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Oxido-reductive regulation of vascular remodeling by receptor tyrosine kinase ROS1
Ziad A. Ali, … , Thomas Quertermous, Euan A. Ashley
Ziad A. Ali, … , Thomas Quertermous, Euan A. Ashley
Published November 17, 2014
Citation Information: J Clin Invest. 2014;124(12):5159-5174. https://doi.org/10.1172/JCI77484.
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Research Article Article has an altmetric score of 38

Oxido-reductive regulation of vascular remodeling by receptor tyrosine kinase ROS1

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Abstract

Angioplasty and stenting is the primary treatment for flow-limiting atherosclerosis; however, this strategy is limited by pathological vascular remodeling. Using a systems approach, we identified a role for the network hub gene glutathione peroxidase-1 (GPX1) in pathological remodeling following human blood vessel stenting. Constitutive deletion of Gpx1 in atherosclerotic mice recapitulated this phenotype of increased vascular smooth muscle cell (VSMC) proliferation and plaque formation. In an independent patient cohort, gene variant pair analysis identified an interaction of GPX1 with the orphan protooncogene receptor tyrosine kinase ROS1. A meta-analysis of the only genome-wide association studies of human neointima-induced in-stent stenosis confirmed the association of the ROS1 variant with pathological remodeling. Decreased GPX1 expression in atherosclerotic mice led to reductive stress via a time-dependent increase in glutathione, corresponding to phosphorylation of the ROS1 kinase activation site Y2274. Loss of GPX1 function was associated with both oxidative and reductive stress, the latter driving ROS1 activity via s-glutathiolation of critical residues of the ROS1 tyrosine phosphatase SHP-2. ROS1 inhibition with crizotinib and deglutathiolation of SHP-2 abolished GPX1-mediated increases in VSMC proliferation while leaving endothelialization intact. Our results indicate that GPX1-dependent alterations in oxido-reductive stress promote ROS1 activation and mediate vascular remodeling.

Authors

Ziad A. Ali, Vinicio de Jesus Perez, Ke Yuan, Mark Orcholski, Stephen Pan, Wei Qi, Gaurav Chopra, Christopher Adams, Yoko Kojima, Nicholas J. Leeper, Xiumei Qu, Kathia Zaleta-Rivera, Kimihiko Kato, Yoshiji Yamada, Mitsutoshi Oguri, Allan Kuchinsky, Stanley L. Hazen, J. Wouter Jukema, Santhi K. Ganesh, Elizabeth G. Nabel, Keith Channon, Martin B. Leon, Alain Charest, Thomas Quertermous, Euan A. Ashley

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Figure 9

Catalytic and backdoor cysteines inhibit ROS1 deactivation in vitro and in vivo.

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Catalytic and backdoor cysteines inhibit ROS1 deactivation in vitro and ...
(A) Cys/Ser site-directed mutagenesis of catalytic Cys 463 and backdoor Cys 333 and Cys 367 were performed and phosphatase activity was assessed with and without stimulation. C367S did not affect phosphatase activity, whereas C333S led to a 90% reduction in phosphatase activity, and C463S abolished all activity compared with wild-type. *P < 0.05 compared with wild-type; **P < 0.05 compared with C333. (B) SHP-2 time-dependent inactivation by H2O2 and reactivation by DTT. Full-length SHP-2 was oxidized or glutathiolated and phosphatase activities measured in a continuous assay, after which DTT was added. Both oxidation and glutathiolation could induce deactivation of SHP-2; however, recovery following addition of the reducing agent DTT was significantly impaired in the glutathiolation group. *P < 0.05 compared with H2O2 + DTT. (C) SHP-2 is glutathiolated in vivo in experimental animals. Western blotting for glutathiolated proteins in vessel homogenate identified increased band intensity in Gpx1–/– Apoe–/– mice compared with Gpx1+/+ Apoe–/–. Balloon angioplastied vessels were homogenized and immunoprecipitated for ROS1 or SHP-2 under nonreducing conditions. The immunoprecipitated protein was immunoblotted with anti-GSH antibody. Blotting of immunoprecipitate identified the presence of glutathiolated SHP-2, but also ROS1, suggesting a physical interaction in vivo. (D) Increased ROS1 activity in Gpx1–/– Apoe–/– mice in vivo. Balloon angioplastied vessels harvested 28 days postprocedurally were assessed for Ros1 phosphorylation. Levels of ROS1 phosphorylation were higher in Gpx1–/– Apoe–/– mice (P < 0.05) without differences in ROS1 protein.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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