Lysophosphatidylcholine and carotid intima-media thickness in young smokers: a role for oxidized LDL-induced expression of PBMC lipoprotein-associated phospholipase A2?

Abstract

Background: Although cigarette smoking has been associated with carotid intima-media thickness (CIMT) the mechanisms are yet not completely known. Lysophosphatidylcholine (lysoPC), a main product of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, appears to be a major determinant of the pro-atherogenic properties of oxidized LDL (oxLDL) and to induce proteoglycan synthesis, a main player in intimal thickening. In this study we assessed whether cigarette smoking-induced oxidative stress may influence plasma Lp-PLA2 and lysoPC and Lp-PLA2 expression in peripheral blood mononuclear cells (PBMC), as well as the relationship between lysoPC and CIMT.

Methods/results: 45 healthy smokers and 45 age and sex-matched subjects participated in this study. Smokers, compared to non-smokers, showed increased plasma concentrations of oxLDL, Lp-PLA2 and lysoPC together with up-regulation of Lp-PLA2 (mRNA and protein) expression in PBMC (P<0.001). Plasma Lp-PLA2 positively correlated with both lysoPC (r=0.639, P<0.001) and PBMC mRNA Lp-PLA2 (r=0.484, P<0.001) in all subjects. Moreover CIMT that was higher in smokers (P<0.001), positively correlated with lysoPC (r=0.55, P<0.001). Then in in vitro study we demonstrated that both oxLDL (at concentrations similar to those found in smoker's serum) and oxidized phospholipids contained in oxLDL, were able to up-regulate mRNA Lp-PLA2 in PBMC. This effect was likely due, at least in part, to the enrichment in oxidized phospholipids found in PBMC after exposure to oxLDL. Our results also showed that in human aortic smooth muscle cells lysoPC, at concentrations similar to those found in smokers, increased the expression of biglycan and versican, two main proteoglycans.

Conclusions: In smokers a further effect of raised oxidative stress is the up-regulation of Lp-PLA2 expression in PBMC with subsequent increase of plasma Lp-PLA2 and lysoPC. Moreover the correlation between lysoPC and CIMT together with the finding that lysoPC up-regulates proteoglycan synthesis suggests that lysoPC may be a link between smoking and intimal thickening.

Figure 1. Correlation between plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) and oxidized LDL (oxLDL) and between plasma LpPLA2 and lysophosphatidylcholine (lysoPC).

(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.

Figure 2. Lipoprotein-associated phospholipase A2 (Lp-PLA2) mRNA and protein expression in peripheral blood mononuclear cells (PBMC) of non-smokers and smokers and correlation between Lp-PLA2 expression in PBMC and plasma Lp-PLA2.

(A) mRNA was analyzed by quantitative Real-Time PCR. Normalized gene expression levels were given as the ratio between the mean value for the Lp-PLA2 gene and that for beta-actin in each sample. Data are presented as mean+SD. *P<0.001 versus non-smokers. (B) Representative Western blot analysis for Lp-PLA2 and the average quantification obtained by densitometric analysis of all the samples (20 subjects per group, randomly selected). (C) Correlation between Lp-PLA2 mRNA expression in PBMC and plasma concentrations of Lp-PLA2 in the whole population of subjects studied.

Figure 3. Carotid intima-media thickness (CIMT) in non-smokers and smokers and correlation between CIMT and plasma concentrations of lysophosphatidylcholine (lysoPC).

(A) Mean and maximal CIMT values in non-smokers and smokers, *P<0.001 versus non-smokers. Data are presented as mean+SD. (B) Correlation between CIMT and plasma concentrations of lysoPC in the whole population of subjects studied.

Figure 4. Dose-response effect of native LDL (nLDL), oxidized LDL (oxLDL), oxidized phospholipids (oxPAPC), and of lysophosphatidylcholine (lysoPC) on mRNA expression of Lp-PLA2.

(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.

Figure 5. Representative mass spectrometry (MS) analysis of PBMC extract.

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 m/z), B) POVPC (594.4 m/z) and C) PGPC (610.4 m/z). 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 m/z).

Figure 6. Dose-response effect of lysophosphatidylcholine (lysoPC) on biglycan and versican mRNA expression.

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.

Figure 7. Dose-response effect of lysophosphatidylcholine (lysoPC) on biglycan protein expression.

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.