In VSMCs, vascular injury induces activation of ROS1 through either a yet-unidentified ligand (ligand X) or endogenous phosphorylation. (A) Concomitant vascular injury also leads to decreased transcription of GPX1 (i), which leads to a decrease in GPX1 enzymatic activity during a time of increased GSH synthesis, resulting in increased intracellular GSH (ii). Under oxidative conditions, high GSH leads to s-glutathiolation of proteins, including the ROS1 regulatory protein tyrosine phosphatase SHP-2 (iii). (B) s-Glutathiolation deactivates SHP-2, which in turn leads to sustained phosphorylation and activation of ROS1. Sustained phosphorylation of ROS1 amplifies its ability to activate kinases involved in proliferation, migration, and apoptosis through downstream target phosphorylation (iv).

(A) Relative area of atherosclerosis in the aortic root was significantly increased in Gpx1^–/– Apoe^–/– mice, but addition of genistein to inhibit ROS1 abolished this increase, while crizotinib did not. Scale bar: 50 μm. (B) Gpx1^–/– Apoe^–/– mice 4 weeks following BAS developed significantly greater neointimal hyperplasia and in-stent stenosis compared with control Gpx1^+/+ Apoe^–/–. Addition of genistein or crizotinib decreased neointimal hyperplasia to levels observed with the mTOR inhibitor rapamycin. AdGRX1 decreased neointimal hyperplasia to levels similar to those seen with crizotinib in Gpx1^–/– Apoe^–/– vessels, but not Gpx1^+/+ Apoe^–/–, suggesting deglutathiolation of SHP-2 as the mechanism. Scale bar: 50 μm. Black arrowheads denote neointima. Although overall neointimal cell density was higher in Gpx1^–/– Apoe^–/– mice, there was no difference in cell density when expressed per unit area between groups; however, addition of genistein, crizotinib, or rapamycin significantly decreased cell density per unit area in both experimental groups. Treatment with AdGrx1 reduced cell density only in Gpx1^–/– Apoe^–/– mice. *P < 0.05 compared with control; ^†P < 0.05 compared with AdGFP. n = 8–15/group. (C) Assessment of reendothelialization revealed that the ROS1 inhibitor crizotinib did not impair endothelial regeneration 4 weeks following BAS, while rapamycin did in both Gpx1^–/– Apoe^–/– Tie2-LacZ⁺ mice and control Gpx1^+/+ Apoe^–/– Tie2-LacZ⁺ mice. *P < 0.05 versus crizotinib. n = 6–10 mice/group. Gray arrowheads denote neointima. Black arrowheads denote LacZ-positive endothelial cells. Scale bar: 50 μm.

(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.

(A) HEK293 cells transfected with myc-tagged ROS1 and incubated with biotinylated GSH-ethyl-ester induced cysteine activation and s-glutathiolation of many proteins inhibitable by the reducing agent DTT. (B) s-Glutathiolation of ROS1 was not detected following immunoprecipitation; however, other GSS-protein conjugates were detected. Peptide mass fingerprinting showed that SHP-1 and SHP-2 were coimmunoprecipitated with ROS1, and (C) immunoprecipitation identified s-glutathiolation of SHP-2. LC-ESI-MS/MS identified the sites of glutathiolation. Extracted ion chromatograms of the SHP-2 peptides (D) Q450-R469 and (E) S326-R343 in the native form (top panel, control), s-glutathiolated form (middle panel), and reduced form (bottom panel) identify the catalytic Cys 463 and backdoor Cys 333 as the sites of s-glutathiolation. Insets are the representative MS/MS spectra of the Q450-R469 and S326-R343 peptide, providing site-specific localization of s-glutathiolation in the case of the modified forms. Upper insets represent the specific B and y ions as detected in the MS/MS spectra, showing inter-residue bond breakage generating b₁₃ and y₇ ions for Cys 463 and b₈ and y₁₁ for Cys 333, providing absolute confirmation. (F) (Upper panel and inset on left) Schematic representation of SHP-2 showing catalytic Cys 463 and backdoor Cys 333 and Cys 367 in the active PTP domain. (Inset on right) Modeling of s-glutathiolation of catalytic Cys 463 depicted by the spheres, demonstrating a physical presence within the active phosphatase domain of the enzyme.

