Lysophospholipase D and Elevated Lysophosphatidyl Choline as Sources of Elevated Aqueous Humor LPA Following Corneal Injury

Abstract

Abstract: : Purpose: The cornea is bathed by the aqueous humor, which contains various growth factors that may participate in the healing process following corneal injury. We previously found that lysophosphatidic acid (LPA), an important growth factor present in animal sera, is accumulated in rabbit aqueous humor following corneal freeze wounds. In the current study, we examined the possibility that soluble lysophospholipase D (lysoPLD), an enzyme that generates LPA from lysophosphatidylcholine (LPC), is relevant to this injury–induced increase in aqueous humor LPA concentration. Methods: Rabbit corneas were freeze wounded, and aqueous humor samples were collected 24 h following injury. LysoPLD activity was measured by the enzymatic determination of choline concentration following incubation of aqueous humor samples with exogenous LPC. Lysophospholipids present in aqueous humor were analyzed by liquid chromatography–electrospray ionization mass spectrometry. Results: Aqueous humor samples from both control and injured corneas showed a high, but similar degree of lysoPLD activity. LysoPLD enzymatic properties, such as sensitivity to inhibition by bivalent ion chelators (EDTA and o–phenanthroline), metal–ion requirement, potentiation by Co²⁺, and pattern of relative activities for various lysophospholipids, resembled those of lysoPLD found in human plasma, which was recently identified by us as autotaxin (a member of the ecto–nucleotide pyrophosphatase/phosphodiesterase–I family). Autotaxin was originally identified as a tumor cell–motility stimulating protein found in conditioned medium of human melanoma cells. Mass spectrometry experiments determined that levels of four major molecular species of LPC, predominant substrates of lysoPLD in aqueous humor, were 5–13 times higher in the aqueous humor samples from injured eyes than those in control eyes. Conclusions: These results suggest that lysoPLD is constitutively released into rabbit aqueous humor, with no injury–induced increase in release following freeze–wounding. They also show that wound–induced increases in LPA are likely due to an increase in the aqueous humor concentration of several LPC species, which are being catalyzed to form LPA by the constitutively active lysoPLD. An elevated aqueous humor LPA concentration likely plays a role in corneal wound healing.

Keywords: cornea: basic science • aqueous • lipids