Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC121
The lipid mediator sphingosylphosphorylcholine regulates inflammatory functions in rat vascular smooth muscle cells
C. Wirrig1, G. F. Nixon1
1. School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.
Sphingolipids are naturally occurring cellular mediators which can regulate vascular function. Two sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), are integral components of lipoproteins and also released from platelets. S1P effects are mediated by S1P receptors while the mode of action of SPC is unknown. Sphingolipids can regulate inflammation but their role in vascular smooth muscle cell (VSMC) inflammation is unclear. Our aim was to determine the inflammatory effects of sphingolipids on VSMC. To assess vascular adhesion of immune cells we used an in vitro co-culture system of primary cultured rat aortic VSMC and Sprague Dawley rat bone marrow-derived macrophages (BMDM). BMDM were primed with 10 ng/ml tumour necrosis factor (TNF)-α for 24 h in all experiments and co-cultured with VSMC for 15 min. Adherent fluorescently-labelled BMDM were counted. Incubation of VSMC (but not BMDM) with 5 μM S1P or 10 μM SPC for 24 h did not change adhesion of BMDM to VSMC. In contrast, TNF-α treatment of VSMC for 24 h increased adhesion (1.8 ± 0.1 fold increase compared to control, mean ± s.e.m., n=19). This increase was inhibited by treatment of VSMC with SPC (0.9 ± 0.1 fold increase compared to TNF alone, p<0.05 ANOVA, n=19), but not S1P. Incubation of BMDM with SPC did not reduce TNF-α-induced adhesion. We examined the potential mechanisms involved in the anti-adhesive effect of SPC on VSMC. Protein expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule 1 in VSMC was increased by TNF-α but not altered by SPC (n=15). SPC increased protein expression of the prostaglandin producing enzyme cyclooxygenase (COX)-2 in VSMC after 24 h incubation assessed by immunoblotting (1.9 ± 0.3 fold increase, n=13). This was significantly enhanced when SPC and TNF-α were added simultaneously (3.8 ± 0.4 fold increase compared to SPC alone, p<0.05, n=13). The selective COX-2 inhibitor NS-398 (5 µM) had no effect on the SPC-induced decrease in BMDM adhesion. COX-2 is therefore unlikely to be involved. Finally, as SPC is lipophilic it may interact with lipid raft areas of the membrane. Incubation of VSMC for 1 h with the cholesterol sequestering agent methyl β-cyclodextrin (MβCD, 1 mM) did not alter TNF-α induced adhesion of BMDM. The anti-adhesive effect of SPC on VSMC binding to BMDM was, however, significantly inhibited by 1 h incubation with MβCD (TNF alone - 2.9 ± 0.3 fold increase, SPC+TNF - 1.7 ± 0.1 fold increase, SPC+TNF+ MβCD - 3.2 ± 0.3 fold increase, p<0.05, n=5-8). This indicates that SPC may require lipid rafts to induce this effect. In conclusion, despite inducing pro-inflammatory mediators (such as COX-2), SPC has an anti-inflammatory effect via reducing adhesion of macrophages to VSMC. This may occur via lipid raft interactions.
Where applicable, experiments conform with Society ethical requirements