Androgens deciency impairs thoracic aortic vascular function by promoting smooth muscle cell proliferation via an autophagy dependent manner

Androgens play an essential role in maintaining vascular hemostasis via the regulation of autophagy, but the underlying mechanisms remain unknown. Here we investigated the effects of androgen deciency in a rat castration model and the effects of androgen treatment in human umbilical vein smooth muscle cells (HUVSMCs). Male Sprague–Dawley rats were divided into two groups: (A) control group, (B) castration group. The vasodilation and vasoconstriction effect of dihydrotestosterone (DHT) were detected on rat thoracic aortic rings. Transmission electron microscopy was used to determine the levels of autophagy. Masson’s trichrome staining, MTT assay and ow cytometry were performed to detect the levels of HUVSMC cell proliferation.


Abstract Background
Androgens play an essential role in maintaining vascular hemostasis via the regulation of autophagy, but the underlying mechanisms remain unknown. Here we investigated the effects of androgen de ciency in a rat castration model and the effects of androgen treatment in human umbilical vein smooth muscle cells (HUVSMCs). Male Sprague-Dawley rats were divided into two groups: (A) control group, (B) castration group. The vasodilation and vasoconstriction effect of dihydrotestosterone (DHT) were detected on rat thoracic aortic rings. Transmission electron microscopy was used to determine the levels of autophagy. Masson's trichrome staining, MTT assay and ow cytometry were performed to detect the levels of HUVSMC cell proliferation.

Results
Androgen de ciency led to signi cantly enhance in the vasoconstriction and reductions in the vasodilatation of rat thoracic aortic rings. Treatment with rapamycin or 3-MA attenuated and enhanced, respectively, the vasorelaxation effect of androgen de ciency. Autophagy was signi cantly increased in the endothelial cells and SMCs of the thoracic aortic rings of model rats. Moreover, androgen de ciency induced excessive proliferation of SMCs. Treatment of HUVMSCs with high concentrations of DHT increased the percentage of cells in G0/G1 phase and reduced the percentages of cells in the G2 phase and S phase.

Conclusions
It was concluded that vascular injury resulting from androgen de ciency is mediated, in part, by increased autophagy. By promoting SMCs proliferation, androgen de ciency results in alteration of the aortic structure and aggravates vascular injury. Androgen treatment can inhibit the excessive proliferation of SMCs.

Background
Androgens, a type of steroid hormones, function in both physiological conditions and pathophysiological states via non-DNA binding-dependent actions 1 . They have multiple actions in their target organs, particularly in the process of sex differentiation and sexual maturation. However, recent studies have provided evidence that androgens are also involved in many diseases, including those of the cardiovascular system. In a clinical study, administration of androgen suppression therapy in patients with prostate cancer increased patients' risk of fatal cardiovascular disease and myocardial infarction 2 .
Testosterone is the principal member of the androgen family, and low serum testosterone levels were found to be common among patients with coronary disease and to negatively impact their survival [3][4][5] .
Androgens were also shown to have vasodilatory effects in different vascular beds, leading to disruption of vascular homeostasis 6 . One study showed androgen deprivation can reduce the angiogenic capability in mice 7 .
The endothelial cell lining of blood vessels is known to be a crucial regulator of vascular homeostasis, angiogenesis, and vascular remodeling. Upon vascular injury, the endothelium mediates the recruitment of pro-in ammatory leukocytes through expression of multiple cytokines and cell adhesion molecules 8, 9 . Activation of the endothelium causes vascular smooth muscle cells (VSMCs) to display an altered phenotype and subsequently to migrate and proliferate within the intimal compartment, with the result being the generation of a bromuscular plaque 10,11 . VSMC phenotypic modulation is an adaptive response for vascular homeostasis maintenance, but excessive stimuli-induced uncontrolled VSMC adaptation has been shown to lead to severe vascular injury 12,13 . The effects of androgens on the phenotype of VSMCs remains unclear at present. Although it has been demonstrated that both low and high concentrations of testosterone can increase cardiovascular risk by promoting excessive proliferation of VSMCs 14 , the underlying mechanisms remain largely unknown.
In our previous research, we found in the corpus cavernosum of a castration rat model that the smooth muscle content was signi cantly decreased and collagen deposition was remarkably increased 15 .
Moreover, it was shown that low androgens levels induce corporeal brosis by promoting apoptosis and inhibiting autophagy 16 . These results suggest that androgens may exert an essential role in maintaining vascular hemostasis by counter-regulating autophagy. Furthermore, inhibition of dihydrotestosterone (DHT) by long-term treatment with 5a-reductase inhibitors decreased the rate of autophagy and increased the rate of apoptosis via decreased LC3-and Beclin 1 expression 17 . Despite these investigations of the roles of androgens in vascular disease, the effects of androgens on thoracic aortic vascular function and the underlying mechanisms remain largely unclear.
In the present study, we investigated the effects of androgen (testosterone) de ciency on vasomotoricity in the thoracic arteries of the castrated rat model and determined the underlying mechanisms by which androgens counter-regulate autophagy in human umbilical vein smooth muscle cells (HUVSMCs).

