Autotaxin and IOP Figure 7. Intracameral injections of ATX inhibitor decreases IOP in rabbits. To determine the effects of intracameral delivery of S32826 on IOP, Dutch belted adult male rabbits (one eye from each animal, n = 4) were intracamerally injected with 5 ml of 100 mM drug. IOP was monitored in drug treated and contralateral (untreated) control eyes using a pneumotonometer. Drug treated eyes showed a significant decrease in IOP from the baseline (by 6–7 mm Hg) at 24 and 48 hrs based on paired t-test. This IOP lowering effect continued for more than 96 hrs, with IOP gradually returning to basal value by120 hrs post- injection. Further, IOP in drug treated eyes was significantly reduced as compared to IOP of the contralateral untreated eyes and eyes treated with sham control (PBS containing 2%DMSO) based on the Wilcoxon Rank Sum test of differences in medians. For the sham treatment, we used the contralateral eyes of the same animals in which we tested the effects of drug, with the sham treatment being performed 2 weeks after completion of the drug treatments (n = 4 for drug treated, sham treated and untreated controls). However, at the 96 hr time point, IOP was recorded in only two of the four rabbits from the drug treated and untreated control groups. *P,0.05. doi:10.1371/journal.pone.0042627.g007 present in abundant amounts in human AH. ATX, a secretory protein, which was initially characterized as autocrine factor involved in cell motility, proliferation, differentiation and survival, has been identified as an extracellular LysoPLD which generates LPA and to a lesser extent S1P [24,25,26,36,37,38]. LPA is a bioactive lipid that in turn regulates several cellular processes including Rho GTPase regulated cell adhesion, contraction and migration by acting through different G-protein coupled receptors [39]. Importantly, abnormal regulation of the ATX-LPA axis has been shown to be associated with various pathological conditions including cancer and fibrosis [24,37]. Our analysis of AH samples derived from POAG and control (cataract) patients revealed significantly elevated levels of LysoPLD activity (expressed per unit of total protein) in POAG patients. Although there was a significant difference in racial demogra- phy between the glaucoma and control specimens tested in this study, it is unlikely that the 1.8 fold elevation in the LysoPLD activity that we noted in POAG patients relative to age-matched cataract control samples is solely related to differences in the racial demography. Further, there is no known currently available information on racial differences in gene expression profiles of TM or other ocular tissues of the anterior segment of the eye. However, it is important to note that POAG is more prevalent in African Americans and Hispanics relative to Caucasians [40,41]. While it would have been ideal to determine and confirm whether the observed elevated levels of LysoPLD activity in the AH of POAG patients is associated with elevated levels of LPA, the product of ATX, we could not however, establish this association due to inadequate amounts of AH obtained from each patient. Based on abundant levels of ATX in AH, and elevated LysoPLD activity in the AH of POAG patients, we hypothesized that inhibition of ATX in the eye would likely impact AH outflow through the trabecular meshwork, and thereby IOP as well. To set the stage for addressing this hypothesis, we first performed in vitro studies using a relevant cell type, confirming that TM cells derived from humans and other species express and secrete ATX. Additionally, both secretion and expression of ATX by TM cells was found to be responsive to cyclic mechanical stretch, further supporting the mechanosensitive nature of TM cells and the impact of mechanotransducing stimuli on TM cell physiology [8,14]. To test whether inhibition of ATX in the AH influences IOP, we evaluated the effects of a well characterized small molecular weight water soluble pharmacological inhibitor of ATX (S32826) [29] on IOP using topical and intracameral drug delivery in rabbits. Both routes of S32826 administration were found to cause significant reductions in IOP in these animals. Since the topical route of administration requires use of millimolar concentrations of S32826, we could not use DMSO to dissolve the drug. Dissolution of drug in sterile filtered water resulted in a solution with an alkaline pH of ,8–9. Since the drug precipitates out of solution at neutral pH, we used pH adjusted water as sham control in our experiments. S32826 treatment resulted in a dose - dependent ocular hypotensive effect on IOP in rabbits relative to control (sham-treated) eyes. This decrease in IOP was also significant relative to basal IOP in the same animal, and lasted for a minimum of 24 hrs post-treatment in rabbits treated twice a day with 10 mM S32826. It was noted that topical application of alkaline water (sham control) per se caused an upward shift in IOP values by ,2 to 2.5 mm Hg. The reason for this effect of the sham treatment is not clear. Topical drug application, especially at the 10 mM concentration, was found to cause a transient conjunctival hyperaemia, a response which appears to be associated with drug precipitation on the cornea and conjunctival tissue. However, the decrease in IOP in drug treated eyes was not related to conjunctival hyperaemia since IOP was found to be significantly less in the drug treated animals even after 24 hrs after drug application, by which time there was no evidence of hyperaemia or drug precipitation. To substantiate the ocular hypotensive effects of S32826 and to circumvent both drug precipitation and resulting conjunctival hyperaemia noted with topical drug delivery in rabbit eyes, S32826 was delivered into the eye anterior chamber by PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e42627