Following balloon angioplasty and treatment with high concentrations of GSH, primary aortic VSMC proliferation was inhibited by siROS1 in Gpx1^+/+ Apoe^–/– mice, while Gpx1^–/– Apoe^–/– cells were not affected. Adenoviral overexpression of GRX1 inhibited proliferation in both groups, while siGRX1 increased proliferation in Gpx1^+/+ Apoe^–/– VSMCs, suggesting proliferation was mediated by protein s-glutathiolation. Representative images of cell proliferation (green fluorescence) in Gpx1^+/+ Apoe^–/– mice treated with GSH and siROS1 or AdGRX1. *P < 0.05 vs. control; ^†P < 0.05 vs. AdGFP. Scale bar: 20 μm.

(A) Vascular injury led to a significant increase in the expression of enzymes involved in biosynthesis of GSH, suggesting vascular injury as a major determinant of GSH reductive stress in Gpx1^–/– Apoe^–/– vessels following angioplasty. *P < 0.05. (B) These findings were confirmed in human in-stent neointima. The expression of enzymes involved in biosynthesis of GSH was significantly increased compared with human internal mammary artery harvested at the time of coronary artery bypass surgery. *P < 0.05. Fold change in mRNA versus GAPDH. (C) mRNA levels of enzymes involved in the metabolic fate of GSH showed absence of expression of Gpx1 in Gpx1^–/– Apoe^–/– vessels and an increase in expression of the deglutathiolating enzyme Grx1. *P < 0.05. Fold change in mRNA versus GAPDH. Gsr, glutathione reductase; G6pd, glucose6-phosphate dehydrogenase; Gst, glutathione S-transferase; Ggt, glutathione hydrolase.

(A) ROS1 activity, assessed semiquantitatively, expressed as phosphorylated ROS1, was detectable in aortic lysates of Gpx1^–/– Apoe^–/– mice. *P < 0.05 versus Gpx1^+/+ Apoe^–/– P-ROS1; ^†P < 0.05 versus Gpx1^+/+ Apoe^–/– GSH. (B and C) Deficiency of GPX1 in Gpx1^–/– Apoe^–/– mice that underwent balloon angioplasty led to a time-dependent increase in intracellular GSH levels and GSH/GSSG ratios with a (D) corresponding increase in ROS1 phosphorylation indicative of increasing ROS1 activity. *P < 0.05 compared with same time point; **P < 0.05 across time points. n = 3–6/group.

(A) Transcriptional profiling of human coronary artery atherectomy specimens of control (AHA class I lesions), de novo atherosclerosis (AHA class III–V lesions), or in-stent stenosis identified that ROS1 expression was increased in atherosclerosis and furthermore in in-stent stenosis. *P < 0.01 vs. control; ^#P < 0.05 vs. atherosclerosis. n = 55 atherosclerosis/34 in-stent stenosis. (B) mRNA levels of Ros1 in Gpx1^+/+ Apoe^–/– mice were dramatically higher in aorta that had undergone BAS compared with uninjured aorta harvested at 28 days. *P < 0.001. n = 4/group. (C) HCASMCs were grown under both serum-fed proliferative and serum-starved nonproliferative phenotypes. ROS1 expression was minimal, but was upregulated as these cells differentiated into “contractile” phenotype and further by confluence, returning to contractile phenotype levels following passage. ROS1 expression was significantly decreased by siROS1. *P < 0.05 vs. (+) serum; **P < 0.05 vs. confluent; ^†P < 0.05 vs. (–) serum. (D) In primary aortic VSMCs from experimental animals that underwent angioplasty, proliferation was higher in Gpx1^–/– Apoe^–/– VSMCs compared with control Gpx1^+/+ Apoe^–/– VSMCs. siROS1, genistein, and crizotinib decreased proliferation in Gpx1^–/– Apoe^–/– mice, but not in Gpx1^+/+ Apoe^–/– controls, indicating that the phenotype was dependent on GPX1 deficiency. Daidzein, the structural analogue of genistein with similar antioxidant effects but no effect on tyrosine kinase inhibition, did not inhibit proliferation in Gpx1^–/– Apoe^–/– cells. Addition of siROS1 to Daidzein showed a reduction in proliferation similar to that of siROS1 alone. *P < 0.05 vs. Gpx1^+/+ Apoe^–/–; ^†P < 0.05 vs. baseline Gpx1^–/– Apoe^–/–.

