regulation of AH outflow and potentially IOP [15,17,23]. Although these observations implied the significance of LPA and SIP in the regulation of AH outflow, we currently know very little about how the levels of these bioactive lipids are regulated in AH, and the different molecular mechanisms involved in the generation of these lipids in the AH. To explore these questions, we recently performed quantitative proteomic analysis of human AH derived from cataract surgery patients with normal IOP. This initial analysis revealed that ATX [24], which is a well characterized secretory protein with lysophospholipase D (LysoPLD) activity, was one of the abundant proteins in human AH. Since this protein has been considered a major source for extracellular LPA [25,26], and LPA has in turn been demonstrated to influence AH outflow facility [15], in this study we investigated the role of ATX in IOP using a small molecular weight chemical inhibitor of this protein. This study reports that inhibition of ATX LysoPLD activity by topical and intracameral delivery of a chemical inhibitor significantly decreases IOP, suggesting that ATX is a potential therapeutic target for lowering IOP in glaucoma patients. Results ATX is an abundant protein of human aqueous humor Towards our objective to identifying extracellular factors regulating AH outflow and IOP, an LC-MS (liquid chromatog- raphy- mass spectrometry) proteomic analysis was performed to identify abundant proteins of human AH, using one mg of pooled AH total protein derived from 8 individual donors who underwent cataract surgery. This analysis identified eighty nine non- redundant proteins with high confidence based on 2 or more matching peptides. Individual proteins were quantified by comparison of signal intensity, using an internal standard of yeast alcohol dehydrogenase (label free). Table S1 lists AH proteins identified by proteomic analysis, together with their respective Swiss-Prot protein accession number, protein common name and relative concentration (fmol/1 mg total protein). Of the different easily detectable proteins present in the human AH sample, ATX, also known as Ectonucleotide pyrophosphatase/phosphodiesterase (ENNP2), was found to be one of 25 most abundant proteins (Table S1). To independently confirm the presence of ATX in AH by an alternative means, we performed immunoblotting analysis of AH using a rabbit polyclonal anti-ATX antibody. Immunoblotting analysis of 10 mg of human AH total protein derived from two independent donors (aged 40 and 52 years) identified multiple immunopositive bands with molecular mass ranging from 65 to 250 kDa, confirming the presence of ATX isoforms in different proteolytically and postranslationally processed forms (Fig. 1A). Protein blots of the AH sample were also probed with primary antibody which was pre-blocked with antigen specific peptide or secondary antibody alone, to confirm specificity of the primary antibody used for immunoblotting, and confirmed lack of non- specific reactions in the observed results. We also confirmed the presence of ATX in mouse and porcine AH by immunoblot analysis (Fig. 1B) and comparative analysis revealed interesting differences and similarities in the profiles of ATX isoforms between the human, mouse and porcine species. Elevated levels of LysoPLD activity in the AH of POAG patients To measure LysoPLD activity in human AH, we utilized a modified fluorogenic enzyme-coupled assay protocol [27] using a commercially available Amplex Red PLD Assay kit. Human AH samples derived from 20 different cataract and POAG patients Autotaxin and IOP Figure 1. Detection of ATX in the AH from human and other species. A. Immunoblot analysis of AH (10 mg protein) derived from two different human donors (aged 40 and 52 years) revealed multiple immunopositive bands with molecular mass ranging between 65 to 250 kDa. B. Detection of ATX by immunoblot analysis of mouse and porcine AH (10 mg protein). Similar to human AH, both mouse and porcine exhibit ATX immunopositive bands with molecular mass ranging from 95 to 250 kDa. doi:10.1371/journal.pone.0042627.g001 were tested for LysoPLD activity. While age and sex were matched between the cataract and glaucoma groups, race demography was found to be significantly (p,0.05) different between the two groups based on both Fisher’s exact and Chi-Square tests. As shown in Figure 2A, the racial composition of the POAG sample group consisted of 75% African American, 15% Caucasian/white and 5% Hispanic patients. On the other hand, the cataract control group comprised of 85% Caucasian/white and 15% African American patients. LysoPLD activity measured in these specimens was expressed as micromoles of H2O2 generated per milligram of AH protein, with the activity measured for POAG patients being significantly elevated (by 1.8 fold, n = 20), as compared to the cataract control specimen values, based on the Wilcoxon Rank Sum test of differences in medians with one-way Chi-Square approximation (Fig. 2B and 2C). Figure 2C provides Box-and- whisker plots of the AH LysoPLD activity of glaucoma and cataract controls, with the box showing median and interquartile ranges. ATX is expressed and secreted by TM cells Since ATX was easily detectable in human AH samples, we hypothesized that this protein is expressed and secreted by cells of the AH outflow pathway. RT-PCR analysis confirmed expression of all three isoforms of ATX (a, b and c, Fig. 3A) [28] in primary cultures of HTM cells derived from three different donors (aged 33, 54 and 75 years; representative data from a single TM cell strain (donor) is shown). Examination of the cellular distribution profiles of ATX in primary cultures of HTM cells by immuno- fluorescence analysis using a polyclonal antibody specific for ATX (Fig. 3B) reveals a distinct vesicular/punctate distribution pattern, which is a characteristic feature of secretory proteins. Immuno- blotting analysis of ATX protein profiles in cell lysates (20 mg protein) derived from primary cultures of HTM and porcine TM cells (Fig. 3C and 3D, respectively) revealed multiple immunopo- sitive bands with molecular mass ranging from 55 to 250 kDa. On the other hand, the HTM cell conditioned media (10 mg protein) exhibited a single immunopositive band with a molecular mass of ,95 kDa (Fig. 3C). Compared to the HTM cells, the PTM cell conditioned media (10 mg) showed the 95 kDa protein similar to PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42627