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

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

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

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

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

Biometric and echocardiographic parameters of rats fed the control or HS diet.

<p>Data are the means ± SD.</p>*<p>P<0.05, **P<0.01 vs. control fed group.</p><p>SBP, systolic blood pressure; DBP, diastolic blood pressure; IVSd, Interventricular septal end-diastolic dimension; LVEdD or LVEsD, left ventricular end-diastolic or systolic diameter; LVPWd, left ventricular posterior wall thickness in diastole; EF, ejection fraction; FS, fractional shortening; E/A, transmitral flow ratio.</p

FA and glucose oxidations in SCD1-overexpressed cardiac myocytes.

<p>(A): FA oxidation was significantly increased in rat neonatal cardiac myocytes treated with 100 µM palmitic acid (PA). (B): On the other hand, glucose oxidation was significantly reduced in cardiac myocytes treated with PA (100 µM). (C): PA (50 to 100 µM) induced excessive FA oxidation was attenuated in cardiac myocytes transduced with a MOI of 20 of Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). (D): PA (50 to 100 µM) -induced suppression of glucose oxidation recovered in cardiac myocytes transduced with Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). BSA was used as a vehicle. Each sample was counted in a scintillation counter (cpm) and data are shown as the mean ± SD. **P<0.01 vs. Ad-LacZ in each PA concentration. (E); Phosphorylation levels of AMPK and ACC decreased in cardiac myocytes transduced with an MOI of 20 of Ad-SCD1 in comparison with control cells transduced with Ad-LacZ. Oligomycin (1 µM) was used as an AMPK and ACC activator.</p

Heart tissue fatty acid composition of rats fed the control or HS diet for 3 months.

<p>Data are the means ±SD, and are presented as percent of total fatty acids.</p>*<p>P<0.05, **P<0.01 vs. control fed group.</p