OxLDL-induced EGFR tyrosine phosphorylation and inhibition by antioxidants and tyrosine kinase inhibitors. Cells ((A – E) ECV-304 endothelial cells; (F) GM-08133A smooth muscle cells; (G) B82LK+ cells transduced with wild type EGFR) were incubated with oxLDL (200 μg apoB ml⁻¹) for the indicated time, then harvested and used for Western blots, labelled with anti-phosphotyrosine and anti-EGFR antibodies. EGF (10 nM for 15 min) was used as maximal positive control. (A – C) Time course of EGFR autophosphorylation induced by oxLDL in the absence (A), or presence (B,C) of antioxidants, caffeic acid (100 μM) or trolox (100 μM). (D,E) Inhibition of the oxLDL-induced EGFR autophosphorylation by increasing concentrations of caffeic acid (D) or trolox (E) in ECV-304 EC. Cells were co-incubated for 5 h with or without antioxidants and oxLDL. (F,G) Inhibition of the oxLDL-induced EGFR autophosphorylation at 5 h, by caffeic acid (100 μM), trolox (100 μM), α-tocopherol (100 μM) or tyrphostins A25 (5 μM) or A46 (20 μM), in GM-08133A smooth muscle cells (F) or in B82LK+ cells (transduced with and overexpressing wild type EGFR) (G). Representative data of 3 – 5 experiments.

OxLDL- and 4-HNE-induced EGFR derivatization and activation. Lack of effect of antioxidants. (A,B) ECV-304 cells were incubated (for 3 h) with or without oxLDL (200 μg apoB ml⁻¹) and with or without caffeic acid (100 μM) or trolox (100 μM) or with 4-HNE (1 μM). Then cells were harvested and lysed and EGFR was immunoprecipitated. In (A), free amino groups were labelled by [³H]-NSP, EGFR was resolved by SDS – PAGE and the radioactivity of the 170 kDa bands was counted. Mean±s.e.mean of three experiments. In (B) EGFR was immunoprecipitated, resolved by SDS – PAGE and 4-HNE adducts was detected by polyclonal antibodies anti-4-HNE-protein (K5-4412) or anti-EGFR antibodies. (C) ECV-304 cells were incubated for 1 h with or without 4-HNE (1 μM) and with or without caffeic acid (100 μM) or trolox (100 μM) or α-tocopherol (100 μM), then harvested and lysed. Immunoprecipitated EGFR was resolved by SDS – PAGE and labelled with anti-phosphotyrosine and anti-EGFR antibodies. Representative data of four experiments. In B and C, semi-quantitative evaluation of Western blot spots was performed by densitometry.

Cellular oxidative stress triggered by H₂O₂ induces EGFR activation in ECV-304 EC. (A) Time course of intracellular peroxides, monitored by using the fluorescence of the oxidation-sensitive H₂DCFDA fluorogenic probe, in cells incubated with H₂O₂ (200 μM). Mean±s.e.mean of three experiments. (B,C) Cells were incubated with 200 μM H₂O₂ for 3 h (B and C) or oxLDL (200 μg apoB ml⁻¹) for 5 h (C) and without (−) or with (+) antioxidants, caffeic acid (100 μM) or trolox (100 μM) (B) or PDTC (100 μM) (C). Western blot were revealed by anti-phosphotyrosine and anti-EGFR antibodies. In (B) and (C), representative data of three experiments.

Evaluation of PTPase activity and EGFR dephosphorylation in cells incubated with oxLDL and antioxidants. (A,B) PTPase activity was evaluated in ECV-304 EC preincubated (or not) with oxLDL (200 μg apoB ml⁻¹ of oxLDL) for 1 h (A) or 5 h (B) with (+) or without (0) antioxidants caffeic acid, C or trolox, T. (C,D) ECV-304 EC were preincubated with (+) or without (−) oxLDL (5 h preincubation with 200 μg apoB ml⁻¹ of oxLDL), then were incubated with EGF (2 nM for 5 min) and finally were chased (without EGF) for 30 min with (+) or without (−) tyrphostin AG1478 (100 nM) (C), and, when indicated, with antioxidants caffeic acid, C or trolox, T (added during preincubation of cells with oxLDL) (D). Western blot were revealed by anti-phosphotyrosine and anti-EGFR antibodies. In (C) and (D), representative data of three experiments.

