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. 2010 Jun 15;12(12):1355-69.
doi: 10.1089/ars.2009.2874.

Cigarette-smoke-induced oxidative/nitrosative stress impairs VEGF- and fluid-shear-stress-mediated signaling in endothelial cells

Indika Edirisinghe  1 , Gnanapragasam Arunachalam, Chelsea Wong, Hongwei Yao, Arshad Rahman, Richard P Phipps, Zheng-Gen Jin, Irfan Rahman
Affiliations

Cigarette-smoke-induced oxidative/nitrosative stress impairs VEGF- and fluid-shear-stress-mediated signaling in endothelial cells

Indika Edirisinghe et al. Antioxid Redox Signal. .

Retraction in

Abstract

VEGF receptor 2 (VEGFR2), a tyrosine kinase receptor, is activated by VEGF and fluid shear stress (FSS), and its downstream signaling is important in the regulation of endothelial functions, such as cell migration, endothelium-dependent relaxation, and angiogenesis. Cigarette smoke (CS) is known to cause oxidative/nitrosative stress, leading to modifications of tyrosine kinase receptors and impaired downstream signaling. We hypothesized that CS-induced oxidative/nitrosative stress impairs VEGF- and FSS-mediated VEGFR2 activation, leading to endothelial dysfunction. Human lung microvascular endothelial cells and human umbilical vein endothelial cells were treated with different concentrations of cigarette smoke extract (CSE) to investigate the VEGF- or FSS-mediated VEGFR2 phosphorylation and its downstream signaling involved in endothelial function. CSE treatment impaired both VEGF- and FSS-mediated VEGFR2 phosphorylation, resulting in impaired endothelial nitric oxide synthase (eNOS) phosphorylation by Akt. CS-derived reactive oxygen/nitrogen species react with VEGFR2, rendering VEGFR2 inactive for its downstream signaling. Pretreatment with nitric oxide scavenger (PTIO), reactive oxygen species scavengers (combination of SOD with catalase), and N-acetyl-L-cysteine, significantly attenuated the CSE-induced impairment of VEGF-mediated Akt and eNOS phosphorylation. These findings suggest that CSE-induced oxidative/nitrosative stress impairs VEGF- and FSS-mediated endothelial cell function and has important implications in the pathogenesis of CS-induced pulmonary and cardiovascular diseases associated with endothelial dysfunction.