Effects of androgen de ciency on thoracic arterial vasomotricity
Rat thoracic aortic rings harvested from the castration group exhibited enhanced endothelium-mediated contraction (Fig. 1A) and reduced endothelium-mediated relaxation (Fig. 1B, C) upon exposure to PE and acetylcholine Ach or to SNP, respectively, compared with those from the control group. These results suggest that the vascular endothelial cells and smooth muscle cells in the thoracic aortic rings were injured following castration of the rats.
To investigate whether this DHT-induced vascular injury was associated with autophagy, the rat thoracic aortic rings were pre-treated with rapamycin (autophagy promoter) or 3-MA (autophagy inhibitor) before treatment with PE and then increasing concentrations of DHT. The results showed that the androgeninduced vascular relaxation was signi cantly attenuated following treatment of the thoracic aortic rings with rapamycin (Fig. 2). By contrast, the thoracic aortic rings treated with 3-MA showed stronger vasodilation than those treated with rapamycin. These ndings suggest that autophagy is involved in the vascular injury induced by androgen de ciency.

Effect of androgen de ciency on autophagy in vascular endothelial cells and VSMCs
The morphology of the vascular endothelial cells and VSMCs within the aortic rings from each group of rats was observed by TEM. Compared with samples from the control group, those from the castration group showed increases in the number and size of vacuoles and lipid droplets in the cytosol (Fig. 3B) as well as an increase in autophagosomes (yellow arrows, Fig. 3A). Quantitative analysis indicated that the numbers of vacuoles ( Fig. 3C, P < 0.001) and autophagosomes ( Fig. 3D, P < 0.001) were signi cantly increased in the vascular endothelial cells and VSMCs of thoracic aortic samples from the castration group compared with the control group. Correspondingly, the relative area of autophagosomes within the cytoplasm also was remarkably greater for the castration group compared with the control group ( Fig. 3E, P < 0.001). These ndings suggest that androgen de ciency promotes autophagy among vascular endothelial cells and VSMCs in the rat castration model.

Regulatory role of androgens in VSMCs
The staining of hematoxylin-eosin (HE) was employed to evaluate the histological changes of thoracic aortic, and the histological changes between the castration group (

Effect of androgen treatment on HUVSMC proliferation
To investigate the proliferation of HUVSMCs exposed to androgen, HUVSMCs in culture were treated with different concentrations of DHT (0, 100, or 200 nM). The results showed that the HUVSMC proliferation rate was signi cantly inhibited by DHT treatment, and this inhibitory effect increased with increasing DHT concentration (Fig. 5A). To con rm this observation, ow cytometric analysis was performed to evaluate the cell cycle distribution among HUVSMCs treated with DHT. After exposure to DHT in culture, the percentage of HUVSMCs in the G0/G1 phase was signi cantly increased, whereas those in the G2 phase and S phase were markedly reduced (Fig. 5B). These results indicated that a high concentration of androgen altered the cell cycle progression of HUVSMCs by increasing the percentage of cells in G0/G1 phase and reducing the percentages of cells in G2 phase and S phase (Fig. 5B, C).

