Chrysin restores PDGF-induced inhibition on protein tyrosine phosphatase and reduces PDGF signaling in cultured VSMCs  Huey-Ming Lo, Min-Wen Wu, Shiow-Lin Pan, Chieh-Yu Peng, Pi-Hui Wu, Wen-Bin Wu  Journal of Nutritional Biochemistry  Volume 23, Issue 6, Pages 667-678 (June 2012) DOI: 10.1016/j.jnutbio.2011.03.011 Copyright © 2012 Elsevier Inc. Terms and Conditions

Fig. 1 Effect of chrysin on PDGF-induced proliferation, chemotaxis, and signal transduction in VSMCs. (A) VSMCs were treated with PBS, vehicle (DMSO), or chrysin (10 and 20 μM) in serum-free medium containing PBS or PDGF-BB (10 ng/ml) for 48 h. Cell proliferation assay was performed as described in Methods (n=3-4). (B) VSMCs were seeded in the upper chamber in the presence of vehicle or chrysin. The inserts were assembled with the lower chamber, which was filled with serum-free (basal) or PDGF-containing medium in the presence of vehicle or chrysin for 30 min. After an additional incubation for 3 h, migrated VSMCs were analyzed by microscopy (n=3-5). ⁎P<.05 and ⁎⁎⁎P<.001 versus control. (C) Concentration- and time-dependent effects of chrysin on PDGF signaling. VSMCs were treated with chrysin and followed by PDGF-BB (10 ng/ml) stimulation for an additional 10 min (left panel) or for the indicated time points (right panel). Quantitative analysis of data by densitometry was performed (n=3-4). ⁎P<.05 versus control. Journal of Nutritional Biochemistry 2012 23, 667-678DOI: (10.1016/j.jnutbio.2011.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Fig. 2 Effect of different chrysin treatments on PDGF signaling. (A) VSMCs were pretreated (Pre-Tx), treated (Tx), or post-treated (post-Tx) with chrysin (CS) and stimulated with PDGF-BB (BB, 10 ng/ml) for 10 min as shown in the diagram. w/o wash: without wash. (B) VSMCs were stimulated with PDGF-BB. During PDGF stimulation, vehicle or chrysin was added at the indicated time points. Cells were collected, lysed, and protein phosphorylation and expression was analyzed by Western blotting. Data were quantified by densitometry and expressed as a bar graph (n=3). ⁎P<.05 and ⁎⁎P<.01 versus control. Journal of Nutritional Biochemistry 2012 23, 667-678DOI: (10.1016/j.jnutbio.2011.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Fig. 3 Effect of chrysin, NAC and EGCG on PDGF signaling in VSMCs and fibroblasts. (A) VSMCs or (B) fibroblasts were simultaneously stimulated with PDGF-BB and the indicated compounds for 10 min or were stimulated with PDGF-BB for 10 min and followed by the addition of the indicated compounds at the last 1 min (post addition). Protein phosphorylation and expression were analyzed by Western blotting and quantitative analysis by densitometry was shown (n=3). ⁎P<.05 and ⁎⁎P<.01 versus control. Journal of Nutritional Biochemistry 2012 23, 667-678DOI: (10.1016/j.jnutbio.2011.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Fig. 4 Effect of chrysin on H2O2 signaling, intracellular ROS production, NADPH oxidase activation, and PDGF binding to cells. (A) VSMCs were pretreated with vehicle (veh) or chrysin for 1 h and followed by the addition of H2O2 (3 mM) for 5 min. PDGFR-β and do wnstream protein phosphorylation and expression were determined by Western blotting (n=4). Quantitative analysis of protein tyrosine phosphorylation was shown on the right panels. ⁎P<.05 versus control. (B) VSMCs were treated with PDGF-BB (PDGF, 10 ng/ml) in the presence of vehicle (veh), chrysin (20 μM), or EGCG (20 μM) for 10 min. Cells were immediately analyzed by fluorescence microscopy. The representative data (in duplicate) from an experiment were shown (n=5-6). (C) VSMCs were treated with PDGF-BB (10 ng/ml) in the presence of vehicle or chrysin (20 μM) for the indicated times. P47 phox level (p47 phox ratio to PDGFRβ or tubulin) from this result was shown. (D) Effect of chrysin and EGCG on PDGF-BB binding to fibroblasts and VSMCs. Cells incubated with fluorescein-labeled PDGF in the presence of vehicle (control), chrysin or EGCG were analyzed by flowcytometry The obtained mean fluorescence intensity reflected the extent of PDGF binding to cells (n=3-4). ⁎⁎P<.01 versus control. Journal of Nutritional Biochemistry 2012 23, 667-678DOI: (10.1016/j.jnutbio.2011.