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Figures

FIG. 1.
FIG. 1.
CSE impaired VEGF-mediated VEGFR2 phosphorylation and its downstream signaling in endothelial cells. (A) CSE- or VEGFR2-inhibitor–treated cells (2 h) were incubated with VEGF, and the levels of phosphorylated and total VEGFR2, Akt, and eNOS were measured with immunoblotting. The VEGFR2 blot showed two bands, a 230-kDa, fully glycosylated functional receptor, and a 200-kDa, semiglycosylated nonfunctional receptor. Phosphorylation was seen only in the fully glycosylated functional receptor. CSE treatments downregulated VEGF-mediated VEGFR2 phosphorylation and its downstream signaling in a concentration-dependent manner. Pretreatment of cells with NVP-AAD777 (1 μM) abolished the VEGF-mediated VEGFR2 phosphorylation. Histograms represent mean ± SEM of the percentage of VEGFR2 (B), Akt (C), and eNOS (D) phosphorylation compared with respective control experiments (n = 3). **p < 0.01; ***p < 0.001 vs. control group. p-VEGFR2 = phosphorylated VEGFR2 (Tyr 1175); p-Akt = phosphorylated Akt (Ser 473); p-eNOS = phosphorylated eNOS (Ser 1177).
FIG. 2.
FIG. 2.
CSE-impaired fluid shear stress–mediated VEGFR2 phosphorylation and its downstream signaling in endothelial cells. (A) Fluid shear-stress force (12 dyn/cm2) was applied to CSE-treated HMVEC-Ls (2 h), as described in Materials and Methods. The levels of phosphorylated and total VEGFR2, Akt, and eNOS were measured with immunoblotting. The VEGFR2 blot showed two bands: a 230-kDa, fully glycosylated functional receptor, and a 200-kDa, semiglycosylated nonfunctional receptor. Phosphorylation was seen only in the fully glycosylated functional receptor. CSE treatment downregulated the shear stress–mediated VEGFR2 phosphorylation and its downstream signaling in a concentration-dependent manner. Histograms represent the mean ± SEM of the percentage of VEGFR2 (B), Akt (C), and eNOS (D) phosphorylation compared with the respective control experiments (n = 3). **p < 0.01 vs. control group. p-VEGFR2 = phosphorylated VEGFR2 (Tyr 1175); p-Akt = phosphorylated Akt (Ser 473); p-eNOS = phosphorylated eNOS (Ser 1177).
FIG. 3.
FIG. 3.
CSE caused nitration of tyrosine residue in VEGFR2 in endothelial cells. (A) HMVEC-Ls were treated with CSE (0.1–0.5%), NO donor (Deta-NONOate, 50 μM), NO scavenger (PTIO, 100 μM), or a combination of CSE with NO scavenger. VEGFR2 was immunoprecipitated, and immunoblot was performed to determine the nitration of tyrosine residues of VEGFR2. CSE treatment increased the level of nitrated tyrosine residues on VEGFR2 in a concentration-dependent manner. Pretreatment with NO scavenger (PTIO) attenuated the CSE-induced nitration of tyrosine on VEGFR2. (B) Histograms represent the mean ± SEM of the percentage of nitrated tyrosine residues in VEGFR2 compared with those in the control experiments (n = 3). **p < 0.01; ***p < 0.001 vs. control group.+++p < 0.001 vs. CSE-alone–treated group. (C) HMVEC-Ls were treated with CSE (0.5%), NAC (1 mM), or a combination of CSE with NAC. VEGFR2 was immunoprecipitated, and immunoblot was performed to determine the nitration of tyrosine residues of VEGFR2. CSE increased the level of nitrated tyrosine residues on VEGFR2. Pretreatment with NAC attenuated the CSE-induced nitration of tyrosine on VEGFR2. (D) Histograms represent the mean ± SEM of the percentage of nitrated tyrosine residues in VEGFR2 compared with those in the control experiments (n = 3). ***p < 0.001 vs. control group.+++p < 0.001 vs. CSE-alone–treated group.
FIG. 4.
FIG. 4.
Nitric oxide scavenger (PTIO) attenuated the CSE-induced impairment of VEGF-mediated VEGFR2 phosphorylation and its downstream signaling in endothelial cells. (A) HMVEC-Ls were treated with CSE (0.5%) and incubated with VEGF in the presence or absence of the NO scavenger (PTIO, 100 μM), and the levels of phosphorylated and total VEGFR2, Akt, and eNOS were measured with immunoblotting. CSE-induced impairment of VEGF/VEGFR2 signaling was attenuated in presence of the NO scavenger (PTIO). Histograms represent the mean ± SEM of the percentage of VEGFR2 (B), Akt (C), and eNOS (D) phosphorylation compared with the respective control experiments (n = 3). **p < 0.01; ***p < 0.001 vs. control group; ++p < 0.01 vs. CSE-alone–treated group. p-VEGFR2 = phosphorylated VEGFR2 (Tyr 1175); p-Akt = phosphorylated Akt (Ser 473); p-eNOS = phosphorylated eNOS (Ser 1177).
FIG. 5.
FIG. 5.
ROS scavengers (SOD and catalase) attenuated the CSE-induced impairment of VEGF-mediated VEGFR2 phosphorylation and its downstream signaling in endothelial cells. (A) HMVEC-Ls were treated with CSE (0.5%) in the presence or absence ROS scavengers (SOD and catalase, 400 U/ml each), and incubated with VEGF, and the levels of phosphorylated and total VEGFR2, Akt, and eNOS were measured with immunoblotting. CSE-induced impairment of VEGF/VEGFR2 signaling and its downstream signaling were attenuated by ROS scavengers. Histograms represent the mean ± SEM of the percentage of VEGFR2 (B), Akt (C), and eNOS (D) phosphorylation compared with the respective control experiments (n = 3). **p < 0.01; ***p < 0.001 vs. control group;++p < 0.01; +++p < 0.001 vs. the CSE-alone–treated group. p-VEGFR2 = phosphorylated VEGFR2 (Tyr 1175); p-Akt = phosphorylated Akt (Ser 473); p-eNOS = phosphorylated eNOS (Ser1177).
FIG. 6.
FIG. 6.
Effect of N-acetyl-l-cysteine on CSE-induced impairment of VEGF-mediated phosphorylation of VEGFR2 and its downstream signaling in endothelial cells. (A) HMVEC-Ls were treated with CSE (0.5%) in the presence or absence of NAC (1 mM), and incubated with VEGF, as described in Materials and Methods. The levels of phosphorylated and total VEGFR2, Akt, and eNOS were measured with immunoblotting. CSE-induced impairment of VEGF/VEGFR2 and its downstream signaling were attenuated in the presence of NAC. Histograms represent the mean ± SEM of the percentage of VEGFR2 (B), Akt (C), and eNOS (D) phosphorylation compared with respective control experiments (n = 3). **p < 0.01 vs. control group;+p < 0.05; ++p < 0.01 vs. CSE-alone–treated group. p-VEGFR2 = phosphorylated VEGFR2 (Tyr 1175); p-Akt = phosphorylated Akt (Ser 473); p-eNOS = phosphorylated eNOS (Ser 1177).
FIG. 7.
FIG. 7.
Effect of nitric oxide scavenger (PTIO) and ROS scavengers (SOD + catalase) on CSE-induced impairment of FSS-mediated phosphorylation of Akt and eNOS in endothelial cells. (A) HUVECs were treated with CSE (0.25%) in the presence or absence of NO scavenger (PTIO, 100 μM) and ROS scavengers (combination of SOD, 400 U/ml, and catalase, 400 U/ml); thereafter, fluid shear-stress force (12 dyn/cm2) was applied, as mentioned in Materials and Methods. The levels of phosphorylated and total Akt and eNOS were measured by using immunoblotting. Histograms represent the mean ± SEM of the percentage of Akt (B) and eNOS (C) phosphorylation compared with the respective control experiments (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 vs. control group.++p < 0.01 vs. CSE-alone–treated group. p-Akt = phosphorylated Akt (Ser 473); p-eNOS = phosphorylated eNOS (Ser 1177).
FIG. 8.
FIG. 8.
Effect of N-acetyl-l-cysteine on CSE-induced impairment of FSS-mediated phosphorylation of Akt and eNOS in endothelial cells. (A) HUVECs were treated with CSE (0.25%) in presence or absence of N-acetyl-l-cysteine (1 mM); thereafter, fluid shear-stress force (12 dyn/cm2) was applied, as mentioned in Materials and Methods. Phosphorylated and total protein levels of Akt and eNOS were measured with immunoblotting. CSE-induced impaired Akt and eNOS signaling were attenuated in presence of N-acetyl-l-cysteine. Histograms represent the mean ± SEM of the percentage of Akt (B) and eNOS (C) phosphorylation compared with respective control experiments (n = 3). **p < 0.01; ***p < 0.001; vs. control group. +++p < 0.001 vs. CSE-alone–treated group. p-Akt = phosphorylated Akt (Ser 473). p-eNos = phosphorylated eNos (ser1177).
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