Figure 6.

OA and LA attenuate ER stress and increase LOX expression in VSMCs. A, the expression of LOX relative to GAPDH in ECs and VSMCs treated for 24 h with or without AT-II (100 nm) (n = 4). B, the expression of LOX relative to GAPDH in ECs and VSMCs treated for 24 h with or without TGFβ1 (5 ng/ml) (n = 4). C, expression of PLA2G5 in ECs that were pretreated with PLA2G5-specific siRNA or nonsilencing negative control siRNA and then treated for 24 h with or without AT-II (n = 4). D–F, expression of LOX in VSMCs treated for 24 h with the supernatant (sup) of ECs. D, ECs were pretreated with a PLA2G5-specific or control siRNA and then treated for 24 h with or without AT-II (n = 4). E, ECs were treated for 24 h with or without AT-II in the presence or absence of 100 nm varespladib, a pan-sPLA₂ inhibitor (n = 4). F, ECs were treated for 24 h with or without AT-II in the presence or absence of 10 μm AACOCF₃, a cPLA₂α inhibitor (n = 3). G, effect of OA or LA on the expression of LOX relative to GAPDH in VSMCs treated for 24 h with or without TGFβ1 (n = 3). H, LOX activity in supernatant of VSMCs treated for 24 h with or without TGFβ1 in the presence or absence of OA or LA (100 μm) (n = 4). I, expression of LOX in VSMCs treated for 24 h with or without TGFβ1 (0.2–5 ng/ml) in the presence or absence of OA or LA (100 μm) (n = 4). J, expression of TGFB1 relative to GAPDH in ECs treated for 24 h with or without AT-II (100 nm) in the presence or absence of OA or LA (100 μm) (n = 4). K, expression of TGFB1 in VSMCs treated for 24 h with or without TGFβ1 (5 ng/ml) in the presence or absence of OA or LA (100 μm) (n = 4). L, expression of PLA2G5 in ECs treated for 24 h with or without TNFα (10 ng/ml) or VEGF (10 ng/ml) (n = 4). Shown are immunoblot analysis of Bip protein, with GAPDH as an internal control (M), and densitometric analysis of Bip protein relative to GAPDH (n = 4) (N) in VSMCs treated for 24 h with or without TGFβ1 in the presence (+) or absence (−) of OA or LA (100 μm). O and P, effect of OA or LA on the expression of GATA3 (O) or LOX (P) in VSMCs treated for 24 h with or without TGFβ1 in the presence or absence of TGN (10 nm) (n = 3). Q and R, mRNA expression of GATA3 (Q) and LOX (R) in VSMCs that were pretreated with GATA3-specific siRNAs (#1 and #2) or control siRNA and then treated for 24 h with or without TGFβ1 (n = 4). S, a schematic summary of ECM stabilization by OA or LA in VSMCs. Whereas TGFβ1 induces LOX expression through canonical signaling pathways, it also induces ER stress leading to up-regulation of GATA3, which prevents LOX expression as a counter-regulatory arm. OA and LA counteract this process by attenuating TGFβ1-induced ER stress. *, p <0.05; **, p <0.01; ns, not significant by one-way ANOVA followed by Tukey's multiple-comparison test. Data are presented as mean ± S.E. (error bars) of the indicated number (n) of biological replicates.