OxLDL-induced recruitment of EGFR substrates (i.e. activation of the EGFR signalling pathway) and inhibition by antioxidants. ECV-304 cells were incubated with oxLDL (200 μg apoB ml⁻¹) for 5 h (or 10 nM EGF for 15 min, used as maximal control) in the presence or absence of caffeic acid (100 μM) or trolox (100 μM). Cells were lysed in solubilization buffer (the used solubilization buffer allows co-immunoprecipitation of SH₂-proteins bound to EGFR) and (1.5 mg cell protein) were immunoprecipitated with anti-EGFR antibody (LA22, overnight at 4°C). Then, anti-EGFR immunoprecipitates were recovered on protein G-sepharose (1 h at 4°C), washed, eluted and analysed by SDS – PAGE. The spots were revealed by immunoblotting with anti-phosphotyrosine, anti-EGFR, anti-PLCγ, anti-SHP2 or anti-SHC antibodies. Representative data of three experiments.

OxLDL induce oxidative stress in ECV-304 EC. Inhibition by caffeic acid or trolox. (A) Time course of the intracellular oxidative stress (squares) and extracellular liberation of H₂O₂ (circles) induced by oxLDL. Cells were incubated with oxLDL (200 μg apoB ml⁻¹) and, at the indicated time, cellular H₂DCFDA fluorescence was monitored, and H₂O₂ released in the cultured medium was determined by scopoletin fluorescence quenching method, as indicated in the Methods section. (B,C) Inhibition by caffeic acid or trolox of the intracellular oxidative stress (B) and extracellular liberation of H₂O₂ (C). Cells, were incubated for 3 h with or without oxLDL (200 μg apoB ml⁻¹) and with or without caffeic acid (100 μM) or trolox (100 μM). Then, the intracellular cellular oxidative stress (H₂DCFDA) and extracellular liberation of H₂O₂ (scopoletin) were measured as in Figure 5A. Fluorescence is expressed as arbitrary units. Mean±s.e.mean of 3 – 4 experiments.

Caffeic acid and tyrphostin A46 (but not trolox) inhibit EGF-induced EGFR activation (inhibition of the EGFR tyrosine kinase). (A) B82LK+ cells, pre-incubated for 1 h with or without caffeic acid (100 μM), trolox (100 μM) or tyrphostin A46 (20 μM) before treatment with EGF (2 nM for 20 min) (Co, untreated control). EGFR tyrosine phosphorylation was visualized on Western blots labelled with anti-phosphotyrosine antibody. (B) Lack of influence of caffeic acid or trolox on the association (binding and uptake) of ¹²⁵I-EGF to ECV-304 EC. Cells were pre-incubated for 3 h with or without oxLDL (200 μg apoB ml⁻¹) and caffeic acid (100 μM) or trolox (100 μM); then a tracer amount of ¹²⁵I-EGF (70,000 c.p.m. ml⁻¹) was added for variable periods of time (up to 60 min). Pre-incubation conditions: black circles, no addition; empty squares, oxLDL (200 μg apoB ml⁻¹); empty circles, oxLDL and caffeic acid (100 μM); empty triangles, oxLDL and trolox (100 μM). (C,D) Caffeic acid (but not trolox) inhibits the in vitro EGF-induced tyrosine phosphorylation (C) and tyrosine kinase activation (D) of immunopurified EGFR. EGFR purified by immunoprecipitation from unstimulated B82LK+ cells, was incubated without (control, Co) or with EGF (5 nM) and without or with caffeic acid (100 μM) or trolox (100 μM) or genistein (25 μM) in the phosphorylation buffer for 15 min. In (C), EGFR tyrosine phosphorylation was visualized on Western blot labelled with anti-phosphotyrosine and anti-EGFR antibodies. In (D), EGFR tyrosine kinase activity was evaluated under the same conditions as in (C) but in the presence of [γ-³³P]-ATP and poly Glu-Tyr (as indicated in Methods). In (A) and (C), representative data of three experiments. In (B) and (D), Mean±s.e.mean of three experiments.