Sphingosine-1-Phosphate (S1P)-Related Response of Human Conjunctival Fibroblasts After Filtration Surgery for Glaucoma

Abstract

Citation: Aoyama-Araki Y, Honjo M, Uchida T, et al. Sphingosine-1-phosphate (S1P)-related response of human conjunctival fibroblasts after filtration surgery for glaucoma. Invest Ophthalmol Vis Sci. 2017;58:225858: -226558: . DOI:10.1167 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 chromatographytandem 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.

Autotaxin–Lysophosphatidic Acid Pathway in Intraocular Pressure Regulation and Glaucoma Subtypes

“…Quantification of the lysophospholipids was performed as previously described in detail. 29,30 Briefly, a total of 20 lL AH sample per patient was mixed with a 10-fold volume of methanol and an internal standard and then used for the LC-MS/MS analysis. Then, 20 lL methanol extract was separated using a Nanospace LC (Shiseido, Tokyo, Japan) equipped with a C18 CAPCELL PAK ACR column (1.5 3 250 mm; Shiseido) using a gradient of solvent A (5 mM ammonium formate in water) and solvent B (5 mM ammonium formate in 95% [v/v] acetonitrile).…”

Role of the Autotaxin-LPA Pathway in Dexamethasone-Induced Fibrotic Responses and Extracellular Matrix Production in Human Trabecular Meshwork Cells

“…mRNA levels were quantified by qRT-PCR of cDNA with SYBR Premix Ex Taq II and the Thermal Cycler Dice Real-Time System II (TaKaRa BIO, Inc.) with the DDCt method, as previously described. 46 The sequences of PCR primers used were as follows: GAPDH: forward 5 0 -GAGTCAACGGAT TTGGTCGT-3 0 and reverse 5 0 -TTGATTTTGGAGGGATCTCG-3 0 ; ATX: forward 5 0 -ACAACGAGGAGAGCTGCAAT-3 0 and reverse 5 0 -AGAAGTCCAGGCTGGTGAGA-3 0 ; fibronectin: forward 5 0 -AAACCAATTCTTGGAGCAGG-3 0 and reverse 5 0 -CCAT AAAGGGCAACCAAGAG-3 0 ; COL1A1: forward 5 0 -CAGCCGCTT CACCTACAGC-3 0 and reverse 5 0 -TTTTGTATTCAATCAC TGTCTTG-3 0 ; and COL4A1: forward 5 0 -TAGAGAGGAGCGAGAT GTTC-3 0 and reverse 5 0 -GTGACATTAGCTGAGTCAGG-3 0 . Target gene expression was normalized to that of GAPDH mRNA.…”

“…The expression of phosphorylated myosin light chain (MLC) and cofilin was determined by using Western blot analysis, as previously described. 14,46 Briefly, after serum starvation, cells were stimulated with Dex and/or inhibitors, and cell lysates were collected. Protein concentrations of the cell lysates were measured by using a bicinchoninic acid protein assay kit, and equal amounts of protein samples were separated on 4% to 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes.…”

Evaluation of Lysophospholipid Measurement in Cerebrospinal Fluid Samples using Liquid Chromatography–Tandem Mass Spectrometry

“…Some classes of lysoPL reportedly exist in blood (Yatomi et al, ), urine (Geoffroy et al, ), cerebrospinal fluid (CSF) (Kulakowska et al, ), aqueous fluid (Aoyama‐Araki et al, ), and sperm (Nimptsch et al, ) and can be measured using liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Baker et al, ; Emoto et al, ; Yatomi et al, ; Yin et al, ). Actually, we have measured several lysoPL and their molecular species (lysophosphatidylcholine [lysoPtdCho], lysoPtdOH, lysophosphatidylethanolamine [lysoPtdEtn], lysophosphatidylglycerol [lysoPtdGro], lysophosphatidylinositol [lysoPtdIns], lysophosphatidylserine [lysoPtdSer], and S1P) in blood samples (Yatomi et al, ; Yin et al, ), ascites (Emoto et al, ), and aqueous humor (Aoyama‐Araki et al, ) using a simultaneous lysoPL quantification system and LC–MS/MS.…”

“…LysoPL are emerging as important bioactive lipid mediators in various fields of human disease such as cancer (Yung et al, 2014) and atherosclerosis (Kurano and Yatomi, 2018), because some classes of lysoPL, such as sphingosine 1-phosphate (S1P) or lysophosphatidic acid (lysoPtdOH), exert potent biological properties through G protein-coupled receptors (GPCR). Some classes of lysoPL reportedly exist in blood (Yatomi et al, 1995), urine (Geoffroy et al, 2005), cerebrospinal fluid (CSF) (Kulakowska et al, 2014), aqueous fluid (Aoyama- Araki et al, 2017), and sperm (Nimptsch et al, 2014) and can be measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Baker et al, 2001;Emoto et al, 2017;Yatomi et al, 1997;Yin et al, 2013). Actually, we have measured several lysoPL and their molecular species (lysophosphatidylcholine [lysoPtdCho], lysoPtdOH, lysophosphatidylethanolamine [lysoPtdEtn], lysophosphatidylglycerol [lysoPtdGro], lysophosphatidylinositol [lysoPtdIns], lysophosphatidylserine [lysoPtdSer], and S1P) in blood samples (Yatomi et al, 1997;Yin et al, 2013), ascites (Emoto et al, 2017), and aqueous humor (Aoyama-Araki et al, 2017) using a simultaneous lysoPL quantification system and LC-MS/MS.…”