Dose-response effect of lysophosphatidylcholine (lysoPC) on biglycan protein expression.

<p>Effect of increasing concentrations (0-50 µM) of lysoPC on biglycan protein expression after incubation with (A) quiescent and (C) proliferating human aortic SMCs for 24 h. Data are the average quantification obtained by densitometric analysis of all the samples, expressed as the density ratio of target to control (beta-actin) in arbitrary units x10. Figure also shows a representative Western blot analysis (B and D) of three independent experiments for biglycan.</p

Representative mass spectrometry (MS) analysis of PBMC extract.

<p>PBMC were incubated with native LDL (nLDL) or oxidized LDL (oxLDL) (100 µg/ml) for 18 h. Indicated peaks correspond to A) lysoPC (496.1 <i>m/z</i>), B) POVPC (594.4 <i>m/z</i>) and C) PGPC (610.4 <i>m/z</i>). MS analysis of PBMC extract (1/10 with 10 mM ammonium acetate in methanol) was performed on an ion trap mass spectrometer where ions were generated with an electrospray ionization source at 350° C, and spectra were acquired in the positive mode (total ion range 480–640 <i>m/z</i>).</p

Effect of serum derived from non-smokers and smokers and of lipoprotein-depleted serum (LPDS) derived from smokers on intracellular GSH concentration, on intracellular reactive oxygen species (ROS) and on nitric oxide (NO) formation in human umbilical vein endothelial cells (HUVECs).

<p>Confluent HUVECs were incubated without and with 10, 30 and 50% serum derived from non-smokers and smokers and with the corresponding LPDS derived from smokers for 12 hours. Figure shows intracellular GSH concentration (A), intracellular ROS (B) and cumulative basal and bradykinin-stimulated NO production, evaluated by measuring levels of nitrite in the supernatants (C). Data are presented as mean±SD of measurements performed in triplicate in four different occasions; *P<0.01 versus control (no addition of serum derived from the subjects) non-smokers' serum and smokers' LPDS.</p

Dose-response effect of native LDL (nLDL), oxidized LDL (oxLDL), oxidized phospholipids (oxPAPC), and of lysophosphatidylcholine (lysoPC) on mRNA expression of Lp-PLA2.

<p>(A) Dose-response effect of nLDL, oxLDL and oxPAPC (0-100 µg/ml) and (B) dose-response effect of lysoPC (0-50 µM) on mRNA expression of Lp-PLA2 after incubation with PBMC for 6 h. Normalized gene expression levels were given as the ratio between the mean value for Lp-PLA2 gene and that for the beta-actin in each sample. Data represent the mean±SD of measurements performed in triplicate in four different occasions. *P<0.01 versus basal, †P<0.01 versus oxPAPC.</p

Dose-response effect of lysophosphatidylcholine (lysoPC) on biglycan and versican mRNA expression.

<p>Effect of increasing concentrations (0-50 µM) of lysoPC on (A) biglycan and on (B) versican mRNA expression after incubation with quiescent and proliferating human aortic SMCs for 24 h. Normalized gene expression levels were given as the ratio between the mean value for target gene and that for the beta-actin in each sample. Data represent the mean±SD of measurements performed in triplicate in four different occasions. * P<0.01 versus quiescent; †P<0.01 versus basal.</p

Effect of increasing concentrations of oxPAPC on intracellular GSH concentration, on intracellular reactive oxygen species (ROS), and on nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs).

<p>Confluent HUVECs were incubated without and with increasing concentrations (25–150 µg/mL) of oxPAPC for 6 hours. Figure shows intracellular GSH concentration (A), intracellular ROS (B) and cumulative basal and bradykinin-stimulated NO production, evaluated by measuring levels of nitrite in the supernatants (C). Data are presented as mean±SD of measurements performed in triplicate in four different occasions. *P<0.01 versus oxPAPC 0 e 75 µg/mL. # P<0.01 versus basal NO.</p

Correlation between plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) and oxidized LDL (oxLDL) and between plasma LpPLA2 and lysophosphatidylcholine (lysoPC).

<p>(A) Correlation between plasma Lp-PLA2 and oxLDL and (B) correlation between plasma Lp-PLA2 and lysoPC concentrations in the whole population of subjects studied.</p

Endothelial function and correlation between flow-mediated vasodilation (FMD) and concentrations of GSH in peripheral blood mononuclear cells (PBMC) of non-smokers and smokers.

<p>FMD and glyceryl trinitrate (GTN)-induced vasodilation in non-smokers and smokers (A); correlation between FMD and intracellular concentrations of GSH in PBMC of non-smokers and smokers (B). Data are presented as mean±SD; FMD and GTN are expressed as maximal percentage change in brachial artery dilation. *P<0.001 versus non-smokers.</p

Effect of increasing concentrations of oxPAPC on Nrf2, GCLC and HO-1 expression in human umbilical vein endothelial cells.

<p>mRNA (A–C) was analysed by quantitative Real-Time PCR. Normalised gene expression levels were given as the ratio between the mean value for the target gene and that for beta-actin in each sample. Results are presented as the mean±SD of measurements performed in triplicate.*P<0.01 versus oxPAPC 0 and 150 µg/mL. Figure (D) shows a representative Western blot analysis of three independent experiments for nuclear Nrf2, GCLC and HO-1.</p

Effect of serum derived from non-smokers and smokers and of lipoprotein-depleted serum (LPDS) derived from smokers on Nrf2, GCLC and HO-1 expression in human umbilical vein endothelial cells (HUVECs).

<p>Confluent HUVECs were incubated without and with increasing amounts (10, 30 and 50%) of serum derived from smokers (serum S 10, serum S 30, serum S 50), with 50% serum derived from non-smokers (serum N–S 50) and with 50% LPDS derived from smokers (LPDS S 50) for 12 hours. mRNA for Nrf2, GCLC and HO-1 (A–C) was analysed by quantitative Real-Time PCR. Normalised gene expression levels were given as the ratio between the mean value for the target gene and that for beta-actin in each sample. Results are reported as the mean±SD of measurements performed in triplicate. *P<0.01 versus non-smokers and LPDS. (D) shows a representative Western blot analysis of three independent experiments for nuclear Nrf2, for GCLC and HO-1.</p