Fig 2: OSS induces NOX4-dependent oxidative inactivation of VEGFR2(A) HAECs were cultured under LSS or OSS for 2 days using a cone-and-plate viscometer. The cells were stimulated with VEGF-A (25 ng/mL) for 10 min. VEGFR2 was immunoprecipitated (IP) and subjected to the in vitro kinase assay (KA). The precipitated VEGFR2 and cell lysate were immunoblotted (IB) for equal loading.Data in the graph are mean ± SD of the 32P radioactivity normalized to the intensity of VEGFR2 band (n = 3, *p < 0.005; two-way ANOVA with Tukey’s honest significant difference [HSD] post hoc test).(B) Basal mRNA levels of NOX isoforms in HAECs and HUVECs. Data in the graph are mean ± SD of fold increase versus NOX1 level (n = 4).(C) NOX2/4 expression under static and shear stress conditions. HAECs were placed in the indicated culture conditions for 48 h. Data in the graph are mean ± SD of fold increase versus static condition (n = 3, *p < 0.001; NS, not significant; Student’s t test).(D) HAECs were adapted to LSS or OSS for 48 h and treated with either vehicle control (Veh) or NOX inhibitor (APX115) for 1 h. Cells were subjected to intracellular ROS assay using dichlorofluorescein (DCF) dye. Brightfield (BF) images show live cells. Data in the graph are mean ± SEM of DCF fluorescence averaged from 60 to 80 cells (n = 3, *p < 0.005; Student’s t test).(E) HAECs were transfected with control or NOX4 siRNA for 18 h and exposed to OSS. The cells were then stimulated with VEGF-A (25 ng/mL) for 10 min. VEGFR2 was immunoprecipitated (IP) and subjected to the in vitro kinase assay (KA). The precipitated VEGFR2 and cell lysate were immunoblotted (IB) for equal loading. Data in the graph are mean ± SD of the 32P radioactivity normalized to the intensity of VEGFR2 protein (n = 3, *p < 0.01; two-way ANOVA with Tukey’s HSD post hoc test). A representative immunoblot is shown (n = 3). See also Figures S3 and S4.

Fig 3: Oxidation-resistant mutant of VEGFR2 prevents the disturbed flow-induced endothelial damage and arterial hyperplasia(A) Levels of endothelial NOX2 and NOX4 expression in the sham-operated and partially ligated LCAs. Data in the graph are mean ± SEM of fold change of relative NOX mRNA level versus the level in the contralateral RCA (n = 3, *p < 0.001; NS, not significant; Student’s t test).(B) Neointimal hyperplasia in the partially ligated LCAs of WT and VEGFR2C1206S KI mice. Carotid vessels were isolated at 3 and 5 weeks after partial ligation surgery. Untreated contralateral RCA is the normal vessel as control. Data in the graph are mean ± SEM of percent of intimal lesion area to medial area in the LCAs (n = 6–8, *p < 0.01, **p < 0.005; two-way ANOVA with Tukey’s honest significant difference [HSD] post hoc test).(C and D) En face IF staining in untreated contralateral RCAs and partially ligated LCAs of WT and VEGFR2C1206S KI mice. Carotid vessels were isolated at 2 weeks after partial ligation surgery. VEGFR2 and cleaved caspase-3 were independently immunostained. Data in the graph are mean ± SEM of the fluorescence intensity normalized to that of VEGFR2 (n = 5 per group, *p < 0.005, **p < 0.001; two-way ANOVA with Tukey’s HSD post hoc test). DAPI labels nuclei (blue).(E) Vascular permeability in untreated contralateral RCAs and partially ligated LCAs of WT and VEGFR2C1206S KI mice. Experiments were performed at 5 weeks after partial ligation surgery. Data in the graph are mean ± SEM of the light absorbance of Evans blue dye per mg protein (n = 3, *p < 0.01; two-way ANOVA with Tukey’s HSD post hoc test).(F) Schematic model depicting a blood flow-dependent redox regulation of VEGFR2 activation in the arterial endothelium. See also Figures S5 and S6.