(A) Vascular superoxide production, measured by DHE fluorescence, in Gpx1^–/– Apoe^–/– mice was higher than in the Gpx1^+/+ Apoe^–/– mouse medial layer (red fluorescence), but not the endothelium or adventitia. Elastic laminae exhibit green autofluorescence. Scale bar: 5 μm. *P < 0.01. n = 6/group. (B) Aortic superoxide production using lucigenin chemiluminescence was higher in Gpx1^–/– Apoe^–/– compared with Gpx1^+/+ Apoe^–/– mice without a contribution by the eNOS inhibitor L-NAME, suggesting minimal contribution from endothelium. *P < 0.01. n = 7/group. (C) Net NO levels were significantly decreased in Gpx1^–/– Apoe^–/– compared with Gpx1^+/+ Apoe^–/– mice that underwent BAS using Fe-DETC EPR. L-NAME did not contribute to net NO levels. n = 6/group. (D) Immunoblotting for eNOS protein in aortic lysates revealed no difference in eNOS band intensity normalized to GAPDH control between groups. n = 6–8.

In Gpx1^–/– Apoe^–/– and Gpx1^+/+ Apoe^–/– mice additionally expressing β-gal (LacZ) targeted to endothelial cells (Tie2), en face macroscopic vessel images identify (A) stents struts (black arrowheads), endothelium (blue stain) on the luminal surface, and (B) areas with no endothelium (gray arrowheads). Scale bar: 0.5 mm. (C) Reendothelialization was impaired in Gpx1^–/– Apoe^–/– Tie2-LacZ⁺ mice 4 weeks following BAS. Left panel shows high-power en face microscopy images of the luminal surface of the stented vessel. Right panel shows cross-sectional histomorphometry. *P < 0.05. n = 6–10 mice/group. White arrowheads denote stent struts; black arrowheads denote endothelial cells. Scale bar: 50 μm.

(A) Transcriptional profiling of human coronary atherectomy specimens of control (AHA class I lesions), atherosclerosis (AHA class III–V lesions), or in-stent stenosis identified that GPX1 expression was incrementally lower in atherosclerosis and in-stent stenosis. *P < 0.01 vs. control; ^#P < 0.05 vs. atherosclerosis. n = 55 atherosclerosis/34 in-stent stenosis. (B) Relative area of atherosclerosis in the aortic root and lesion burden in aorta were increased in Gpx1^–/– Apoe^–/– mice compared with Gpx1^+/+ Apoe^–/– mice (*P < 0.02, n = 8–15/group). Black scale bar: 50 μm. White scale bar: 0.5 mm. (C) mRNA levels of Gpx1 in Gpx1^+/+ Apoe^–/– mice were lower in aorta that had undergone BAS compared with uninjured aorta harvested at 28 days. *P = 0.05. n = 4/group. (D) Gpx1^–/– Apoe^–/– mice 4 weeks following BAS developed significantly greater neointimal hyperplasia compared with control Gpx1^+/+ Apoe^–/–. *P < 0.05. Although overall neointimal cell density was higher in Gpx1^–/– Apoe^–/– mice, there was no difference in cell density when expressed per unit area between groups. Scale bar: 50 μm. Black arrowheads denote neointima.