Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension
Pin-I Chen, … , Brian J. Feldman, Marlene Rabinovitch
Pin-I Chen, … , Brian J. Feldman, Marlene Rabinovitch
Published January 26, 2017
Citation Information: JCI Insight. 2017;2(2):e90427. https://doi.org/10.1172/jci.insight.90427.
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Research Article Cell biology Vascular biology

Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension

Abstract

Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by γH2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1α, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome c oxidase 4 isoform switch. Mitochondrial oxidative phosphorylation is inappropriately enhanced and, as a result of impaired electron transport and mitochondrial ROS increase, caspase-3 is activated and DNA damage is induced. In mice given binge doses of METH followed by hypoxia, HIF1α is suppressed and pulmonary artery DNA damage foci are associated with worse pulmonary vascular remodeling. Thus, chronic AMPH/METH can induce DNA damage associated with vascular disease by subverting the adaptive responses to oxidative stress.

Authors

Pin-I Chen, Aiqin Cao, Kazuya Miyagawa, Nancy F. Tojais, Jan K. Hennigs, Caiyun G. Li, Nathaly M. Sweeney, Audrey S. Inglis, Lingli Wang, Dan Li, Matthew Ye, Brian J. Feldman, Marlene Rabinovitch

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Figure 2

Amphetamine exacerbates genotoxic stress–induced DNA damage.

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Amphetamine exacerbates genotoxic stress–induced DNA damage. (A) Commerc...
(A) Commercially available human pulmonary artery endothelial cells (PAECs) were treated in full medium with indicated doses of amphetamine (AMPH) daily for 3 days, and then stimulated with doxorubicin (Dox, 0.2 μg/ml) for 24 hours in the presence of vehicle (Veh) or AMPH. Cell lysates were immunoblotted for γH2AX and β-actin (for normalization). (B) PAECs were treated with 1 mM AMPH daily for 3 days, and then stimulated with Dox in the presence of Veh or AMPH for 6 hours. Cells were analyzed by comet assay as described for Figure 1. Scale bar: 60 μm. (C) Cells treated as in B were immunostained for γH2AX (red), pDNA-PK (green), and DAPI (blue). Ten to 15 confocal images with at least 40 cells per image per condition were analyzed using ImageJ. Scale bar: 15 μm. (D) PAECs were treated daily for 3 days with the indicated dose of AMPH, and then cultured with Veh or AMPH under 0.5% O2 hypoxia (Hx) or normoxia (Nx) for 48 hours. Cell lysates were assayed as in A. (E) Comet assay of PAECs treated with 0.5 mM AMPH and Hx/Nx as in D. Scale bar: 60 μm. (A and D) Dot plots represent mean ± SEM, n = 3–4. (B, C, and E) Box-and-whisker plots represent values within the interquartile range (boxes) and the minimum to maximum (whiskers). The line within the box shows the median. n = 3 independent experiments with 3 to 5 replicates per experiment. (A–C) *P < 0.05, **P < 0.005 vs. Veh; ##P < 0.005 vs. Dox+Veh. (D and E) *P < 0.05, **P < 0.005, ***P < 0.0005 vs. Nx+Veh; #P < 0.05, ##P < 0.005, ####P < 0.0001 vs. Hx+Veh or Vec+Hx+Veh; &P < 0.05, &&P < 0.005, &&&&P < 0.0001 vs. Hx+AMPH or Vec+Hx+AMPH, by 2-way ANOVA, Bonferroni’s post-test.