Discussion
In the present study, we found that androgen de ciency in the rat castration model led to enhanced endothelium-mediated contraction (Fig. 1A) and reduced endothelium-mediated relaxation (Fig. 1B, C) of rat thoracic arteries. Moreover, androgen-induced vasorelaxation could be attenuated by pre-treatment of the rat thoracic aortic rings with rapamycin, a promoter of autophagy, whereas pre-treatment with 3-MA, an autophagy inhibitor signi cantly increased vasodilation in the thoracic aortic rings. Furthermore, increases in the number and size of vacuoles and lipid droplets were observed in the cytosol of cells with the thoracic aortic rings of the model rats, along with the increased appearance of autophagosomes and apoptotic bodies in the endothelium and VSMCs. Masson's trichrome staining showed an increase in the area of smooth muscle in thoracic aortic rings of the model rats. Additionally, high concentrations of DHT inhibited HUVSMC proliferation in vitro.
The incidence rates of hypertension and coronary artery disease have long been known to be higher in men than in women [18][19][20] , but only recently has evidence begun to accumulate indicating that low concentrations of androgens are associated with vascular injury. Notably, male patients receiving androgen deprivation therapy have an increased risk of cardiovascular disease 21 , while exogenous testosterone supplementation protects cardiac myocytes 22 . Experiments in male mice showed that testosterone protects against atherosclerosis by targeting thymic epithelial cells 21 . In addition, androgens were shown to play an antihypertensive role in spontaneously hypertensive rats, while androgen de ciency could trigger the development of hypertension 23 . The vasorelaxing effect of androgens has been previously identi ed in various types of isolated vascular beds, but few studies have examined their effects on thoracic arteries. Research has shown though that different types of blood vessels have differing sensitivity to androgens 6 . For example, the coronary artery is more sensitive to androgen-mediated vasorelaxation 6 . In the present study, we investigated the effects of an androgen, DHT, on isolated thoracic aortic rings from male castrated rats. Adding to the previous observations that androgens exert a relaxing effect on both phenylephrine-and KCl-induced contraction in the mesenteric artery and thoracic aorta of male hypertensive rats 24 , we further con rmed in the thoracic aortic rings that androgen de ciency led to exhibited enhanced PE-induced contraction and reduced Ach-and SNPinduced vasorelaxation. These results indicate that androgen de ciency can cause vascular injury and negatively affect vascular homeostasis, with additional research needed to elucidate the underlying mechanisms.
We observed in thoracic aortic rings that DHT-induced vascular relaxation could be attenuated by treatment with rapamycin and DHT-induced vasodilation could be increased by 3-MA. In the cytosol of cells with in the thoracic aortic rings, the number and size of lipid droplets were increased in the rat castration model. We previously found in this model that the number of autophagosomes is decreased in cavernous smooth muscle cells 16 . In contrast, the present study revealed increased numbers of vacuoles, autophagosomes, and apoptotic bodies in specimens from the castration group. These ndings demonstrate that androgen de ciency led to increased autophagy in the thoracic arteries. The discrepancy between the ndings of the previous study and our present study are likely due to apoptosis among endothelial cells as a result of vascular injury.
In response to endothelial cell dysfunction, VSMCs, the main components of the arterial vessel wall, undergo a phenotypic switch from a contractile phenotype to a synthetic phenotype that is essential for vascular remodeling 25 . This VSMC phenotypic modulation is regulated by autophagy, and a previous study reported that the expression of the autophagy marker Beclin 1 is increased in shear stress-induced VSMC phenotypic modulation 26 . Autophagy plays an essential role in maintaining cellular homeostasis in physiological conditions 27,28 and participates in various physiological cellular processes as well as some pathological processes. A relationship between autophagy and cell proliferation has been demonstrated in many conditions 29,30 . Cell proliferation is controlled by progression through the cell cycle. The percentage of cells in the S phase were increased and the percentage of those in the G0/G1 phases were decreased when cell proliferation was enhanced. Previous research found that transitions from the G1 into the S phase of pulmonary arterial smooth muscle cell were enhanced under hypoxic conditions. And the enhancement of pulmonary arterial smooth muscle cell proliferation is related to the activation of autophagy 31 .
Androgens were shown to be crucial for the survival and proliferation of cells within the prostate gland 32 , and adrenal androgen dehydroepiandrosterone sulfate can inhibit VSMC proliferation 33 . Our results demonstrated that androgen de ciency lead to increased proliferation of VSMCs, and treatment of HUVSMCs with high concentrations of DHT increased the percentage of cells in G0/G1 phase and reduced the percentage of cells in G2 phase and S phase. These results revealed that androgens de ciency could markedly enhance the proliferation of HUVSMCs, and the enhancement of HUVSMCs proliferation may related to the activation of autophagy.
A major limitation of this current study is that we did not evaluate HUVSMC phenotypes. Moreover, how HUVSMC autophagy affects vascular homeostasis remains largely unknown. Additional studies are necessary to explore the direct effects of androgen-induced autophagy on HUVSMC phenotypes and vascular function both in vitro and in vivo.

