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

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.

Figure 1. 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.

Figure 2. Androgens promote VSMC calcification and regulate osteogenic gene expression.

VSMCs were cultured with high phosphate (3 mM P_i; filled bar) or control (1 mM P_i; white bar) for up to 9 days. Calcium content (μg/mg protein) of cells treated with (a) testosterone (1–100 nM) (n = 4) or (b) DHT (1-100 nM) (n = 4). Fold change in the mRNA expression of (c) Alpl (d) Osterix and (e) Mgp following 48 hr treatment with testosterone (white bar) or DHT (filled bar) (n = 6). Results are presented as mean +/− S.E.M *P < 0.05; **P < 0.01; ***P < 0.001 compared with control.

Figure 3. Androgens promote P_i-induced calcification via the AR.

(a) AR protein expression in SM-ARKO VSMCs versus WT control cells in the presence or absence of testosterone (100 nM) (Images were cropped from original scans, and gels were performed under the same experimental conditions. Unprocessed original scans are shown in Suppl Fig. 1). Fold change in the mRNA expression of (b) Esr2 and (c) Esr1 in SM-ARKO VSMCs versus WT control cells (n = 5). (d) Calcium content (μg/mg protein) of SM-ARKO and WT VSMCs cultured with high phosphate (3 mM P_i) for 9 days and treated with testosterone (100 nM) (n = 6). Results are presented as mean + /− S.E.M *P < 0.05 compared with control.

Figure 4. Altered mRNA expression of osteogenic genes in SM-ARKO VSMCs.

Fold change in the mRNA expression of (a) Osterix (b) Alpl and (c) Mgp in SM-ARKO VSMCs versus WT control cells (n = 5). Results are presented as mean + /− S.E.M ***P < 0.001 compared with control.

Figure 5. Testosterone has no effect on apoptosis of VSMCs.

Testosterone or DHT (100 nM) treatment does not (a) reduce VSMC viability (n = 6) or (b) induce apoptosis (n = 6).