Growth factor-like phospholipids generated after corneal injury

Károly Liliom1,2, Zhiwei Guan1, Jih-Lie Tseng3, Dominic M. Desiderio3,4,5, Gábor Tigyi1, and Mitchell A. Watsky1

1 Department of Physiology and Biophysics, 3 The Charles B. Stout Neuroscience Mass Spectrometry Laboratory, 4 Department of Neurology, and 5 Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163; and 2 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study provides evidence that growth factor-like glycerophosphate mediators of the lysophosphatidic acid (LPA) family are present in the aqueous humor and the lacrimal gland fluid of the rabbit eye. By use of a combination of HPLC, two-dimensional TLC, mass spectrometry, and the Xenopus oocyte bioassay, the LPA-like phospholipids LPA, cyclic PA, alkenyl-glycerophosphate (GP), lysophosphatidylserine, and phosphatidic acid were detected as physiological constituents of the fluids bathing the cornea. Corneal injury resulted in an increased production of some of these mediators. Alkenyl-GP, a novel member of the LPA family, has been identified in postinjury aqueous humor, establishing that it is generated endogenously. LPA and its homologues were found to be mitogenic in freshly dissociated keratocytes from uninjured corneas. There appears to be a link between the occurrence of LPA responsiveness in keratocytes activated by injury and the increase in LPA-like activity in aqueous humor. These data suggest that LPA and its homologues are involved in maintaining the integrity of the normal cornea and in promoting cellular regeneration of the injured cornea.

lysophosphatidic acid; lipid mediator; chloride current; wound healing; keratocyte; lacrimal gland; aqueous humor

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE CORNEA IS BATHED on its external surface by the tear layer and on its internal surface by the aqueous humor (AH). One component of the tear layer is the protein-rich fluid produced by the lacrimal glands. After corneal injury, it is apparent that growth factors and growth factor-like compounds, generated in lacrimal gland fluid (LGF) and AH, influence the healing process. In the cornea, wound healing involves the proliferation and migration of cells to fill the injured area (41).

In a previous study we demonstrated that serum activates a Cl- current in keratocytes (ker-ICl) dissociated from injured but not from uninjured corneas (39). In the same study we demonstrated that the growth factor-like lipid mediator lysophosphatidic acid (LPA) mimicked the effects of whole serum, activating ker-ICl solely in keratocytes from injured corneas. Because the concentration of LPA in serum is sufficiently high to activate ker-ICl (3-10 µM) (5), we proposed that LPA-like lipids, at least in part, were the serum factors responsible for the activation of ker-ICl. In other cell types, LPA activates Cl- currents through a receptor-mediated pathway (10, 26, 35). Given that LPA is ineffective in eliciting ker-ICl from control uninjured corneas, it would appear that LPA receptors are either not expressed or not functional in keratocytes isolated from uninjured corneas (39, 40). On the other hand, hyposmotic stress is also capable of activating ker-ICl, but, unlike serum and LPA, it is effective in cells from normal and injured corneas (39). Activation of ker-ICl by hyposmotic stimulus apparently utilizes a receptor or signaling pathway distinct from that activated by serum and LPA receptors. Nonetheless, this observation suggests that the ker-ICl channel is expressed in uninjured and injured keratocytes and that the hypotonically activated ker-ICl may be involved in cell volume control. The responsiveness of LPA after injury underlines the potentially important role of this group of lipid mediators in wound healing.

LPA is a lipid mediator abundant in vertebrate serum, where it is generated from activated platelets (5, 8, 36) and other cells (7). LPA elicits a broad spectrum of actions in a variety of cells that range from simple unicellular organisms to mammals (see Ref. 12 for review). In some cell types, LPA stimulates DNA synthesis and cell division (38); in other cells, LPA is antimitogenic (34). LPA has also been shown to prevent programmed cell death in cardiomyocytes and fibroblasts (44). LPA stimulates cell motility and migration (13, 46), which lends further support for its role in the physiological regulation of wound healing.