Conclusions
In conclusion, androgen de ciency in rats resulting from castration can led to signi cant enhanced vasoconstriction and reduced vasodilatation through the induction of autophagic vascular endothelial cell death. Autophagy is essential for androgen de ciency-induced vascular injury in rat thoracic arteries. In addition, androgen de ciency altered structural features of the thoracic aorta and aggravated vascular injury by promoting proliferation of VSMCs. The results of the present study provide insight toward a better understanding of the effects of androgens in vascular diseases.

Transmission electron microscopy (TEM)
The harvested rat aortic rings were xed in glutaraldehyde for 2 h followed by post-xation in a 1% osmic acid solution at 4 °C for 2 h. The samples were then embedded in epoxy resin 618 embedding solution (TAAB Laboratories Equipment, Berks, UK). Thin sections were cut using an LKB V ultra-thin microtome (LKB, Sweden) and stained with lead citrate. Cytoplasmic vacuoles, lipid droplets, and autophagosomes were visualized by TEM (Philips CM-120, Eindhoven, The Netherlands). The degree of autophagy was quanti ed according to the number of autophagosomes as well as the ratio of autophagic vesicles to the total cytoplasmic area.
Masson's trichrome staining According to our previously described protocol 17

Flow cytometric analysis
HUVSMCs were seeded in 6-well culture plates and dispersed into a single cell suspension after treatment with DHT (0, 50, or 200 nM). Aliquots of 1 million cells in 10 µl were stained with uorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) for 10-15 min at room temperature protected from light. Then 400 µl buffer solution was added to each sample, and all liquid in each sample was transferred to a ow analysis tube. Flow cytometry was used to analyze the cell cycle distribution within each sample.

Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). Data were compared among the groups by one-way analysis of variance (ANOVA) using SPSS software (SPSS21.0, Inc., Chicago, IL, USA Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by grants from the National Natural Science Foundation of China (81771578) and the project of Science and Technology Commission of Shanghai Municipality (14ZR1426200). The founder is the corresponding author of this article, and he designed and supervised this investigation.
Authors' contributions ZMG: designed and supervised this investigation. ZNN, LM, GFB and WXJ: performed this investigation, and they contributed equally to this work. DF and SYF contributed to the data collection. ZS, CSW and DQ: provided technical or material support. All authors read and approved the nal manuscript.  Autophagy of vascular endothelial cells and vascular smooth muscle cells (VSMCs) within rat thoracic arteries. Transmission electron microscopy (TEM) showed that the numbers of vacuoles, lipid droplets (LDs), and autophagosomes (yellow arrows) were greater in the vascular endothelial cells and VSMCs of the thoracic arteries of specimens harvested from the castrated rats (B) than in those from control rats (A). Numbers of vacuoles (C) and autophagosomes (D) in vascular endothelial cells and VSMCs of specimens from the control and castration groups. (E) Ratio of the area of autophagosomes to the total cytoplasmic area in cells within specimens from each group. n=3 in each group. *P<0.01, #P<0.001.

Figure 4
Representative images of rat thoracic aortic rings from the control and castration groups stained with hematoxylin-eosin (HE, A, B) and Masson's trichrome stain (C, D) to reveal structural features.