Autotaxin and IOP Figure 5. Topical application of ATX inhibitor (S32826) decreases IOP in rabbits. Topical application of S32826 (dissolved in water, pH,8– 9) decreased IOP in a dose- and time- dependent manner in Dutch-Belted adult male rabbits, as determined using pneumotonometer under waking conditions. A. 10 mM S32826 exhibited the maximum and statistically significant effect in lowering IOP (by 5–6 mm Hg; n = 9) with drug application at 0 and 5 hrs (indicated with arrows, 35 ml62) relative to sham controls receiving pH adjusted water. The IOP-lowering effect of drug lasted for a minimum of 24 hrs, after which, the changes in IOP began to normalize and were similar to control values by 48 hrs after initial drug application. B. The effects of S32826 (10 mM) on net change in IOP from basal values in the same animals. Net IOP values were significantly reduced from basal values, and between 2 to 24 hours following drug administration, based on a paired t-test. Further, IOP in the drug treated eyes was significantly reduced as compared to IOP in the control eyes based on the Wilcoxon Rank Sum test of differences in median’s (n = 9). C and D. Effects of 5 and 2 mM S32826 on IOP with a daily (single morning dose, 35 ml62; n = 4) dose of drug application, respectively. Relative to the 2 mM concentration of drug, administration of 5 mM drug resulted in a significant drop in IOP starting from 2 hrs post drug injection as compared the control values, with the IOP lowering effect lasting till the end of the study (5 days later). While eyes treated with 2 mM S32826 exhibited IOP values that were significantly reduced relative to control values throughout the 5-day study period, based on the Wilcoxon Rank Sum test of differences in medians, significance was not consistent across all time points as shown in the figure. In all these experiments, sham treatment alone increased IOP; the difference was not significant relative to the basal IOP values in any of the experiments. *P,0.05. doi:10.1371/journal.pone.0042627.g005 found in the floating membranous material shed from drug treated cells belonged to the class of proteins involved in membrane trafficking, exocytosis and membrane repair mechanisms, and included Annexin-2, 5 and 1, myosin-9 and 10, filamin A, radixin, actin, lysozyme C, cathepsin G, a2-macroglobulin, various ECM molecules, heat shock proteins-60, 70 and 90, myoferlin, 14-3-3 adaptor protein zeta/delta, protein S100-09/08, serotransferin, peptidyl-prolyl cis-trans-isomerase, cystatin-A, protein disulfide isomerise and vimentin. The protein profile of membranous material derived from drug treated HTM cells was similar regardless of whether cells had been maintained under serum starved conditions or in medium containing 0.5% FBS. Influence of Cyclic Mechanical Stretch on ATX Expression and Secretion in TM cells TM cells are mechano-sensitive and fluctuations in IOP are thought to influence paracrine and autocrine activity of these cells and thereby influence the homeostatic mechanisms involved in maintenance of normal AH outflow and IOP [8]. To explore the possible involvement of ATX in the effects of cyclic mechanical stretch, serum starved confluent cultures of porcine TM cells (passage 4) were subjected to 17 hrs of cyclic mechanical stretch with one cycle/second with 10% final stretch. In this experiment we used porcine TM cells due to limited availability of HTM cells. Cells exposed to these conditions exhibited significantly elevated levels of ATX both in the conditioned media (Fig. 10A and 10B) and in cell lysates (Fig. 10C and 10D), relative to unstretched control cells as determined by immunoblotting analysis. Figure 10A and 10C show the cyclic stretch-induced changes in ATX protein PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42627