonstrates a role for testosterone in vascular calcification.

Zhu D, Hadoke PW, Wu J, Vesey AT, Lerman DA, Dweck MR, Newby DE, Smith LB, MacRae VE - Sci Rep (2016)

Bottom Line: Testosterone-induced calcification was blunted in VSMC-specific AR-ablated (SM-ARKO) VSMCs compared to WT.Consistent with these data, SM-ARKO VSMCs showed a reduction in Osterix mRNA expression.Androgen signalling may represent a novel potential therapeutic target for clinical intervention.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, EH25 9RG, UK.

ABSTRACT
Vascular calcification powerfully predicts mortality and morbidity from cardiovascular disease. Men have a greater risk of cardiovascular disease, compared to women of a similar age. These gender disparities suggest an influence of sex hormones. Testosterone is the primary and most well-recognised androgen in men. Therefore, we addressed the hypothesis that exogenous androgen treatment induces vascular calcification. Immunohistochemical analysis revealed expression of androgen receptor (AR) in the calcified media of human femoral artery tissue and calcified human valves. Furthermore, in vitro studies revealed increased phosphate (Pi)-induced mouse vascular smooth muscle cell (VSMC) calcification following either testosterone or dihydrotestosterone (DHT) treatment for 9 days. Testosterone and DHT treatment increased tissue non-specific alkaline phosphatase (Alpl) mRNA expression. Testosterone-induced calcification was blunted in VSMC-specific AR-ablated (SM-ARKO) VSMCs compared to WT. Consistent with these data, SM-ARKO VSMCs showed a reduction in Osterix mRNA expression. However, intriguingly, a counter-intuitive increase in Alpl was observed. These novel data demonstrate that androgens play a role in inducing vascular calcification through the AR. Androgen signalling may represent a novel potential therapeutic target for clinical intervention.

No MeSH data available.


Related in: MedlinePlus

Androgen receptor (AR) expression in calcified human cardiovascular tissue and primary murine VSMCs.(a) Calcified human femoral artery was confirmed by alizarin red (I & III, arrows indicate positive alizarin red staining) and von kossa staining (II & IV, arrows indicate positive von kossa staining). (b) AR expression was observed in the calcified tunica media region of human femoral artery tissue (I & II, arrows indicate positive AR staining). (c) Calcified aortic valve was confirmed by alizarin red (I & II, arrows indicate positive alizarin red staining) and von kossa staining (III & IV, arrows indicate positive von kossa staining). (d) Higher AR expression was observed in calcified aortic valve compared to control (I & II, arrows indicate positive AR staining). (e) Immunofluorescence staining showed nucleus staining of AR in murine VSMCs (SMA-red, AR-green, DAPI-blue, arrows indicate colocalisation of AR and DAPI staining). (f) Representative image of western blotting for AR following 100 nM testosterone treatment in VSMCs (Images were cropped from original scans, and gels were performed under the same experimental conditions. Unprocessed original scans are shown in Suppl Fig. 1) and (g) densitometry quantification (n = 3) showed increased expression of AR in VSMCs following treatment with testosterone. (h) Aromatase expression was absent from murine VSMCs (Images were cropped from original scans, and gels were performed under the same experimental conditions. Unprocessed original scans are shown in Suppl Fig. 1). NC=Negative Control. Scale bar = 100 μm. *P < 0.05.
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f1: Androgen receptor (AR) expression in calcified human cardiovascular tissue and primary murine VSMCs.(a) Calcified human femoral artery was confirmed by alizarin red (I & III, arrows indicate positive alizarin red staining) and von kossa staining (II & IV, arrows indicate positive von kossa staining). (b) AR expression was observed in the calcified tunica media region of human femoral artery tissue (I & II, arrows indicate positive AR staining). (c) Calcified aortic valve was confirmed by alizarin red (I & II, arrows indicate positive alizarin red staining) and von kossa staining (III & IV, arrows indicate positive von kossa staining). (d) Higher AR expression was observed in calcified aortic valve compared to control (I & II, arrows indicate positive AR staining). (e) Immunofluorescence staining showed nucleus staining of AR in murine VSMCs (SMA-red, AR-green, DAPI-blue, arrows indicate colocalisation of AR and DAPI staining). (f) Representative image of western blotting for AR following 100 nM testosterone treatment in VSMCs (Images were cropped from original scans, and gels were performed under the same experimental conditions. Unprocessed original scans are shown in Suppl Fig. 1) and (g) densitometry quantification (n = 3) showed increased expression of AR in VSMCs following treatment with testosterone. (h) Aromatase expression was absent from murine VSMCs (Images were cropped from original scans, and gels were performed under the same experimental conditions. Unprocessed original scans are shown in Suppl Fig. 1). NC=Negative Control. Scale bar = 100 μm. *P < 0.05.

Mentions: To investigate the role of the AR in vascular calcification, a critical and advanced phenotype of atherosclerosis, localisation studies were undertaken. Calcification of human femoral artery and aortic valve tissues was confirmed by alizarin red and von kossa staining (Fig. 1a I-IV,c I-IV). Immunohistochemistry established AR expression in calcified areas of human femoral artery and arterial valve tissues (Fig. 1b I-II,d I-II). Additionally, higher expression of AR was observed in calcified human aortic valve compared to control valve tissue (Fig. 1d I-II). These data are the first to show that AR is expressed alongside calcification in human calcified cardiovascular tissue.


