Figure 5: Effects of Lp(a) on Cytokines and Phosphorylated MAPKs in HAVICs

(A) Effects of Lp(a) on release of cytokines and chemokines by HAVICs. Rectangular outlines indicate reference controls. (B) Effects of Lp(a) on phosphorylated MAPKs in HAVICs. Cells were treated with or without 50 μg/ml Lp(a) for 1 h, and total proteins were extracted and used for phosphorylated MAPK profiling using Proteomic Profiler of Human Phospho-MAPK Array (R&D Systems, Minneapolis, Minneapolis) according to the manufacturer’s instructions. Blot images were taken using Chemidoc MP (Bio-Rad Laboratories, Hercules, California). Rectangular outlines indicate reference controls. (C) Densitometry of phosphorylated MAPKs. Intensities of individual molecules on blot (B) were first normalized to the 3 sets of control spots (rectangle boxed spots), and calculated as ratio against non–Lp(a)-treated controls. Total intensities of molecules shown are presented as mean ± SEM. *Significance of Lp(a)-treated vs. nontreated controls, p = 0.002 for p38β/δ/y, p = 0.001 for MKK3/6, p = 0.001 for MSK2, and p = 0.03 for GSK-3α/β. (D) Effect of MAPK and GSK3b inhibition on HAVIC calcification. HAVICs were treated with Lp(a) in the presence or absence of the MAPKP38 inhibitor SB203580 (SB; 2 μmol/l, Sigma-Aldrich Co., LLC., St. Louis, Missouri), GSK3β inhibitor CHIR99021 (CH; 5 μmol/l, Sigma-Aldrich Co., LLC.) or the MEK1/2 inhibitor PD184352 (PD; 2 μmol/l, Sigma-Aldrich Co., LLC.) for 2 weeks in OSM. Total calcium deposition was measured using Alizarin Red S stain and 10% cetylpyridinium chloride extraction protocol, normalized with total protein concentration, and presented as percentage changes against OSM control. One-way ANOVA test showed *p = 0.007, Lp(a)-treated vs. nontreated control; #significant difference between Lp(a)-treated and Lp(a) plus inhibitors (p < 0.00001). MAPK = mitogen-activated protein kinase; other abbreviations as in Figure 1.