Autotaxin and IOP Figure 6. Effects of topical application of S32826 on the rabbit cornea. A. Topical application of 10 mM S32826 (twice daily, 35 ml aliquots) in Dutch-Belted adult male rabbits caused conjunctival hyperemia within the first 2 hrs after drug application, in association with drug precipitation on the cornea and conjunctival tissue as a fine thin film. However, hyperemia and all evidence of drug precipitation disappeared within 24 hrs following drug application. B. Topical effects of S32826 on rabbit corneal histology. Rabbits treated with a topical dose of 5 mM S32826 for 5 days (single daily dose) and which showed a decrease in IOP were enucleated and examined for corneal histological changes relative to sham treated specimens. As shown in the figure, drug application did not cause any apparent change on the integrity of corneal epithelium, endothelium, Bowman’s layer, Stroma or Descemet’s membrane (based on hematoxylin and eosin staining) relative to the control cornea. Bar: 10 mm. Labels: 1. Corneal Epithelium; 2. Bowman’s layer; 3. Corneal Stroma; 4. Descemet’s membrane; 5. Corneal Endothelium. doi:10.1371/journal.pone.0042627.g006 levels in media and cell lysates, respectively. Densitometric scanning of immunoblots from 4 independent experiments showed a significant (p,0.005) increase in secretory (Fig. 10B) and intracellular (Fig. 10D) ATX protein levels in stretched cells as compared to unstretched control cells. Discussion Increased IOP is a well recognized risk factor for development of POAG, which is a blinding disease [3]. Although lowering IOP is the primary treatment available for different types of glaucoma at present and different classes of ocular hypotensive drugs are used in clinical management of IOP in POAG patients, we lack a mechanism-based and targeted therapy for elevated IOP in glaucoma patients [5,6]. As part of our ongoing research to identify and characterize molecular mechanisms that lead to ocular hypertension and regulate AH outflow, we have in this study identified ATX/LysoPLD, which is a major source for extracellular LPA, as an abundant protein of human AH and found that AH LysoPLD activity is abnormally elevated in POAG patients. Significantly, topical or intracameral delivery of a small molecular weight chemical inhibitor of ATX (S32826) results in lowering IOP in normal rabbits. Collectively, these observations reveal the importance of the ATX-LPA axis in homeostasis of IOP and its potential as a therapeutic target for development of novel ocular hypotensive agents to treat glaucoma patients. To our knowledge, this is the first report on the possible therapeutic importance of the ATX-LPA axis in treatment of ocular hypertension in glaucoma patients. The AH contains various extracellular factors of both protein and non-protein makeup which influence trabecular meshwork cell physiology, phagocytic function, contractile responses, auto- crine activity and extracellular matrix turnover, several of which have been found to influence AH outflow and IOP [3,8,18,31]. Further, there is convincing experimental and clinical evidence in support of the contention that changes in levels of certain physiologically relevant factors present in the AH might be linked to increased resistance to AH outflow and elevated IOP [11,18,19,32]. To identify such AH factors, we and other investigators have begun to analyze the protein composition of AH derived from the human subjects using recent technical advancements in proteomics capabilities. In our previous studies we demonstrated a role for bioactive lipids (LPA and SIP) and lipid metabolizing enzymes (iPLA2) in regulating AH outflow through the trabecular meshwork [15,33]. Therefore, we were particularly interested in understanding the role played by AH lipid metabolizing enzymes and exploring the relevance of changes in the levels of these proteins in normal and glaucoma patients. While it is known that ATX is present in human AH, [34,35] our quantitative proteomics analysis, revealed that this protein is PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42627