In Xenopus oocytes, serum with response thresholds in excess of a 106-fold dilution (33) and LPA-like lipids in subnanomolar concentrations (20, 35) elicit oscillatory Cl- currents (oo-ICl). This LPA-elicited oo-ICl shows a highly reproducible dose-response relationship in oocytes from different frogs, making it a sensitive and simple tool for measuring LPA-like phospholipids using standard two-electrode voltage-clamp recording (32, 35). The oo-ICl is evoked by LPA-activated G protein-coupled cell surface receptors, stimulating the phosphoinositide-Ca2+ second messenger system, resulting in a transient elevation of intracellular Ca2+. This increase in intracellular Ca2+, in turn, opens Ca2+-sensitive Cl- channels in the oocyte plasma membrane (33, 35). LPA was identified as the major serum factor responsible for stimulating this Cl- current (35).

LPA is only one member of a larger glycerophosphate family found to act as a mediator of different cellular activities. For example, eight other lipids that can activate Cl- currents in the oocyte assay have been detected in serum (35). Although the structure of these endogenous lipids remains to be elucidated, synthetically prepared analogs of LPA, including lysophosphatidylserine (LPS) (35) and 1-acyl-sn-glycero-2,3-cyclic phosphate (cyclic PA) (25), have been shown to activate oo-ICl. We recently identified 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP), in which the hydrocarbon side chain is linked with a vinyl-ether bond to the glycerol backbone, as a potent agent in mobilizing intracellular Ca2+ through the activation of distinct subtypes of LPA receptors in the Xenopus oocyte (29; unpublished observations). Moreover, using cyclic PA, a receptor subtype-specific agonist, we demonstrated the simultaneous expression of at least two different LPA receptors (20). Recently, we cloned one of these receptors that shows a high affinity and selectivity for LPA (10). In light of these findings, LPA-like lipids and their receptors are emerging as a novel lipid mediator-receptor system that may potentially be involved in the physiological regulation of wound healing and regeneration.

The goal of the present study was to determine whether LPA and/or other LPA-like lipid mediators were present in LGF and AH after corneal injury to the rabbit eye. Our data provide evidence that growth factor-like glycerophosphate mediators are indeed present in AH and LGF. By use of a combination of the Xenopus oocyte bioassay, HPLC, two-dimensional TLC, and mass spectrometry (MS), the growth factor-like phospholipids LPA, cyclic PA, alkenyl-GP, LPS, and phosphatidic acid (PA) were detected as physiological constituents of the biologic fluids bathing the cornea. LPA was also found to be mitogenic in keratocytes freshly dissociated from the uninjured cornea. There appears to be a link between the occurrence of LPA responsiveness in keratocytes activated by injury (39) and the increase in LPA-like activity in AH. These data suggest that LPA and its homologues are involved in maintaining the integrity of the normal cornea and in promoting regeneration of the injured cornea.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animal rights. Rabbits were treated in accordance with the "Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research" as well as the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.

Rabbit corneal injury. The procedures have been reviewed and approved by the Animal Care and Use Committee of the University of Tennessee, Memphis. Animals were housed in an American Association for Accreditation of Laboratory Animal Care-accredited facility. Rabbit corneas were injured by transcorneal freezing (40). Briefly, rabbits were put under general anesthesia with xylazine (5 mg/kg) and ketamine (35 mg/kg), and corneas were anesthetized locally with proparacaine (0.5%). Butorphanol tartarate (0.2 mg/kg) was used as a postsurgical analgesic. Corneal freeze wounds were made in both eyes by placement of a liquid nitrogen-cooled brass probe with a 3-mm tip onto the epithelial surface for 40 s. This procedure results in the death of all corneal cells in an ~6- to 7-mm-diameter area, with minimal disruption of the stroma. Rabbits were divided into three groups: control (no wounds), 24-h wound healing, and 90-h wound healing. Each group contained three rabbits, with samples taken separately from the left and right eye.

LGF and AH collection. LGF was collected by cannulating the lacrimal duct of control and injured animals (4, 37). After the prescribed wound-healing period, rabbits were anesthetized as described above. Lacrimal glands were cannulated, and pilocarpine (200 µg/kg iv) was administered to stimulate lacrimal secretion. Lacrimal fluid was collected and placed into preweighed, chilled micropipettes. After lacrimal fluid was collected from both eyes, rabbits were killed and the anterior chambers were punctured using a 27-gauge needle. The average volumes of AH and LGF were 200-300 and 100-200 µl/eye, respectively. Ocular fluids and lipid extracts were stored under N2 gas at -70°C.

