Autotaxin and IOP Figure 9. ATX inhibitor-S32826 induced changes in cultured human TM cells. A. Primary cultures of human TM cells cultured either on gelatin-coated glass coverslips or in plastic petridishes were treated with S32826 (10 mM) in the absence of serum or in the presence of 0.5% fetal bovine serum for 24 hrs. Based on phase contrast microscopic examination, treatment of TM cells cultures with drug caused the appearance of membranous floating clumps (arrows) in culture medium, without any noticeable changes in cell morphology and cell adhesion relative to control cells treated with DMSO vehicle. This response appeared to increase progressively with time. B. The membranous floating material generated from drug (S32826) treated cells (A) was extracted from the cell culture media by centrifugation, rinsed with PBS, dissolved in Laemmli sample buffer, separated by SDS-PAGE and stained with gelcode blue as shown in the figure (two independent samples are shown from each treatment). Subsequently, the predominant protein bands were gel extracted and subjected to mass spectrometry-based identification. This analysis revealed presence of proteins involved in lysosomal exocytosis, trafficking and membrane repair as discussed in the results section. doi:10.1371/journal.pone.0042627.g009 induces formation of actin stress fibers, focal adhesions and myosin light chain phosphorylation and Rho GTPase activation in TM cells [15]. Further, in contrast to the ocular hypotensive effects of the ATX inhibitor S32826 noted in this study, perfusion of LPA has been demonstrated to decrease AH outflow facility, leading to increased IOP [15]. Intriguingly, S32826 showed no detectable effects either on cell morphology or actin cytoskeletal reorganiza- tion of TM cells maintained in serum free media. Instead, drug treated TM cell cultures maintained either under serum-starved conditions or with 0.5%FBS were observed to generate cell- derived membranous floating material with no evidence of a cytotoxic response. The analysis of protein composition of this material by mass spectrometry analysis confirmed presence of proteins involved in membrane trafficking, repair and exocytosis. It is not clear whether inhibition of autotaxin, which is known to act locally at cell surface by interacting with integrins and to produce LPA close to the cell surface receptors, influences membrane recycling properties in TM cells [42,43,44]. In conclusion, this initial study reports the ocular hypotensive effect of an ATX inhibitor in live rabbits, suggesting potential significance of ATX as a therapeutic target for lowering IOP in glaucoma patients. Further studies are necessary to delineate the molecular mechanisms by which inhibition of ATX mediates decrease of IOP. Methods Ethics Statement Animals. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Association for Research in Vision and Ophthalmology. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Duke University School of Medicine (Protocol Number: A094-10-04). Intracameral injections were performed under ketamine/xylazine anesthesia, and all efforts were made to minimize pain and suffering during experiments. Human Subjects. All research involving collection of human aqueous humor samples has been approved by the Duke University School of Medicine’s institutional review board (IRB). PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e42627