Review
Cellular and molecular events in corneal wound healing: significance of lipid signalling

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Abstract

Alterations in the normal healing process after corneal injury can produce undesirable outcomes that range from corneal haze to ulceration and perforation. Lipids play important roles in the complex inflammatory processes that occur after corneal wounding. While some lipid mediators, such as the lipoxygenase derivatives of arachidonic acid, 12-hydroxyeicosatetraenoic acid (12[S]-HETE and 15[S]-HETE), act as second messengers to promote cell proliferation and are possibly involved in the synthesis of other molecules that suppress inflammation, others, such as platelet-activating factor (PAF), exert their actions through specific receptors, play key roles during sustained corneal inflammation (as might occur with chemical burns), and contribute to tissue destruction and neovascularization. PAF is also a strong inducer of selective metalloproteinases (MMPs) that degrade the extracellular matrix. The use of a new PAF antagonist has shown great promise for the treatment of diffuse lamellar keratitis (DLK) and alkali-burned corneas.

Introduction

Lipids were regarded for many years merely as structural components of cell membranes whose main function was to regulate membrane permeability. Today, it is known that lipids form specific domains in membranes and play important roles as second messengers in cell signal transduction. They are involved in cell proliferation, migration, and survival, as well as in inflammation and angiogenesis.

Phospholipids are the basic building blocks of cell membranes and the substrates for production of lipid second messengers. They contain a glycerol backbone, a polar phosphate group attached to the third carbon, and an acyl, alkyl, or alkenyl group attached to the sn-1 and sn-2 positions. The possible combinations of the phosphate group attached to different bases (e.g. choline, ethanolamine, serine, inositol), the acyl (or other) groups at C1 and C2, and the degree of unsaturation give rise to hundreds of different lipid species in a cell. This allows for the formation of lipid rafts and specific lipid–protein interactions. The diverse roles of lipids in cell function and disease that have been discovered have renewed interest in these molecules and aided the development of new and sophisticated methods to determine changes in their composition, launching the new era of ‘lipidomics’ (Forrester et al., 2004).

The cornea is one of the simplest and, at the same time, most fascinating, tissues. It is composed mainly of three different cell layers: the epithelium, composed of 5–6 layers of cells that are very actively proliferating; the stroma, the thickest layer, with its amazing collagen organization in which are embedded the nearly quiescent keratocytes; and the endothelial monolayer, with its important pump systems to regulate the entry of substances from the aqueous humor. Corneal tissue is devoid of vascular elements; therefore, its use in research allows investigators to design experiments that need not accommodate the actions of the vascular cellular components. This simpler structure has allowed us to pose questions that are difficult to answer in more complicated systems (e.g. the nervous system). The first corneal response to injury is the very complex process of inflammation. The outcome of this response has consequences insofar as the integrity and transparency of the tissue. In the avascular cornea, inflammation is characterized by stromal cell infiltration, mainly of polymorphonuclear leukocytes (PMNs) that arrive from the limbal vessels (Paterson et al., 1984, Pfister et al., 1988). These cells are activated and generate a number of bioactive lipids that contribute to the propagation of the response (Leibowitz and Frangie, 1998). I became interested in the significance of lipid mediators in the cornea after injury in the early 1980s, and this review concentrates mainly on the work done in my laboratory on this subject. Papers or reviews that are relevant to some aspects of this discussion are cited, but this is not intended to be a comprehensive review of all the literature on the subject. Space constraints prevent me from quoting all relevant contributions.

Section snippets

Corneal injury induces the release of arachidonic acid and its conversion to eicosanoids: cellular specificity

Being trained in lipid biochemistry, my first project was to determine the lipid composition of the three layers of the cornea, specifically, the fatty acid composition of membrane lipids. There were, at that time, some reports on the composition of phospholipids and their accumulation during aging and in corneas with granular dystrophies (Feldman, 1967, Broekhuyse, 1968, Rodrigues et al., 1983), but overall there was very little information about fatty acid composition or corneal lipid

Selective growth factor signalling in corneal epithelial cell proliferation is mediated by 12/15(S)-HETEs

