Sphingosine-1-phosphate (S1P)-related response of human conjunctival fibroblasts after filtration surgery for glaucoma

PURPOSE. To investigate levels of sphingosine-1-phosphate (S1P) in aqueous fluid samples taken before and after filtration surgery and S1P-induced human conjunctival fibroblast (HCF) responses. METHODS. Levels of S1P and its related sphingophospholipids in aqueous fluid obtained immediately before and after filtration surgery were determined by liquid chromatography– tandem mass spectrometry. HCFs were used for all in vitro experiments. The expression of five S1P receptor subtypes in HCFs was examined by quantitative real-time PCR. The effect of S1P and receptor-specific antagonists on HCF viability and cell migration was assessed by WST-1 assay and scratch migration assay, respectively. Differentiation to myofibroblasts and extracellular matrix production was evaluated by examining changes in F-actin, a-smooth muscle actin (aSMA), and collagen expression with immunocytochemistry, Western blotting, and collagen accumulation assay, respectively. RESULTS. No significant S1P levels in the aqueous fluid samples were detectable immediately before surgery, but postoperative levels of several lysophospholipids, including S1P, dehydro- S1P, and sphingosine, were significantly increased to bioactive concentrations in aqueous fluid in the blebs (P < 0.0001). mRNA expression of the three main S1P receptor subtypes was detected in HCFs. Although S1P levels did not influence HCF proliferation, S1P enhanced cell migration, which could be inhibited by the S1P2 antagonist JTE 013. F-actin, aSMA, and collagen expression was significantly increased by S1P stimulation and was reduced by JTE 013. CONCLUSIONS. Bioactive S1P concentrations were present in the aqueous fluid at the end of filtration surgery. S1P activated HCFs via S1P2 receptors. These results revealed the potential of S1P2 antagonists in preventing scarring after glaucoma filtration surgery.