Fig 4: VEGFR2 is reactivated by the Trx redox system(A) Recombinant mouse VEGFR2 CD (mVEGFR2-CD) bound to nickel beads was incubated with 250 μM H2O2 for indicated times, washed, and subjected to the in vitro kinase assay (KA). Data in the graph are mean ± SD of the relative radioactivity normalized to the amount of recombinant protein (n = 3, *p < 0.05, **p < 0.01, #p < 0.0005; NS, not significant; Student’s t test). CBB, Coomassie brilliant blue.(B) Recombinant mVEGFR2-CD bound to nickel beads was either untreated or pretreated with 250 μM H2O2 for 5 min and incubated with the redox systems for 10 min. Data in the graph are mean ± SD of the relative radioactivity normalized to the amount of recombinant protein (n = 3, *p < 0.001; Student’s t test).(C) HAECs were either untreated or pretreated with 250 μM H2O2 for 5 min and stimulated with VEGF-A (25 ng/mL) for 10 min. VEGFR2 was immunoprecipitated (IP) and subjected to the in vitro kinase assay. One sample (lane 4) was incubated with Trx/TR system for 10 min. The amount of precipitated VEGFR2 was evaluated by immunoblotting (IB). Data in the graph are mean ± SD of the relative radioactivity normalized to the amount of immunoprecipitated protein (n = 3, *p < 0.05, **p < 0.005; Student’s t test).(D) HAECs were pretreated with either control vehicle or auranofin (0.5 μM) for 1 h. The cells were either untreated or treated with 100 μM H2O2 for 5 min and stimulated with VEGF-A (25 ng/mL) for 10 min after 15 min recovery in fresh medium. Data in the graph are mean ± SD of relative intensities of p-VEGFR2 bands normalized to the intensities of tubulin bands (n = 3, *p < 0.05, **p < 0.01, #p < 0.001; NS, not significant; Student’s t test). A representative immunoblot is shown (n = 3). See also Figure S2B–D.

Fig 5: Flow-dependent Cys modification on VEGFR2 by S-nitrosylation and S-oxidation(A) Flow-dependent expression change of NOX4 and eNOS in HAECs. HAECs were cultured in either LSS or OSS for 48 h. LSS-adapted HAECs were transferred to OSS condition and adapted for additional 48 h. Immunoblotting was performed and quantified. Data in the graph are mean ± SD of the band intensities of each protein normalized to that of α-tubulin (n = 3, *p < 0.05, **p < 0.005; Student’s t test). Hyperoxidation of Prx (Prx-SO2/3) was shown by immunoblotting.(B) S-nitrosylation (SNO) of VEGFR2 in the HAECs either untreated or treated with 250 μM S-nitroso-GHS (GSNO) for 30 min. VEGFR2 was immunoprecipitated (IP) and subjected to the biotin-switch assay to measure the SNO levels.(C) HAECs were transfected with either control or eNOS-specific small interfering RNA (siRNA) for 12 h and adapted to LSS for additional 48 h. VEGFR2 was immunoprecipitated (IP) and subjected to the biotin-switch assay to measure the SNO levels.(D) HAECs were transfected with either control or eNOS-specific siRNA for 12 h and then adapted to LSS for 48 h. Cells were pretreated with or without 100 μM H2O2 for 10 min and stimulated with VEGF-A (25 ng/mL) for an additional 10 min. Cell lysates were subjected to immunoblot analysis.(E) VEGFR2 activation in response to treatments with H2O2 and the NO donor, SNAP. HAECs were either untreated, treated with H2O2 alone for 5 min, or treated sequentially with H2O2 (100 μM) and SNAP (250 μM) for 5 min each in the indicated order. VEGFR2 activation was measured by immunoblotting. Data in the graph are mean ± SD of the band intensities of p-VEGFR2 normalized to that of VEGFR2 (n = 3, *p < 0.005; NS, not significant; Student’s t test).(F) HUVECs were pretreated with SNAP (250 μM) for 5 min and stimulated with H2O2 (100 μM) for 5 min. Cells were lysed in denaturing sample buffer with DTT (R) or without DTT (NR) and then resolved on SDS-PAGE gel. VEGFR2 was immunoblotted. The slow- and fast-migrating bands represent reduced (-SH) and oxidized (-S-S-) forms of VEGFR2, respectively.(G) S-nitrosylation of VEGFR2 in HUVECs grown under difference shear stresses. VEGFR2 was immunoprecipitated and treated with either 2 mM DTT or 5 mM sodium ascorbate prior to biotin-switch assay. Controls (—) remained untreated. Data in the graph are mean ± SD of the band intensities of biotin-VEGFR2 normalized to that of VEGFR2 (n = 3, *p < 0.01; NS, not significant; Student’s t test).(H) Differential fluorescence labeling of Cys thiols on VEGFR2. HUVECs grown under difference flow patterns were sequentially labeled using two thiol-selective fluorescent dyes, Cy3 and Cy5, in accordance with a scheme depicting the sequential labeling procedure.(I) S-nitrosylation of the WT and five CS mutants of VEGFR2. HEK293T cells were transfected with each retroviral vector encoding HA-tagged mouse VEGFR2. The cell lysates were either untreated or treated with 25 μM GSNO for 40 min. VEGFR2 was immunoprecipiated and subjected to the biotin-switch assay to measure the SNO levels (n = 3, *p < 0.05, **p < 0.005, #p < 0.001; NS, not significant; Student’s t test). A representative immunoblot is shown (n = 3).