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Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with
Rheure Alves Moreira Lopes1, Karla Bianca Neves2, Cezar Rangel Pestana2
1Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; rheurealves@usp.br.
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.
Insights
Testosterone triggers apoptosis in vascular smooth muscle cells (VSMCs) by increasing reactive oxygen species (ROS) through mitochondria. This process involves the extrinsic apoptotic pathway and androgen receptor activation.
Area of Science:
- Cardiovascular Science
- Cell Biology
- Endocrinology
Background:
- Testosterone has dual effects on the cardiovascular system, with potential harmful impacts.
- Reactive oxygen species (ROS) are implicated in cell death signaling pathways.
- Vascular smooth muscle cells (VSMCs) play a critical role in cardiovascular health.
Purpose of the Study:
- To investigate if testosterone induces apoptosis in VSMCs.
- To determine the role of mitochondria-dependent ROS generation in testosterone-induced VSMC apoptosis.
- To elucidate the specific apoptotic pathways and mechanisms involved.
Main Methods:
- Cultured VSMCs were treated with testosterone and various inhibitors (flutamide, CCCP, MnTMPyP, Z-IETD-FMK).
- ROS generation was measured using lucigenin and dichlorodihydrofluorescein assays.
- Apoptosis was assessed via annexin V and calcein staining, and caspase activation was analyzed by immunoblotting.
Main Results:
- Testosterone significantly increased ROS generation and procaspase-3 activation in VSMCs.
- Testosterone induced apoptosis, evidenced by increased annexin-V and decreased calcein fluorescence.
- Testosterone promoted caspase-8 cleavage and increased gene expression of Fas ligand and TNF-α, indicating extrinsic pathway activation.
Conclusions:
- Testosterone induces apoptosis in VSMCs through the extrinsic apoptotic pathway.
- Mitochondria-generated ROS, mediated by androgen receptor activation, are crucial in this process.
- These findings highlight a mechanism by which testosterone may negatively impact cardiovascular health.
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