Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011;6(12):e28454.
doi: 10.1371/journal.pone.0028454. Epub 2011 Dec 1.

Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor receptor-2 and c-Src

Free PMC article

Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor receptor-2 and c-Src

Monica Lee et al. PLoS One. .
Free PMC article


Background: ADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). We previously demonstrated that c-Src plays a key role in VEGF-induced, ROS-dependent selective activation of PI3K-Akt but not PLCγ-1-ERK1/2 signaling pathways. The aim of the present study was to understand how VEGFR-2-c-Src signaling axis 'senses' NADPH oxidase-derived ROS levels and couples VEGF activation of c-Src to the redox state of ECs.

Methodology/principal findings: Using biotinylated probe that detects oxidation of cysteine thiol (cys-OH) in intracellular proteins, we demonstrate that VEGF induced oxidative modification in c-Src and VEGFR-2, and that reduction in ROS levels using siRNA against p47(phox) subunit of Rac1-dependent NADPH oxidase inhibited this phenomenon. Co-immunoprecipitation studies using human coronary artery ECs (HCAEC) showed that VEGF-induced ROS-dependent interaction between VEGFR-2 and c-Src correlated with their thiol oxidation status. Immunofluorescence studies using antibodies against internalized VEGFR-2 and c-Src demonstrated that VEGF-induced subcellular co-localization of these tyrosine kinases were also dependent on NADPH oxidsase-derived ROS.

