Effects of topically administered S32826 on IOP in rabbits Nine Dutch-Belted normal male rabbits were used in two different experiments (4 and 5 in the first and second experiment, respectively). Prior to drug treatment, IOP was monitored for several days using a pneumotonometer to confirm steady baseline values. Of the three different concentrations (2, 5, and 10 mM) of drug tested topically, the highest dose (10 mM) caused a significant drop in IOP starting 2 hrs and continuing for at least for 24 hrs post-application in all nine rabbits evaluated. IOP values decreased by 10–17% from basal IOPs in the drug treated eyes, and by 15–22% between drug-treated and control eyes (p,0.005, n = 9). Further, this response to drug treatment was consistently observed in three different experiments (twice with the first set of rabbits, and for the third time with a second set of rabbits). Figure 5A shows IOP changes with time, and 5B depicts the net change in IOP values in drug-treated eyes as compared to control eyes. We noted a steady increase or shift in basal IOPs to higher levels in these experiments, although the differences were not significant from the basal value in the control eyes, perhaps due to alkaline sham treatment. Daily topical administration of a 5 mM dose (mornings only) for 5 days also caused a significant, but smaller net decrease in IOP (by ,3 mm Hg between drug and control groups; n = 4) as compared to sham treated controls (Fig. 5C). In contrast, while topical treatment with the 2 mM (daily morning dose for 5 days) concentration of drug did not yield a steady significant drop in IOP between drug treated and control eyes, IOP values in the drug treated rabbits were consistently lower than in control animals by 1.5 to 2 mm Hg (Fig. 5D). Throughout these experiments, topical application of all three doses of drug dissolved in aqueous medium was associated with drug precipitation on the corneal surface as a fine thin film. Further, topical application of the 10 mM dose of drug was observed to cause conjunctival hyperaemia within the first 2 hrs of drug application, which disappeared within 24 hrs, at which time we still noted a significant decrease in IOP in the drug treated animals compared to the controls (Fig. 6A). Based on slit lamp microscopic examination performed on these animals by a board certified glaucoma specialist, drug treated eyes did not exhibit any noticeable inflammation. Further, post-mortem histology analysis of the cornea (Fig. 6B) and iridocorneal angle (not shown) of rabbit eyes treated with 5 mM drug for 5 days revealed no obvious or toxicological changes relative to control eyes. Effects of intracameral administration of S32826 on IOP of rabbits Since topical drug application was observed to result in drug precipitating on the cornea and causing conjunctival hyperaemia, we decided to change the route of administration. The effects of intracameral injections (delivering the drug directly into the anterior chamber of the eye) were assessed by administering S32826 dissolved in phosphate buffered saline (pH 7.4) at a much lower concentration (mM) than the topical dose (mM). Four Dutch- Belted normal rabbits were used for this experiment. Drug was dissolved initially in dimethyl sulfoxide (DMSO) and subsequently diluted with PBS containing 2% DMSO. In each rabbit one eye was injected with 5 ml of 100 mM drug dissolved in PBS with 2% DMSO, while the other eye served as a non-injected control. The effect of vehicle alone was tested by administering 5 ml of PBS containing 2% DMSO into the contralateral eyes of the same rabbits used for evaluating the effect of active drug. Vehicle administration was performed after waiting at least for one week following completion of the drug treatment experiments, and after ensuring that IOP values were confirmed to be similar to basal values. Intracameral delivery of drug did not result in precipitation Autotaxin and IOP as evaluated using a slit lamp microscope. Drug-injected eyes exhibited a significant decrease in IOP (33% (p,0.0036) at 24 hrs, 31.5% (p,0.001) at 48 hrs and 18% (p,0.001) at 72 hrs post drug injection) compared to contralateral non-injected eyes (Fig. 7). Similarly, in comparison to the vehicle injected control eyes, IOP was observed to decrease by 18% (p,0.007) at 24 hrs and 30% (p,0.007) at 48 hrs after drug injection in S32826 treated eyes. Further, IOP values of drug treated eyes were significantly lower relative to basal IOP values (prior to drug injection) measured in the same animals (Fig. 7). At the 96 hr time point post drug injection, we missed recording IOP in two of the animals used in the experiment, resulting in an n = 2 observations for this time point, as indicated in Figure 7. There were no notable ocular abnormalities in any group of animals used in this experiment, either during or after completion of the tests described. Effects of siRNA-based suppression of ATX expression in TM cells To explore the role of ATX in TM cell morphological and contractile properties, we suppressed the expression of this protein in HTM cells using the small interfering RNA (siRNA) approach. As shown in Figure 8A, treatment of HTM cells with ATX specific siRNA resulted in significant decreases in the intracellular levels of ATX protein relative to cells treated with scrambled FITC- conjugated siRNA and untreated control cells (n = 4). As can be seen from the figure, ATX siRNA treatment affected the levels of different isoforms of ATX. Based on densitometric analysis using b-tubulin as internal standard, the protein levels of a predominant isoforms of ATX (125 kDa) was reduced by 70% in ATX siRNA treated HTM cells (Fig. 8B), while some isoforms (,100 kDa) were decreased to undetectable levels in HTM cells treated with ATX siRNA (Fig. 8A), relative to control cells treated with scrambled FITC-siRNA. Although the ATX siRNA treated HTM cells did not reveal any obvious changes in cell morphology, F- actin staining by TRITC-phalloidin revealed decreased levels of actin stress fibers compared to the scrambled FITC-siRNA treated controls (Fig. 8C). Additionally, we observed a significant (p,0.03; n = 4) decrease in the levels of phosphorylated myosin light chain, a regulatory subunit of myosin II activity, in HTM cells treated with ATX siRNA, based on immunoblotting analysis (Fig. 8D) and semi-quantitative measurement using densitometric scanning (Fig. 8E). Effects of S32826 on cultured TM cells To determine the effects of S32826 on cultured TM cells, confluent cultures of HTM cells (derived from two different donors) maintained under serum free conditions or in the presence of 0.5% fetal bovine serum (FBS) were treated with 5 or 10 mM S32826 for 24 hrs along with respective sham treated controls. Time dependent observations based on phase contrast microscopy revealed presence of large floating membranous clumps in drug- treated cell culture dishes starting as early as 30 minutes from the time of drug addition, and relative to sham treated cells (Fig. 9A. 10 mM drug; arrows). However, drug treatment did not cause changes in cell morphology or cytotoxic response, as assessed by propidium iodide and fluorescein diacetate in live cell labeling (data not shown), [15] or changes in actin stress fiber distribution (data not shown). However, since floating membranous material was consistently noted in drug treated cell cultures in three independent experiments, the material was collected by centrifu- gation, rinsed with PBS, dissolved in Laemmli sample buffer, and separated by SDS-PAGE. Predominant protein bands were then gel extracted and subjected to mass spectrometry-based identifi- cation (Fig. 9B) [30]. Intriguingly, the majority of the proteins PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42627