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 31, 2017
Citation Information: JCI Insight. 2017;2(2):e90427. doi:10.1172/jci.insight.90427.
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Categories: 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 1

Unrepaired DNA damage in PAECs isolated from D&T-PAH patients.

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Unrepaired DNA damage in PAECs isolated from D&T-PAH patients. Pulmo...
Pulmonary artery endothelial cells (PAECs) were isolated from lung explants of unused control donor (Donor-Ctl, n = 4) and from patients with pulmonary arterial hypertension (PAH) associated with amphetamine (AMPH), methamphetamine (METH) or anorexigens (D&T-PAH, n = 4, including 3 AMPH-PAH and 1 anorexigen-PAH), as described under Methods. PAECs were treated in full medium with vehicle (Veh) or doxorubicin (Dox) for 6 hours. A separate set of Dox-treated cells was allowed to recover (Dox-recovery) for 12 hours in A, or for 6 hours in B. (A) Cells were analyzed by alkaline comet assay. Representative images show intact DNA (comet head), and comet tails, indicative of damaged DNA. The percentage of DNA in the tail of 100 to 150 cells per subject was analyzed using ImageJ. Scale bar: 60 μm. (B) Cells were immunostained for γH2AX and DAPI to assess DNA damage. Representative images show DNA damage foci in the nuclei. Scale bar: 30 μm. Number of γH2AX foci per cell was quantified using ImageJ. Ten to 15 confocal images were assessed per subject. 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 = 4, Donor-Ctl PAECs, and n = 4, D&T-PAH PAECs, each line of PAEC has 3–4 replicate images. ****P < 0.0001 vs. Donor-Ctl+Veh; ##P < 0.005 and ####P < 0.0001 vs. Donor-Ctl+Dox; &&&&P < 0.0001 vs. D&T-PAH+Dox; $$$$P < 0.0001 vs. Donor-Ctl+Dox-recovery, by 2-way ANOVA, Bonferroni’s post-test.