Conclusion/significance: These results demonstrate that VEGF induces cysteine oxidation in VEGFR-2 and c-Src in an NADPH oxidase-derived ROS-dependent manner, suggesting that VEGFR-2 and c-Src can 'sense' redox levels in ECs. The data also suggest that thiol oxidation status of VEGFR-2 and c-Src correlates with their ability to physically interact with each other and c-Src activation. Taken together, these findings suggest that prior to activating downstream c-Src-PI3K-Akt signaling pathway, VEGFR-2-c-Src axis requires an NADPH oxidase-derived ROS threshold in ECs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. VEGF-induced activation of c-Src, Akt, but not VEGFR-2, PLCγ-1 and ERK1/2 requires NADPH oxidase-derived ROS.
(A) Protein extracts from HCAEC transfected with control siRNA (Scram-si) or si-p47phox were subject to Western blots as described in Materials and methods. Serum-starved HCAEC were treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the phospho-specific antibodies as shown. Blots shown are representative of three independent experiments. (B) Same as in (A) except, the membranes were probed for phosphorylation of Y1175 VEGFR-2, Y783 PLCγ-1 and p42/44 ERK1/2. Anti-β-actin antibody was used as loading control.
Figure 2
Figure 2. c-Src and VEGFR-2 are oxidized in VEGF-treated HCAEC in the presence of NADPH oxidase-derived ROS.
(A) Schematic presentation depicting cysteinyl-labeling assay to determine oxidative modification in intracellular proteins. Non-oxidized protein thiols are alkyalated by IAA, oxidized thiols are reduced back to SH-moiety by DTT and subsequently biotinylated by IAP. Biotinylated proteins are then pulled-down by streptavidin-agarose followed by Western blots. (B) Upper panel: cysteinyl labeling assay to Identify thiol oxidation of proteins in VEGF-treated (50 ng/ml for 2 mins) HCAEC lysates using biotinylated IAP probe. HCAEC were transfected with Scram-si or si-p47phox as indicated. After cell lysis in the presence of IAA followed by DTT treatment and IAP labeling, 1.5 mg biotinylated protein lysates were subject to immunoprecipitation using Streptavidin-agarose beads and immunoblotted using anti-VEGFR-2 and anti-c-Src antibodies. Lower panel: Western blot for VEGFR-2 using 50 µg of parallel HCAEC lysates as loading control. (B) Quantitative analyses of oxidized VEGFR-2 (upper panel) and c-Src (lower panel). Bar graph shows quantitative densitometric analysis of three independent cysteinyl labeling assays (as in A) using NIH J image (-fold change expressed in mean ± S.E.M.). *p<0.05 was considered statistically significant.
Figure 3
Figure 3. VEGF-induced interaction between VEGFR-2 and c-Src requires NADPH oxidase-derived ROS.
(A) Co-immunoprecipitation (co-IP) assay using 1.2 mg protein lysates of HCAEC that were transfected with Scram-si or si-p47phox and treated without or with VEGF (50 ng/ml for 5 min). IP was carried out using anti-VEGFR-2 antibody followed by immunoblotting using anti-c-Src (upper panel) and anti-VEGFR-2 (lower panel) antibodies. (B) Quantitative analyses of VEGFR-2-bound c-Src. Bar graphs show quantitative densitometric analysis of three independent experiments using NIH image J (-fold change expressed in mean ± S.E.M.). *p<0.05 was considered statistically significant.
Figure 4
Figure 4. VEGF induces subcellular co-localization of c-Src and internalized VEGFR-2 in an ROS-dependent manner.
HCAEC transfected with control (Scram-si) (A) or si-p47phox (B) were immunofluorescently double labeled for internalized VEGFR-2 (green) and c-Src (red). VEGFR-2 on HCAEC was labeled with single chain E-tagged antibody (scFvA7, Fitzerald) as described in Materials and methods. After incubation with VEGF (50 ng/ml for 10 min), in order to remove the antibody from the cell surface, cells were placed on ice and acid washed. In permeabilized and fixed HCAEC, VEGFR-2 was detected with an AlexaFluor488-conjugated secondary antibody and is shown in green. c-Src was labeled with AlexaFluor647-conjugated secondary antibody (red) and nuclei with DAPI (blue). (B) Bar graphs show image analysis for colocalization events using the NIH Image J plugin (as described in Materials and methods). The graphs present the number of colocalization events normalized for the number of total VEGFR-2–positive immunofluorescence signals. Values are the mean of three experiments ± S.E.M., each containing numbers obtained from five random fields. *p<0.05 was considered statistically significant.
Figure 5
Figure 5. Proposed model: thiol oxidation may help propagate signal transduction from VEGFR-2 to downstream c-Src.
(A) VEGF activation of VEGFR-2 and downstream PLCγ-1-ERK1/2 signaling pathway appears to be independent of ROS levels in ECs. (B) VEGF induces NADPH oxidase-derived ROS, which in turn oxidizes VEGFR-2 and c-Src. Thiol oxidation of these two tyrosine kinases appears to correlate with VEGF-induced activation of c-Src, and also with the sub-cellular colocalization and interaction between VEGFR-2 and c-Src. Dependence of VEGF-induced thiol oxidation and activation of c-Src on NADPH oxidase-derived ROS render downstream activation of PI3K-Akt redox-sensitive in HCAEC. In this model, VEGFR-2 and/or c-Src act as endothelial redox-sensors that determine whether downstream PI3K-Akt signaling pathway should be activated or not.

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles


    1. Ray R, Shah AM. NADPH oxidase and endothelial cell function. Clin Sci (Lond) 2005;109:217–226. - PubMed
    1. Fichtlscherer S, Dimmeler S, Breuer S, Busse R, Zeiher AM, et al. Inhibition of cytochrome P450 2C9 improves endothelium-dependent, nitric oxide-mediated vasodilatation in patients with coronary artery disease. Circulation. 2004;109:178–183. - PubMed
    1. Halcox JP, Schenke WH, Zalos G, Mincemoyer R, Prasad A, et al. Prognostic value of coronary vascular endothelial dysfunction. Circulation. 2002;106:653–658. - PubMed
    1. Irani K. Oxidant signaling in vascular cell growth, death, and survival : a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ Res. 2000;87:179–183. - PubMed
    1. Kunsch C, Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ Res. 1999;85:753–766. - PubMed

Publication types

MeSH terms