Effects of PA, EPA and inhibitors of ACS and NF-κB on the expression of bone-related genes in HASMC.

<p>HASMC were treated with PA (250 µM) for 1 day in the presence or absence of EPA (15 µM), triacsin C (Tri C; 2 µM) or hypoestoxide (Hypo; 100 µM). The expression of BMP-2, Msx2 and osteopontin (OPN) mRNAs was determined by real-time PCR and normalized to GAPDH. The control values are shown as 1.0. All values expressed by arbitrary unit (AU) are presented as the mean ± S.E. (n = 4). #p<0.05, ###p<0.001 vs. control, **p<0.01, ***p<0.001 vs. PA.</p

PA induces NF-κB activation.

<p>A, HASMC were treated with PA (250 µM) for 1 day with or without EPA (15 µM). Either ACSL3 siRNA or nonsilencing control (NC) siRNA was transfected before PA treatment. Expression of nuclear phospho-NF-κB (p65) protein was determined by Western blot analysis. B, HASMC were transfected with pNF-κB-Luc and then treated with PA (250 µM) for 1 day with or without EPA (15 µM) or Tri C (2 µM). The luciferase activity of the NF-κB reporter was measured according to the manufacturer’s protocol. All values expressed by AU are presented as the mean ± S.E. (n = 6). ###p<0.001 vs. control, **p<0.01, ***p<0.001 vs. PA.</p

Knock-down and overexpression of ACSL3 attenuates and augments PA-induced expression of osteoblastic genes in HASMC.

<p>A, HASMC were transfected with either ACSL3 siRNA or nonsilencing negative control (NC) siRNA, and then cells were treated with PA (250 µM) for 1 day. Expressions of ACSL3, BMP-2, Msx2 and OPN mRNA were determined by real-time PCR and normalized to GAPDH. B, HASMC were infected with Ad-LacZ or Ad-ACSL3 and then treated with PA (250 µM) for 1 day. Expression of ACSL3, BMP-2, Msx2 and OPN mRNAs were determined by real-time PCR and normalized to GAPDH. The control values are shown as 1.0. All values expressed by AU are presented as the mean ± S.E. (n = 4–6). ##p<0.01, ###p<0.001 vs. control+NC siRNA or control+Ad-LacZ, *p<0.05, ***p<0.001 vs. PA+NC siRNA or PA+Ad-LacZ.</p

Schematic diagram of possible effects of PA and EPA on osteoblastic differentiation and calcium deposition in HASMC.

<p>Schematic diagram of possible effects of PA and EPA on osteoblastic differentiation and calcium deposition in HASMC.</p

PA increases ACS activity and ACSL3 mRNA level in HASMC.

<p>HASMC were treated with PA (250 µM) for 1 day in the presence or absence of EPA (15 µM). A, ACS activity in cell homogenates was determined as described in Materials and Methods. B, Expression of ACSL1, 3 and 4 mRNAs was determined by real-time PCR and normalized to GAPDH. C, The expression of ACSL1, 3 and 4 protein was determined by Western blot analysis. All values expressed by AU are presented as the mean ± S.E. (n = 3–9). ##p<0.01, ###p<0.001 vs. control, *p<0.05, **p<0.01, ***p<0.001 vs. PA (except for C).</p

The expression of ACSL3 in normal and calcified human carotid artery.

<p>A, Immunohistochemistry for ACSL3 and SM α-actin in normal (upper panel) and calcified human carotid artery (lower panel). Magnification ×100. B to D, Immunofluorescent labeling for ACSL3 (red or green), (B) SM α-actin (green), (C) CD68 (red) or (D) Msx2 (green) and DAPI (blue) in normal and calcified human carotid artery. Magnification ×100 and ×200 (only calcified human carotid artery in D).</p

High-concentration PA induced calcium deposition and caspase activation in HASMC.

<p>HASMC were treated with PA (1 mM) for 1 day with or without EPA (15 µM), Tri C (2 µM), Hypo (100 µM) or Z-VAD-FMK (Z-VAD; 100 µM). ACSL3 siRNA were transfected before PA treatment. A, Calcium deposition was determined by the methylxylenol blue method and normalized to protein content. B, Calcium deposition was detected using von Kossa-staining. C, Caspase-3/7 activity was determined as described in Materials and Methods, and normalized to protein content. All values are presented as the mean ± S.E. (n = 3–14). ###p<0.001 vs. control or control+NC siRNA, **p<0.01, ***p<0.001 vs. PA or PA+NC siRNA.</p