TY - JOUR
T1 - Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement
AU - Lopes, Rheure Alves Moreira
AU - Neves, Karla Bianca
AU - Pestana, Cezar Rangel
AU - Queiroz, André Lima
AU - Zanotto, Camila Ziliotto
AU - Chignalia, Andréia Z.
AU - Valim, Yara Maria
AU - Silveira, Leonardo R.
AU - Curti, Carlos
AU - Tostes, Rita C.
PY - 2014/6/1
Y1 - 2014/6/1
N2 - 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 (2ΔΔCt, 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.
AB - 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 (2ΔΔCt, 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.
KW - Apoptosis
KW - Mitochondria
KW - ROS generation
KW - Testosterone
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U2 - 10.1152/ajpheart.00809.2013
DO - 10.1152/ajpheart.00809.2013
M3 - Article
C2 - 24658017
AN - SCOPUS:84901717244
SN - 0363-6135
VL - 306
SP - H1485-H1494
JO - American Journal of Physiology - Heart and Circulatory Physiology
JF - American Journal of Physiology - Heart and Circulatory Physiology
IS - 11
ER -