Increased aqueous autotaxin and lysophosphatidic acid levels are potential prognostic factors after trabeculectomy in different types of glaucoma

Abstract

We explored the potential relevance of aqueous lysophosphatidic acid (LPA) and autotaxin (ATX) levels on postoperative outcomes of trabeculectomy, and the effects of ATX on fibrotic response in cultured human conjunctiva fibroblast (HCF) cells. We enrolled 70 glaucomatous eyes which underwent trabeculectomy, and quantified aqueous LPA and ATX. Those eyes were followed up for 12 months, and postoperative filtering blebs were evaluated using anterior segment optical coherence tomography. Also, the ATX-induced fibrotic changes in HCFs and the effects of an ATX inhibitor were assessed. Measured aqueous ATX and LPA levels were significantly different between glaucoma subtypes. In multivariate analyses, aqueous ATX levels were significantly correlated with the presence of needlings at 1, 3, 6 and 12 months after surgery. Exfoliative glaucoma, whose ATX level was significantly high, showed significantly increased numbers of needlings and a lower cumulative success rate without needlings. An in vitro study showed that fibrotic changes were upregulated by ATX treatment in HCFs, which was significantly suppressed by an ATX inhibitor. We presently demonstrate that aqueous ATX may be a prognostic factor affecting the fibrotic response in HCFs and bleb formation, and inhibition of ATX could be a therapeutic target after trabeculectomy.

Involvement of autotaxin in the pathophysiology of elevated intraocular pressure in Posner-Schlossman syndrome

“…Measurement of AtX, AtX isoforms, and tGf-β1, 2 and 3 in the AH. The level of ATX in the AH was determined using a two-site immunoenzymatic assay with an ATX assay reagent equipped with a Tosoh AIA system (Tosoh, Tokyo, Japan) as described previously 27,28,54,55 . The TGF-β levels in the AH were measured using the Bio-Plex Pro TGF-β Assay (Bio-Rad, CA, USA) following the manufacturer's protocol.…”

“…immunocytochemistry. Immunocytochemistry was performed as previously described 55 . The primary antibodies were Anti-Cytomegalovirus Antibody, clone 8B1.2 (1:2,000; Merck Millipore, Billerica, MA, USA), Anti-ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase family member 2) antibody [5H3] (1:1,000; Abcam, Cambridge, MA, USA), Anti-TGF-β1 antibody (1:100; Sigma-Aldrich), anti-αSMA (1:500; Sigma-Aldrich) and anti-fibronectin [IST-9] (1:400; Abcam).…”

“…Quantitative pcR. The cells were lysed using TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH, USA), and mRNA was isolated using chloroform and isopropyl alcohol as previously described 55 . The mRNA was treated with a PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan) to synthesize cDNA.…”

“…After 1 dpi, cells were collected in RIPA Buffer (Thermo Fisher Scientific) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland), sonicated, and centrifuged. The following protein concentration measurement and SDS-PAGE was performed as previously described 55 . Protein bands were transferred to PVDF membranes (Bio-Rad Laboratories) and the membranes were immersed in Tris-buffered saline with Tween 20 (TBST) containing primary antibody.…”

Effects of ROCK Inhibitors on Apoptosis of Corneal Endothelial Cells in CMV-Positive Posner–Schlossman Syndrome Patients

“…Immunocytochemistry (ICC) was performed as previously described. 34 The cells were grown in chamber slides. The hCECs were fixed in ice-cold 4% paraformaldehyde for 15 minutes 1 day after infection and then permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) for 5 minutes and blocked in 3% BSA for 30 minutes.…”