Statistical analysis In figures, all data are presented as mean 6 SEM. The Wilcoxon Rank Sum test of differences in medians was performed to compare the ATX enzyme activity between glaucoma and cataract controls with one-way Chi-square approximation. IOP changes between drug treated and sham treated rabbits were assessed for significance using the Wilcoxon Rank Sum test. IOP changes within the same group before and after drug treatment was tested using the paired t-test. Racial difference between glaucoma and cataract groups was tested by the Fisher’s exact test. Student’s t-test was used for evaluating significance in biochemical studies performed with cultured cells. A p-value,0.05 was considered significant. A box-and-whisker plot was used to represent the AH LysoPLD activity in glaucoma and cataract control subjects. Supporting Information Table S1 Quantitative proteome of aqueous humor from control (cataract) eyes with proteins listed in order References 1. Quigley HA, Broman AT (2006) The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 90: 262–267. 2. Weinreb RN, Khaw PT (2004) Primary open-angle glaucoma. Lancet 363: 1711–1720. 3. Gabelt BT, Kaufman PL (2005) Changes in aqueous humor dynamics with age and glaucoma.Prog Retin Eye Res 24: 612–637. 4. The AGIS Investigators (2000) The advanced glaucoma intervention study, 6: effect of cataract on visual field and visual acuity. Arch Ophthalmol 118: 1639– 1652. 5. Lee AJ, Goldberg I (2011) Emerging drugs for ocular hypertension. Expert Opin Emerg Drugs 16: 137–161. 6. Toris CB (2010) Pharmacotherapies for glaucoma. Curr Mol Med 10: 824–840. 7. Tamm ER, Fuchshofer R (2007) What increases outflow resistance in primary open-angle glaucoma? Surv Ophthalmol 52 Suppl 2: S101–104. 8. Acott TS, Kelley MJ (2008) Extracellular matrix in the trabecular meshwork. Exp Eye Res 86: 543–561. 9. Clark AF, Wordinger RJ (2009) The role of steroids in outflow resistance. Exp Eye Res 88: 752–759. 10. Fautsch MP, Bahler CK, Vrabel AM, Howell KG, Loewen N, et al. (2006) Perfusion of his-tagged eukaryotic myocilin increases outflow resistance in human anterior segments in the presence of aqueous humor. Invest Ophthalmol Vis Sci 47: 213–221. 11. Gottanka J, Chan D, Eichhorn M, Lutjen-Drecoll E, Ethier CR (2004) Effects of TGF-beta2 in perfused human eyes. Invest Ophthalmol Vis Sci 45: 153–158. 12. Keller KE, Aga M, Bradley JM, Kelley MJ, Acott TS (2009) Extracellular matrix turnover and outflow resistance. Exp Eye Res 88: 676–682. 13. Knepper PA, Mayanil CS, Goossens W, Wertz RD, Holgren C, et al. (2002) Aqueous humor in primary open-angle glaucoma contains an increased level of CD44S. Invest Ophthalmol Vis Sci 43: 133–139. 14. Liton PB, Luna C, Bodman M, Hong A, Epstein DL, et al. (2005) Induction of IL-6 expression by mechanical stress in the trabecular meshwork. Biochem Biophys Res Commun 337: 1229–1236. 15. Mettu PS, Deng PF, Misra UK, Gawdi G, Epstein DL, et al. (2004) Role of lysophospholipid growth factors in the modulation of aqueous humor outflow facility. Invest Ophthalmol Vis Sci 45: 2263–2271. 16. Shepard AR, Millar JC, Pang IH, Jacobson N, Wang WH, et al. (2010) Adenoviral gene transfer of active human transforming growth factor-{beta}2 elevates intraocular pressure and reduces outflow facility in rodent eyes. Invest Ophthalmol Vis Sci 51: 2067–2076. 17. Stamer WD, Read AT, Sumida GM, Ethier CR (2009) Sphingosine-1- phosphate effects on the inner wall of Schlemm’s canal and outflow facility in perfused human eyes. Exp Eye Res 89: 980–988. 18. Wiederholt M, Thieme H, Stumpff F (2000) The regulation of trabecular meshwork and ciliary muscle contractility. Prog Retin Eye Res 19: 271–295. 19. Tripathi RC, Li J, Chan WF, Tripathi BJ (1994) Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2. Exp Eye Res 59: 723–727. 20. Mackay EO, Kallberg ME, Gelatt KN (2008) Aqueous humor myocilin protein levels in normal, genetic carriers, and glaucoma Beagles. Vet Ophthalmol 11: 177–185. 