Abstract
Purpose: :
To determine the molecular mechanisms regulating the production of lipid growth factors (Lysophosphatidic acid (LPA) and Sphingosine-1-phosphate (S1P)) involved in homeostasis of intraocular pressure (IOP).
Methods: :
Human Aqueous humor (AH) derived from cataract patients was analyzed for the presence of autotaxin/Lysophospholipase D, a secretory enzyme generating LPA and S1P extracellularly, by mass spectrometry (Waters MALDI SYNAPT HDMS). Autotaxin enzyme activity was determined in AH derived from patients with primary open angle glaucoma (POAG) and cataract, by in vitro activity analysis. The role of autotaxin in actomyosin organization and myosin II phosphorylation in human trabecular meshwork (HTM) cells was evaluated by targeting autotaxin expression and activity using siRNA technology and a pharmacological inhibitor (S32826), respectively. The effects of mechanical stretch, TGF-β2 and dexamethasone on expression of autotaxin were assessed using HTM cells. The effects of topical application of S32826 on IOP were evaluated using Dutch Belted male rabbits and a pneumotonometer.
Results: :
Autotaxin is identified as a protein that is easily detected by quantitative proteomics analysis of human AH. Two isoforms (α and γ) of autotaxin were detected in the human AH, cultured HTM cells and conditioned media from HTM cells. Autotaxin activity was significantly elevated (by 54%, p< 0.014, n = 13) in AH derived from POAG patients compared to age-matched cataract patients. Topical application of autotaxin inhibitor (2 drops of 10 mM S32826 with a one-time application) decreased IOP significantly (by 27%, p<0.004, n = 4) in rabbits starting from 2 hrs and continued up to 30 hrs post drug application. Autotaxin secretion was significantly stimulated by dexamethasone and mechanically stretching of TM cells. Autotaxin inhibition and suppression in the HTM cells led to cellular relaxation.
Conclusions: :
This study identifies autotaxin, a predominant LPA producing enzyme as an easily detectable protein in human AH. Importantly, autotaxin enzyme activity is elevated in the AH of POAG patients, and inhibition of its activity by a topical pharmacological agent decreases IOP significantly in rabbits. Based on these significant observations, we conclude that the autotaxin-LPA axis is a promising molecular target for lowering IOP in glaucoma patients.
Keywords: intraocular pressure • lipids • trabecular meshwork