Ablation of the androgen receptor from vascular smooth muscle cells demonstrates a role for testosterone in vascular calcification.

Zhu D, Hadoke PW, Wu J, Vesey AT, Lerman DA, Dweck MR, Newby DE, Smith LB, MacRae VE - Sci Rep (2016)

Androgen receptor (AR) expression in calcified human cardiovascular tissue and primary murine VSMCs.(a) Calcified human femoral artery was confirmed by alizarin red (I & III, arrows indicate positive alizarin red staining) and von kossa staining (II & IV, arrows indicate positive von kossa staining). (b) AR expression was observed in the calcified tunica media region of human femoral artery tissue (I & II, arrows indicate positive AR staining). (c) Calcified aortic valve was confirmed by alizarin red (I & II, arrows indicate positive alizarin red staining) and von kossa staining (III & IV, arrows indicate positive von kossa staining). (d) Higher AR expression was observed in calcified aortic valve compared to control (I & II, arrows indicate positive AR staining). (e) Immunofluorescence staining showed nucleus staining of AR in murine VSMCs (SMA-red, AR-green, DAPI-blue, arrows indicate colocalisation of AR and DAPI staining). (f) Representative image of western blotting for AR following 100 nM testosterone treatment in VSMCs (Images were cropped from original scans, and gels were performed under the same experimental conditions. Unprocessed original scans are shown in Suppl Fig. 1) and (g) densitometry quantification (n = 3) showed increased expression of AR in VSMCs following treatment with testosterone. (h) Aromatase expression was absent from murine VSMCs (Images were cropped from original scans, and gels were performed under the same experimental conditions. Unprocessed original scans are shown in Suppl Fig. 1). NC=Negative Control. Scale bar = 100 μm. *P < 0.05.
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Related In: Results  -  Collection

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f1: Androgen receptor (AR) expression in calcified human cardiovascular tissue and primary murine VSMCs.(a) Calcified human femoral artery was confirmed by alizarin red (I & III, arrows indicate positive alizarin red staining) and von kossa staining (II & IV, arrows indicate positive von kossa staining). (b) AR expression was observed in the calcified tunica media region of human femoral artery tissue (I & II, arrows indicate positive AR staining). (c) Calcified aortic valve was confirmed by alizarin red (I & II, arrows indicate positive alizarin red staining) and von kossa staining (III & IV, arrows indicate positive von kossa staining). (d) Higher AR expression was observed in calcified aortic valve compared to control (I & II, arrows indicate positive AR staining). (e) Immunofluorescence staining showed nucleus staining of AR in murine VSMCs (SMA-red, AR-green, DAPI-blue, arrows indicate colocalisation of AR and DAPI staining). (f) Representative image of western blotting for AR following 100 nM testosterone treatment in VSMCs (Images were cropped from original scans, and gels were performed under the same experimental conditions. Unprocessed original scans are shown in Suppl Fig. 1) and (g) densitometry quantification (n = 3) showed increased expression of AR in VSMCs following treatment with testosterone. (h) Aromatase expression was absent from murine VSMCs (Images were cropped from original scans, and gels were performed under the same experimental conditions. Unprocessed original scans are shown in Suppl Fig. 1). NC=Negative Control. Scale bar = 100 μm. *P < 0.05.
Mentions: To investigate the role of the AR in vascular calcification, a critical and advanced phenotype of atherosclerosis, localisation studies were undertaken. Calcification of human femoral artery and aortic valve tissues was confirmed by alizarin red and von kossa staining (Fig. 1a I-IV,c I-IV). Immunohistochemistry established AR expression in calcified areas of human femoral artery and arterial valve tissues (Fig. 1b I-II,d I-II). Additionally, higher expression of AR was observed in calcified human aortic valve compared to control valve tissue (Fig. 1d I-II). These data are the first to show that AR is expressed alongside calcification in human calcified cardiovascular tissue.

Bottom Line: Testosterone-induced calcification was blunted in VSMC-specific AR-ablated (SM-ARKO) VSMCs compared to WT.Consistent with these data, SM-ARKO VSMCs showed a reduction in Osterix mRNA expression.Androgen signalling may represent a novel potential therapeutic target for clinical intervention.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, EH25 9RG, UK.

ABSTRACT
Vascular calcification powerfully predicts mortality and morbidity from cardiovascular disease. Men have a greater risk of cardiovascular disease, compared to women of a similar age. These gender disparities suggest an influence of sex hormones. Testosterone is the primary and most well-recognised androgen in men. Therefore, we addressed the hypothesis that exogenous androgen treatment induces vascular calcification. Immunohistochemical analysis revealed expression of androgen receptor (AR) in the calcified media of human femoral artery tissue and calcified human valves. Furthermore, in vitro studies revealed increased phosphate (Pi)-induced mouse vascular smooth muscle cell (VSMC) calcification following either testosterone or dihydrotestosterone (DHT) treatment for 9 days. Testosterone and DHT treatment increased tissue non-specific alkaline phosphatase (Alpl) mRNA expression. Testosterone-induced calcification was blunted in VSMC-specific AR-ablated (SM-ARKO) VSMCs compared to WT. Consistent with these data, SM-ARKO VSMCs showed a reduction in Osterix mRNA expression. However, intriguingly, a counter-intuitive increase in Alpl was observed. These novel data demonstrate that androgens play a role in inducing vascular calcification through the AR. Androgen signalling may represent a novel potential therapeutic target for clinical intervention.

No MeSH data available.


Related in: MedlinePlus