21. Howell KG, Vrabel AM, Chowdhury UR, Stamer WD, Fautsch MP (2010) Myocilin levels in primary open-angle glaucoma and pseudoexfoliation glaucoma human aqueous humor. J Glaucoma 19: 569–575. Autotaxin and IOP of abundance. Values represent mean 6 standard deviation (SD). (DOCX) Acknowledgments We are grateful to J. Will Thompson, Ph.D., and Nikolai Skiba, Ph.D., for their help with the proteomics analysis and Sandra Stinnett, D.P.H., for her help with statistical analysis. We thank David Epstein, M.D., for his helpful comments and suggestions during the conduction of these studies and manuscript preparation, and Padmanabhan Pattabiraman, Ph.D., for his help in the cell culture studies. Author Contributions Conceived and designed the experiments: PVR. Performed the experi- ments: PI RL CM PC RV. Analyzed the data: PI PVR. Wrote the paper: PI PVR. 22. Pattabiraman PP, Rao PV (2010) Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells. Am J Physiol Cell Physiol 298: C749–763. 23. Kumar J, Epstein DL (2011) Rho GTPase-mediated cytoskeletal organization in Schlemm’s canal cells play a critical role in the regulation of aqueous humor outflow facility. J Cell Biochem 112: 600–606. 24. Moolenaar WH, Perrakis A (2011) Insights into autotaxin: how to produce and present a lipid mediator. Nat Rev Mol Cell Biol 12: 674–679. 25. Umezu-Goto M, Kishi Y, Taira A, Hama K, Dohmae N, et al. (2002) Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production. J Cell Biol 158: 227–233. 26. Tokumura A, Majima E, Kariya Y, Tominaga K, Kogure K, et al. (2002) Identification of human plasma lysophospholipase D, a lysophosphatidic acid- producing enzyme, as autotaxin, a multifunctional phosphodiesterase. J Biol Chem 277: 39436–39442. 27. Gijsbers R, Aoki J, Arai H, Bollen M (2003) The hydrolysis of lysophospholipids and nucleotides by autotaxin (NPP2) involves a single catalytic site. FEBS Lett 538: 60–64. 28. Giganti A, Rodriguez M, Fould B, Moulharat N, Coge F, et al. (2008) Murine and human autotaxin alpha, beta, and gamma isoforms: gene organization, tissue distribution, and biochemical characterization. J Biol Chem 283: 7776– 7789. 29. Ferry G, Moulharat N, Pradere JP, Desos P, Try A, et al. (2008) S32826, a nanomolar inhibitor of autotaxin: discovery, synthesis and applications as a pharmacological tool. J Pharmacol Exp Ther 327: 809–819. 30. Maddala R, Skiba NP, Lalane R III, Sherman DL, Brophy PJ, et al. (2011) Periaxin is required for hexagonal geometry and membrane organization of mature lens fibers. Dev Biol 357: 179–190. 31. Lutjen-Drecoll E (1999) Functional morphology of the trabecular meshwork in primate eyes. Prog Retin Eye Res 18: 91–119. 32. Fleenor DL, Shepard AR, Hellberg PE, Jacobson N, Pang IH, et al. (2006) TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure. Invest Ophthalmol Vis Sci 47: 226–234. 33. Pattabiraman PP, Lih FB, Tomer KB, Rao PV (2012) The role of calcium- independent phospholipase A2gamma in modulation of aqueous humor drainage and Ca2+ sensitization of trabecular meshwork contraction. Am J Physiol Cell Physiol 302: C979–991. 34. Chowdhury UR, Madden BJ, Charlesworth MC, Fautsch MP (2010) Proteome analysis of human aqueous humor. Invest Ophthalmol Vis Sci 51: 4921–4931. 35. Bennett KL, Funk M, Tschernutter M, Breitwieser FP, Planyavsky M, et al. (2011) Proteomic analysis of human cataract aqueous humour: Comparison of one-dimensional gel LCMS with two-dimensional LCMS of unlabelled and iTRAQ(R)-labelled specimens. J Proteomics 74: 151–166. 36. Stracke ML, Krutzsch HC, Unsworth EJ, Arestad A, Cioce V, et al. (1992) Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J Biol Chem 267: 2524–2529. 37. Nakanaga K, Hama K, Aoki J (2010) Autotaxin–an LPA producing enzyme with diverse functions. J Biochem 148: 13–24. 38. Clair T, Aoki J, Koh E, Bandle RW, Nam SW, et al. (2003) Autotaxin hydrolyzes sphingosylphosphorylcholine to produce the regulator of migration, sphingosine-1-phosphate. Cancer Res 63: 5446–5453. 39. Moolenaar WH, van Meeteren LA, Giepmans BN (2004) The ins and outs of lysophosphatidic acid signaling. Bioessays 26: 870–881. PLOS ONE | www.plosone.org 13 August 2012 | Volume 7 | Issue 8 | e42627