Lipoxygenases are enzymes that produce fatty acid hydroxyperoxides by introducing oxygen at different positions along the carbon chain of fatty acids, mainly AA, to form the 5(S)-, 12(S)-, and 15(S)-hydroxyperoxides. These compounds are then rapidly reduced to the corresponding HETES (Fig. 1). The enzymes involved in these conversions are 5-lipoxygenase (5-LOX), 12-LOX, and 15-LOX, which are strongly expressed in leukocytes, platelets, and reticulocytes, respectively, but selective expression

Docosahexaenoic acid and its bioactive products as neuroprotective agents

Injury to the cornea produces damage to corneal nerves, and as a consequence, adverse effects occur, such as delayed wound healing, desiccation of the corneal surface, and alterations in epithelial cell metabolism (for review, see Muller et al., 2003).

Docosahexahenoic acid (DHA) is a polyunsaturated fatty acid with six double bonds that belongs to the omega-3 fatty acid family (Fig. 1). DHA is required for neuronal and retinal photoreceptor functions (for review, see Bazan, 2003). Recently, the

PAF is a key mediator of the inflammatory response that delays corneal wound healing

Another very active lipid in the cornea is PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) (Fig. 1). PAF is synthesized by many types of stimulated cells, and due to its potent actions, has been implicated in the pathogenesis of a variety of diseases (Zimmerman et al., 1996, Zimmerman et al., 2002). It can cause microvascular leakage, vasodilation, and activation of several types of inflammatory cells, such as neutrophils, eosinophils, and macrophages. In cornea, PAF stimulates several

Modulation of corneal extracellular matrix remodelling by PAF

One of the most important mechanisms by which PAF delays epithelial wound closure is by activating, at the transcriptional level, the expression of selective metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) in the cornea (Bazan et al., 1993, Tao et al., 1995, Tao et al., 1996; Fig. 1). The normal process of corneal repair requires degradation and re-synthesis of components of the ECM, such as different types of collagens, gelatins, laminin, and fibronectin.

PAF promotes corneal neovascularization

Slow-release pellets containing PAF implanted in mouse cornea induce a strong angiogenic response. Corneal vessels reaching the micropocket were seen 6 days after implantation (Ma et al., 2004). The PAF-induced neovascularization was significantly reduced in PAF-R-knockout mice and in animals treated with a PAF antagonist. In two types of vascular endothelial cells, human umbilical cord vein endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HMVEC), PAF stimulates cell

PAF activates corneal myofibroblasts' ability to modulate the extracellular matrix

Recent findings that corneal myofibroblasts express the PAF-R (He and Bazan, 2003) led us to investigate what role(s) PAF has in these cells. While stromal keratocytes are nearly quiescent cells that contribute to the maintenance of tissue transparency, myofibroblasts express α-smooth muscle actin (α-SM-actin) and are involved in the contraction of corneal wounds (Jester et al., 1999). PAF induces apoptosis of keratocytes 4 hr after stimulation (Chandrasekher et al., 2002); in contrast,

PAF antagonists inhibit diffuse lamellar keratitis and chemical-induced corneal perforation

The wide variety of responses induced by PAF points toward a central role played by this inflammatory mediator in cornea following pathologic stimulus. Inhibition of the PAF-R is an approach that could attenuate the inflammatory cascade at its initial steps and be beneficial in maintaining the integrity of the tissue (Fig. 2). Several PAF antagonists have been developed and tested for efficacy in different pathologic conditions in the eye as well as in many other tissues (Zimmerman et al., 1996

Conclusions

Here, we have summarized some of the physiological and pathological roles of bioactive lipids in cornea after injury. Inflammation is the first response of corneal tissue to an insult, and during the first hours, the cells of the corneal layers respond by releasing AA from membrane phospholipids and converting this fatty acid into eicosanoids and PAF. If inflammation is more severe and persists, cells that infiltrate the cornea, mainly neutrophils, amplify the response and contribute to the

Acknowledgements

The author's research was supported by United States Public Health Service grants R01 EY04928 and R01 EY06635 from the National Eye Institute, National Institutes of Health, Bethesda, MD.

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