In the present study, we found that androgen deficiency 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 significantly 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 18–20, 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 deficiency could trigger the development of hypertension 23. The vasorelaxing effect of androgens has been previously identified 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 confirmed in the thoracic aortic rings that androgen deficiency led to exhibited enhanced PE-induced contraction and reduced Ach- and SNP-induced vasorelaxation. These results indicate that androgen deficiency 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 findings demonstrate that androgen deficiency led to increased autophagy in the thoracic arteries. The discrepancy between the findings 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 deficiency 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 deficiency 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.