Dasatinib induces lung vascular toxicity and predisposes to pulmonary hypertension
Christophe Guignabert, Carole Phan, Andrei Seferian, Alice Huertas, Ly Tu, Raphaël Thuillet, Caroline Sattler, Morane Le Hiress, Yuichi Tamura, Etienne-Marie Jutant, Marie-Camille Chaumais, Stéphane Bouchet, Benjamin Manéglier, Mathieu Molimard, Philippe Rousselot, Olivier Sitbon, Gérald Simonneau, David Montani, Marc Humbert
Christophe Guignabert, Carole Phan, Andrei Seferian, Alice Huertas, Ly Tu, Raphaël Thuillet, Caroline Sattler, Morane Le Hiress, Yuichi Tamura, Etienne-Marie Jutant, Marie-Camille Chaumais, Stéphane Bouchet, Benjamin Manéglier, Mathieu Molimard, Philippe Rousselot, Olivier Sitbon, Gérald Simonneau, David Montani, Marc Humbert
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Research Article Vascular biology

Dasatinib induces lung vascular toxicity and predisposes to pulmonary hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a life-threatening disease that can be induced by dasatinib, a dual Src and BCR-ABL tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). Today, key questions remain regarding the mechanisms involved in the long-term development of dasatinib-induced PAH. Here, we demonstrated that chronic dasatinib therapy causes pulmonary endothelial damage in humans and rodents. We found that dasatinib treatment attenuated hypoxic pulmonary vasoconstriction responses and increased susceptibility to experimental pulmonary hypertension (PH) in rats, but these effects were absent in rats treated with imatinib, another BCR-ABL tyrosine kinase inhibitor. Furthermore, dasatinib treatment induced pulmonary endothelial cell apoptosis in a dose-dependent manner, while imatinib did not. Dasatinib treatment mediated endothelial cell dysfunction via increased production of ROS that was independent of Src family kinases. Consistent with these findings, we observed elevations in markers of endothelial dysfunction and vascular damage in the serum of CML patients who were treated with dasatinib, compared with CML patients treated with imatinib. Taken together, our findings indicate that dasatinib causes pulmonary vascular damage, induction of ER stress, and mitochondrial ROS production, which leads to increased susceptibility to PH development.

Authors

Christophe Guignabert, Carole Phan, Andrei Seferian, Alice Huertas, Ly Tu, Raphaël Thuillet, Caroline Sattler, Morane Le Hiress, Yuichi Tamura, Etienne-Marie Jutant, Marie-Camille Chaumais, Stéphane Bouchet, Benjamin Manéglier, Mathieu Molimard, Philippe Rousselot, Olivier Sitbon, Gérald Simonneau, David Montani, Marc Humbert

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Figure 5

Dasatinib stimulates mitochondrial ROS production in vivo and in vitro.

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Dasatinib stimulates mitochondrial ROS production in vivo and in vitro. ...
(A) Representative images obtained by confocal microscopic analyses and double labeling with 8-oxo-2′-deoxyguanosine (8-oxo-dG) and the endothelial-specific marker vWF in lungs from vehicle-, dasatinib-, and imatinib-treated rats. (B) Quantifications of the oxidized glutathione/total glutathione ratio and of the protein carbonyl products in lungs and serum of vehicle-, dasatinib-, and imatinib-treated rats, respectively. (C) Representative images and quantification of intracellular ROS generation using fluorogenic probes (CellRox) and dihydroethidium (DHE) in pulmonary ECs treated 30 minutes with vehicle, dasatinib, or imatinib. (D) Gene expression analysis of pro-oxidative enzymes (NADPH oxidase 4 [NOX4], NO synthase 2 [NOS2], heme oxygenase 1 [HMOX1], GA-binding protein α chain [GABPA], flavin-containing monooxygenase 2 [FMO2]) and antioxidative enzymes (catalase [CAT], glutathione peroxidase 1 [GPX1], superoxide dismutase 1 [SOD1]) in lungs of rats treated with vehicle, dasatinib, and imatinib. (E) Mitochondria were labeled with MitoTracker Green (green) and mitochondrial ROS with MitoSOX Red (red). (F–G) Quantitative FACS analysis of the annexin V and propidium iodide (PI) dual labeling and quantification of caspase-3/7 activity in pulmonary ECs pretreated or not for 16 hours with N-acetylcysteine (NAC) at 5 mM and exposed or not to 400 nM dasatinib. (H) Representative images and quantification of the TUNEL staining in pulmonary ECs pretreated or not for 1 hour with NAC at 5 mM and exposed or not to 400 nM dasatinib. Horizontal lines display the mean ± SEM (n = 4–9). *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated cells or -treated rats; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. pulmonary ECs treated with 400 nM dasatinib or rats treated with dasatinib (10×). Scale bars: 20 μm.