Sphingosine-1-Phosphate (S1P)-Related Response of Human Conjunctival Fibroblasts After Filtration Surgery for Glaucoma
Sphingosine-1-Phosphate (S1P)-Related Response of Human Conjunctival Fibroblasts After Filtration Surgery for Glaucoma
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DOI：
10.1167/iovs.16-21288
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发表时间：
2017-04-01
影响因子：
4.4
通讯作者：
Aihara, Makoto
中科院分区：
文献类型：
--
作者：
Aoyama-Araki, Yuka;Honjo, Megumi;Aihara, Makoto
目的.目的：研究青光眼滤过手术前后房水中1-磷酸鞘氨醇（S1 P）的水平及S1 P诱导的人结膜成纤维细胞（HCF）反应。通过液相色谱-串联质谱法测定滤过手术前后即刻获得的水性液体中S1 P及其相关鞘磷脂的水平。HCF用于所有体外实验。实时荧光定量PCR检测5种S1 P受体亚型在HCF中的表达。分别采用WST-1法和划痕迁移法检测S1 P和受体特异性拮抗剂对HCF活力和细胞迁移的影响。通过分别用免疫细胞化学、蛋白质印迹和胶原积累测定检查F-肌动蛋白、α-平滑肌肌动蛋白（α SMA）和胶原表达的变化来评估向肌成纤维细胞的分化和细胞外基质的产生。手术前即刻在房水样本中未检测到显著的S1 P水平，但术后几种溶血磷脂（包括S1 P、脱氢S1 P和鞘氨醇）的水平显著升高至滤过泡中房水中的生物活性浓度（P < 0.0001）。HCF中检测到三种主要S1 P受体亚型的mRNA表达。虽然S1 P水平不影响HCF增殖，但S1 P增强细胞迁移，这可以被S1 P2拮抗剂JTE 013抑制。F-actin、α SMA和胶原蛋白的表达在S1 P刺激后显著增加，而在JTE 013作用下减少。滤过手术结束时，房水中存在生物活性S1 P浓度。S1 P通过S1 P2受体激活HCF。这些结果揭示了S1 P2拮抗剂在预防青光眼滤过手术后瘢痕形成方面的潜力。
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 (alpha SMA), 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, alpha SMA, 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.