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Fig. 5 Chrysin affects PTP activity. (A) Cells were left untreated or treated with PDGF-BB (BB, 10 ng/ml) for 10 min or H2O2 (1 mM) for 5 min in the presence of vehicle or chrysin (20 μM). PTP activity was determined by their abilities to dephosphorylate peptides with tyrosine phosphorylated residues, as described in Methods. PTP activity in each sample was expressed as the percentage of basal (n=3-5). (B) Effect of chrysin on PDGF-induced PTP oxidation (inactivation). Cells were left untreated or treated with PDGF-BB (10 ng/ml) for 10 min in the presence of vehicle or chrysin (20 μM). Cell lysates were prepared in the presence or absence of IAA, and level of TC-PTP oxidation was measured by Ab specific for the oxidized catalytic site of PTP. Data were quantified by densitometry (n=3). (C) Effect of chrysin, NAC and EGCG on vanadate-induced PDGFR signaling. VSMCs were stimulated with PBS or PDGF-BB (10 ng/ml) in the presence of the indicated concentrations of vanadate. PDGFR signaling was analyzed by Western blotting (n=3-4). Journal of Nutritional Biochemistry 2012 23, 667-678DOI: (10.1016/j.jnutbio.2011.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Fig. 6 Effect of intracellular GSH level on PDGF signaling and PTP activity. VSMCs were pretreated with the indicated DEM for 6 h. (A) Cell viability was monitored by microscopic morphological analysis (left panel). After removal of DEM, cells were loaded with GSH-sensitive dye and intracellular GSH content was measured by the fluorescence plate reader (right panel). (B and C) The DEM-pretreated cells were washed with serum-free medium and then treated with PDGF-BB (10 ng/ml) for 10 min in the presence of vehicle, chrysin (CS, 20 μM), NAC (30 mM), or EGCG (20 μM). Protein tyrosine phosphorylation was analyzed by Western blotting (left panel) and densitometry (right panel). Data were expressed as fold of basal (without PDGF stimulation) and the percentage above each bar indicates extent of inhibition of PDGFR phosphorylation. (D) Control and DEM-pretreated cells were stimulated with PDGF-BB for 10 min in the presence of vehicle, chrysin (20 μM), NAC (30 mM), or EGCG (20 μM). The intracellular GSH content (left panel) and PTP activity (right panel) were measured by fluorescence plate reader and PTP assay kit respectively (n=3-4). Journal of Nutritional Biochemistry 2012 23, 667-678DOI: (10.1016/j.jnutbio.2011.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Fig. 7 Chrysin reduces neointima formation in rat carotid balloon injury model. (A) Representative photomicrographs of hematoxylin-eosin-stained sections of uninjured (control) and balloon-injured rat carotid arteries. M: media layer. I: intima. (B) The intima-media-(I/M) ratio and medial area were quantified using image analysis software. Data are presented as mean±S.E.M. (n=3). The ANOVA with Dunnett's post-hoc test was performed. ⁎P<.05 versus balloon-injured group. Journal of Nutritional Biochemistry 2012 23, 667-678DOI: (10.1016/j.jnutbio.2011.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Fig. 8 Schematic diagram of chrysin on PDGF signaling in VSMCs. PDGF binding activates PDGFR and downstream protein tyrosine kinases and increases of intracellular H2O2 production, subsequently leading to oxidation (inactivation) of PTP. The PTPs function as a negative regulator for PDGFR and downstream protein tyrosine enzymes/kinases. Chrysin inhibits PDGF-induced ROS production, relieves PDGF-induced inhibition on PTP activity and affects GSH/glutaredoxin reactivation system (sys). The net reaction is restoring of PTP activity, leading to reduction of PDGF signaling, i.e. increase of dephosphorylated proteins. (+): stimulatory; (-) inhibitory; and (↑↑), increase. Journal of Nutritional Biochemistry 2012 23, 667-678DOI: (10.1016/j.jnutbio.2011.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions