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Am J Physiol Heart Circ Physiol. 2014 Jun 1;306(11):H1485-94. doi: 10.1152/ajpheart.00809.2013. Epub 2014 Mar 21.

Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement.

Author information

1
Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; rheurealves@usp.br.
2
Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil; and.
3
Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil;
4
Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; School of Physical Education and Sports of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil.

Abstract

Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10(-7) mol/l) or vehicle (2-12 h) in the presence of flutamide (10(-5) mol/l), CCCP (10(-6) mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10(-5) mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10(-5) mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome-c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2(ΔΔCt), 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.

KEYWORDS:

ROS generation; apoptosis; mitochondria; testosterone

PMID:
24658017
DOI:
10.1152/ajpheart.00809.2013
[Indexed for MEDLINE]
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