Sphingosine-1-phosphate and toll-like receptor 4-cooperate to induce inflammatory and adhesive responses in human aortic valve endothelial cells

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Purpose: Given the association of Toll-like receptor (TLR)-4 to calcific aortic stenosis and the relevant function of the lipid mediator sphingosine-1-phosphate (S1P) in the cardiovascular system, the putative role of TLR4 ligands and S1P in the induction of pro-inflammatory and pro-adhesive responses in endothelial cells from human aortic (AVEC) and pulmonary valves (PVEC) was investigated. Methods: AVEC and PVEC obtained from n =5 heart transplant patients with no valve disease, and AVEC derived from stenotic aortic valves collected from n = 5 patients with degenerative aortic stenosis and indication for valve replacement were studied. Reverse transcriptase PCR assays were used to evaluate the mRNA expression of TLR and S1P receptors. Cytokine secretion and adhesion molecule expression were evaluated by antibody arrays and Western blot, and adhesion of human monocytes to AVEC monolayers was assayed in vitro. Comparison between stenotic and control aortic valve and between aortic and pulmonar valve were performed. Results: Reverse transcriptase PCR assays revealed the expression of TLRs at the mRNA level in AVEC, being TLR4 the most abundant, as well as the expression of S1P receptors, the S1P1 being the most abundant subtype. As regards pro-inflammatory molecules, the exposure of control AVECs to the bacterial lipopolysaccharide (LPS), a TLR4 ligand, induced the secretion of several cytokines such as interleukin (IL)-6, granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte chemotactic protein-1. Moreover, LPS cooperated with S1P to further up-regulate IL-6 and GM-CSF secretion. In addition, AVEC exposure to LPS induced the expression of the adhesion molecule ICAM-1, which was further up-regulated in the presence of S1P. Interestingly, the effect was more prominent in endothelial cells from the aortic valve than from the pulmonary valve, which rarely undergoes stenosis, and more potent in cells from stenotic valves. The cooperative effect on ICAM-1 up-regulation was inhibited by S1P1 receptor antagonists. In adhesion assays, LPS and S1P cooperated to increase the number of monocytes that adhered to the activated AVEC monolayers, and the effect was more marked in stenotic than in control AVECs. Conclusions: TLR4 and S1P1 receptor cooperate to up-regulate pro-inflammatory/pro-adhesive molecules in AVECs, what might be relevant for the pathogenesis of aortic stenosis given the stronger effect on stenotic aortic valves and the lower effect on pulmonary valves, and could open the way for new therapeutic approaches for the disease.