Effect of 5β-dihydrotestosterone on vasodilator function and on cell proliferation

Abstract

Aging is one of the main factors associated with cardiovascular diseases. Androgens exert beneficial effects on the cardiovascular system and testosterone (TES) replacement therapy improves cardiometabolic risk factors. However, TES is contraindicated in patients with prostate cancer due to its proliferative effects on prostatic tumor cells. Additionally, TES and its reduced metabolites 5α- and 5β-dihydrotestosterone (5α-DHT and 5β-DHT) exert vasodilatory effects. Since androgen levels decrease during aging and 5β-DHT lacks genomic effects, this study is focused on analyzing its effect on vasodilator function and the proliferation rate of prostatic tumor and vascular smooth muscle cells. To study the vascular function, mesenteric arteries from aged-orchidectomized Sprague-Dawley rats were used. Mesenteric segments were divided into one control (without treatment) and three groups with the androgens (10 nM, 30 min) to analyze: acetylcholine- and sodium nitroprusside-induced responses and nitric oxide and superoxide anion production. To analyze cell proliferation, the effect of androgens on cell viability was determined. The results showed that 5β-DHT improves vasodilator function in arteries from aged-orchidectomized rats and induces antioxidant action, while the proliferation rate of the androgen-dependent prostatic tumor cells remains unaltered. These results make 5β-DHT a promising therapeutic agent for the treatment of cardiovascular pathologies.

Fig 1. Effect of androgens on the vasodilator response induced by acetylcholine (ACh).

Effect of 10 nM testosterone (TES), 5α- and 5β-dihydrotestosterone (5α-DHT and 5β-DHT, respectively) on the concentration-response curves to ACh in the mesenteric arteries of aged-orchidectomized rats. Results (mean ± SEM) are expressed as the percentage of the inhibition of the contraction induced by 1 μM noradrenaline. Number of animals is indicated in parenthesis. The statistical significance is indicated in the graph. * p < 0.05 compared with control.

Fig 2. Effect of androgens on the vasodilator response induced by sodium nitroprusside (SNP).

Effect of 10 nM testosterone (TES), 5α- and 5β-dihydrotestosterone (5α-DHT and 5β-DHT, respectively) on the concentration-response curves to SNP in the mesenteric arteries of aged-orchidectomized rats. Results (mean ± SEM) are expressed as the percentage of the inhibition of the contraction induced by 1 μM noradrenaline. Number of animals is indicated in parenthesis. The statistical significance is indicated in the graph.

Fig 3. Effect of androgens on the basal NO release.

Effect of 10 nM testosterone (TES), 5α- and 5β-dihydrotestosterone (5α-DHT and 5β-DHT, respectively) on the basal release of NO in the mesenteric arteries of aged-orchidectomized rats. Results (mean ± SEM) are expressed as the ratio of the release after (b2) and previous (b1) to the androgens incubation and relative to the control condition (= 1). Number of animals: 4. *p < 0.05 compared with control condition.

Fig 4. Effect of androgens on the production of superoxide anion.

Effect of 10 nM testosterone (TES), 5α- and 5β-dihydrotestosterone (5α-DHT and 5β-DHT, respectively) for 30 min on the production of superoxide anion in the mesenteric arteries of aged-orchidectomized rats (A). Results (mean ± SEM) are expressed as the fluorescence emitted by HE (A.U, arbitrary units) per area (μm2) relative to the control condition (= 100). Number of animals: 3. *p < 0.05 compared with the control condition. Representative images of confocal micrographs (B) showing in situ detection of superoxide anion. The statistical significance between 5β-DHT and control condition is indicated in the graph.

Fig 5. Effect of androgens on the proliferation rate of vascular smooth muscle cells.

Effect of incubation with 10 nM testosterone (Test.) for 24, 48 or 72 h (A), 0.2 and 2 nM Test.for 72 h (B) and 2 nM Test., 5α- and 5β-dihydrotestosterone (5α-DHT and 5β-DHT, respectively) for 72 h (C) on the proliferation rate of vascular smooth muscle cells (SV40LT-VSMC). Results (mean ± SEM) are expressed as viability relative to the control condition (= 100). Data represent three independent experiments performed per triplicate.

Fig 6. Effect of androgens on the proliferation rate of androgen-dependent prostatic tumor cells.

Effect of incubation with 0.2 and 2 nM Testosterone (Test.) for 72 h (A) and 2 nM Test, 5α- and 5β-dihydrotestosterone (5α-DHT and 5β-DHT, respectively) for 72 h (B) on the proliferation rate of androgen-dependent prostatic tumor cells, LNCaP. Results (mean ± SEM) are expressed as viability relative to the control condition (= 100). Data represent three independent experiments performed per triplicate. *p < 0.001 compared with control condition; + p < 0.01 compared with 0.2 nM Test.; # p < 0.05 compared with Test; ## p < 0.002 compared with 5α-DHT.

Fig 7. Effect of androgens on the proliferation rate of androgen-independent prostatic tumor cells.

Effect of incubation with 0.2 and 2 nM Testosterone (Test.). for 72 h (A) and 2 nM Test., 5α- and 5β-dihydrotestosterone (5α-DHT and 5β-DHT, respectively) for 72 h (B) on the proliferation rate of androgen-independent prostatic tumor cells, DU145. Results (mean ± SEM) are expressed as viability relative to the control condition (= 100). Data represent three independent experiments performed per triplicate.