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Androgens modulate male-derived endothelial cell homeostasis using androgen receptor-dependent and receptor-independent mechanisms

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Abstract

Background

Sex-related differences in the role of androgen have been reported in cardiovascular diseases and angiogenesis. Moreover, androgen receptor (AR) has been causally involved in the homeostasis of human prostate endothelial cells. However, levels of expression, functionality and biological role of AR in male- and female-derived human endothelial cells (ECs) remain poorly characterized. The objectives of this work were (1) to characterize the functional expression of AR in male- and female-derived human umbilical vein endothelial cell (HUVEC), and (2) to specifically analyze the biological effects of DHT, and the role of AR on these effects, in male-derived HUVECs (mHUVECs).

Results

Immunohistochemical analyses of tissue microarrays from benign human tissues confirmed expression of AR in ECs from several androgen-regulated and non-androgen-regulated human organs. Functional expression of AR was validated in vitro in male- and female-derived HUVECs using quantitative RT-PCR, immunoblotting and AR-mediated transcriptional activity assays. Our results indicated that functional expression of AR in male- and female-derived HUVECs was heterogeneous, but not sex dependent. In parallel, we analyzed in depth the biological effects of DHT, and the role of AR on these effects, on proliferation, survival and tube formation capacity in mHUVECs. Our results indicated that DHT did not affect mHUVEC survival; however, DHT stimulated mHUVEC proliferation and suppressed mHUVEC tube formation capacity. While the effect of DHT on proliferation was mediated through AR, the effect of DHT on tube formation did not depend on the presence of a functional AR, but rather depended on the ability of mHUVECs to further metabolize DHT.

Conclusions

(1) Heterogeneous expression of AR in male- and female-derived HUVEC could define the presence of functionally different subpopulations of ECs that may be affected differentially by androgens, which could explain, at least in part, the pleiotropic effects of androgen on vascular biology, and (2) DHT, and metabolites of DHT, generally thought to represent progressively more hydrophilic products along the path to elimination, may have differential roles in modulating the biology of human ECs through AR-dependent and AR-independent mechanisms, respectively.

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Acknowledgments

This study was supported by the Department of Defense W81XWH-12-1-0341 and FONDECYT Regular (1130051 and 1161115) Grants to A. Godoy and FONDECYT Iniciación (11140255) to P. Sotomayor. V. Torres-Estay was supported by a Ph.D. fellowship from CONICYT. The authors thank Dr. Yusser Olguín from Universidad Andres Bello for the graphic design support.

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    Authors and Affiliations

    1. Department of Physiology, Pontificia Universidad Católica de Chile, Av. Libertador Bernardo O’Higgins 340, Santiago, Chile

      Verónica Torres-Estay, Daniela V. Carreño, Patricia Fuenzalida & Alejandro S. Godoy

    2. Department of Urology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, 14263, USA

      Anica Watts, Gary J. Smith & Alejandro S. Godoy

    3. Department of Urology, Pontificia Universidad Católica de Chile, Av. Libertador Bernardo O’Higgins 340, Santiago, Chile

      Ignacio F. San Francisco

    4. Department of Hematology-Oncology, Pontificia Universidad Católica de Chile, Av. Libertador Bernardo O’Higgins 340, Santiago, Chile

      Viviana P. Montecinos

    5. Center for Integrative Medicine and Innovative Science, Universidad Andres Bello, Santiago, Chile

      Paula C. Sotomayor

    6. Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, 14263, USA

      John Ebos

    7. Department of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, 14263, USA

      John Ebos

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    1. Verónica Torres-Estay
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    Gary J. Smith and Alejandro S. Godoy have contributed equally to this work.

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    10456_2016_9525_MOESM1_ESM.tif

    Supplementary Figure 1. Cellular distribution of AR in mHUVECs. mHUVECs were stimulated for 24 h to vehicle (ethanol) or 1 nM dihydrotestosterone (DHT). Nuclear (N) and cytoplasmic (C) cellular extracts were prepared using the NE-PER kit (ThermoFisher Scientific). Membranes and nuclei were probed using antibodies against AR. Histone H3 (nuclear) and β-tubulin (cytoplasmic) were used as loading controls (TIFF 5416 kb)

    10456_2016_9525_MOESM2_ESM.tif

    Supplementary Fig. 2. FXR expression in mHUVECs. (A) RT-PCR analysis of expression of FXR in HUVECs obtained from 4 different donors and derived from female (lanes 1 and 2) and male (lanes 3 and 4) fetuses. HepG2 cells were used as positive control (lane 5). GAPDH was used as loading control. (B) RT-PCR analysis of expression of FXR directed against four different regions of the FXR gene in HUVECs exposed to vehicle (lanes 1) or 1 nM DHT (lanes 2). HepG2 cells were used as positive control (lanes 3). GAPDH was used as loading control (TIFF 6509 kb)

    Supplementary material 3 (DOCX 17 kb)

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    Torres-Estay, V., Carreño, D.V., Fuenzalida, P. et al. Androgens modulate male-derived endothelial cell homeostasis using androgen receptor-dependent and receptor-independent mechanisms. Angiogenesis 20, 25–38 (2017). https://doi.org/10.1007/s10456-016-9525-6

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