Autotaxin-Lysophosphatidic Acid Axis Is a Novel Molecular Target for Lowering Intraocular Pressure Padma Iyer1, Robert Lalane III1, Corey Morris1, Pratap Challa1, Robin Vann1, Ponugoti Vasantha Rao1,2* 1 Department of Ophthalmology, Duke University School of Medicine, Durham, North Carolina, United States of America, 2 Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina, United States of America Abstract Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non- glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ,1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity in vitro (by .90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (,2 mM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients. Citation: Iyer P, Lalane R III, Morris C, Challa P, Vann R, et al. (2012) Autotaxin-Lysophosphatidic Acid Axis Is a Novel Molecular Target for Lowering Intraocular Pressure. PLoS ONE 7(8): e42627. doi:10.1371/journal.pone.0042627 Editor: Asad U. Khan, Aligarh Muslim University, India Received April 30, 2012; Accepted July 9, 2012; Published August 20, 2012 Copyright: 2012 Iyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Eye Institute, National Institutes of Health grants RO1 EY18950 and EY18950 ARRA stimulus supplement to PVR. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rao00011@mc.duke.edu Introduction Glaucoma, a leading cause of blindness characterized by optic nerve degeneration and progressive visual field loss, is commonly associated with elevated intraocular pressure (IOP) [1]. Ocular hypertension (OH) or elevated IOP is a definite and primary risk factor for primary open angle glaucoma (POAG) [1,2,3]. The risk of developing glaucoma decreases significantly when a 20% reduction in IOP is achieved in patients with OH, and ocular hypotensive therapy remains the mainstay of glaucoma treatment [4,5]. However, while there are several classes of ocular hypotensive drugs available for treatment of glaucoma, there remains a significant unmet medical need for novel, more efficacious and targeted therapy [5,6]. This need requires that we address the gap which currently exists in our understanding of regulation of IOP and AH outflow and identification of physiological and pathological factors that influence IOP and AH outflow in both normal and glaucoma patients. The conventional or trabecular AH outflow pathway is composed of the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT) and Schlemm’s canal (SC). In humans, this pathway represents a predominant route of AH drainage [3]. AH is secreted by non-pigmented epithelial cells that line the ciliary body and flows into the anterior chamber, which then drains through the TM into Schlemm’s canal and the episcleral veins on a continuous basis. While there is a general agreement that decreased AH outflow through the trabecular pathway is the primary cause for increased IOP in glaucoma patients, little is known about the molecular basis for increased resistance to AH outflow through the trabecular pathway [3,7]. Various extracellular factors including TGF-beta, bioactive lipids (lysophosphatidic acid and Sphingosine-1 phos- phate), endothelin-1, myocilin, interleukins, prostaglandins, ste- roids, CD44, matrix metalloproteinases and extracellular matrix proteins have been reported to influence AH outflow and IOP [3,8,9,10,11,12,13,14,15,16,17,18]. Importantly, the levels of some of these factors have been shown to be elevated in the AH of human glaucoma patients [2,13,19,20,21]. In our previous work, we demonstrated that TM and SC cells express different G-protein coupled receptors specific for both lysophosphatidic acid (LPA) and sphingosin-1-phosphate (S1P), and perfusion of enucleated eyes with these lipids was noted to influence AH outflow concomitant with changes in TM cell contractile properties, Rho GTPase activation and expression of extracellular matrix proteins [15,22]. These initial observations were subsequently confirmed by independent investigators and collectively supported the importance of bioactive lipids in PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42627