Electrophysiological recording from Xenopus oocytes. Oocytes were obtained from xylazine-anesthetized adult frogs (Xenopus laevis; Carolina Scientific, Burlington, NC) under aseptic conditions and prepared for the experiments as described previously (33). Briefly, electrophysiological recording was carried out during the first 5 days after defolliculation of the oocytes with collagenase. Oocytes were voltage clamped at -60 mV with a standard two-electrode voltage-clamp amplifier (GeneClamp 500, Axon Instruments, Foster City, CA) during superfusion with frog Na+-Ringer solution (in mM: 120 NaCl, 2 KCl, 1.8 CaCl2, 5 HEPES; pH 7.0) at a flow rate of 5 ml/min. Membrane currents were monitored with a digital oscilloscope (model NIC-310, Nicolet, Madison, WI).

Chemicals. Oleoyl-LPA, dioleoyl-PA, and oleoyl-LPS were purchased from Avanti Polar Lipids (Alabaster, AL) and stored under N2 at -20°C. Palmitoyl cyclic LPA was synthesized as described previously (19). HPLC-grade solvents were purchased from J. T. Baker Chemical (Phillipsburg, NJ) and from Fisher Scientific (Pittsburgh, PA). All other chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified.

HPLC. Lipid extracts were chromatographed on a 5-µm silica-packed semipreparative Microsorb Si column (250 × 10 mm; Rainin Instruments, Woburn, MA). A Waters HPLC system controlled by the Millennium 2010 software package (version 2.1, Waters Instruments, Milford, MA) was used for solvent delivery and injection. Elution of the lipids was monitored by an evaporative light-scattering detector (model IIA, Varex, Burtonsville, MD) through a metering valve, splitting the effluent 1:4 between the detector and a fraction collector, respectively.

A modification of the gradient elution originally described by Bünemann et al. (2) was used. Solvent A was made up of 60:35:4.5:0.5 (vol/vol/vol/vol) chloroform-methanol-water-28% ammonium hydroxide; solvent B consisted of the same compounds with relative proportions of 30:60:9.5:0.5, respectively. Solvent A was applied for 10 min, and a gradient of the two solvents was started by increasing solvent B to 100% over a period of 30 min followed by a 40-min isocratic elution at a flow rate of 2 ml/min throughout the run.

TLC. K6 silica plates, 20 × 20 cm with 250-µm layer thickness, were used (Whatman, Fairfield, NJ). Two-dimensional TLC separations were done using solvents of 6:4:1 (vol/vol/vol) chloroform-methanol-28% ammonium hydroxide in the first dimension and 6:8:2:2:1 (vol/vol/vol/vol/vol) chloroform-acetone-methanol-glacial acetic acid-water in the second dimension (36). Lipid spots were visualized after primulin (Aldrich Chemical, Milwaukee, WI) staining (43). Lipid spots marked with a pencil were scraped off the plate into glass vials, and lipids were eluted from the silica gel with methanol. Extraction was repeated three times for 5 min each, followed by centrifugation. The supernatants were filtered through a 0.2-µm Teflon syringe-filter and dried under nitrogen.

MS. Mass spectra were obtained by using the fast atom bombardment (FAB) technique in the positive and negative mode on a tandem mass spectrometer [AutoSpecQ E1BE2qQ, VG Fisons (now Micromass), Altrincham, UK] and the VG Opus level 1.7f software package for data analysis. A glycerol matrix (1 µl) was applied, and CsI was used for mass calibration.

Assay of LPA-induced DNA synthesis. Keratocytes were dissociated from uninjured corneas taken from rabbits anesthetized as described above. Corneal endothelium and Descemet's membrane were completely peeled off the stroma with a jeweler's forceps. The remaining epithelium and stroma were incubated in 1.2 U/ml dispase (grade II, Boehringer Mannheim, Indianapolis, IN) in MEM for 1 h at 37°C. The epithelium was peeled off, and the stroma was cut into 1- to 2-mm blocks and incubated with 1 mg/ml collagenase (type IA, Sigma Chemical) in MEM for an additional 2 h at 37°C. Cells were triturated with a fire-polished glass pipette and centrifuged at 180 g for 7 min. Freshly dissociated corneal cells were plated on 24-well plates (Sarstedt, Newton, NC) at a density of 5 × 104 cells/well in a 50:50 DMEM/F-12 medium (Cellgrow, Mediatech, Pittsburgh, PA). Wells, in triplicate, received solvent (30 µM BSA in Hanks' buffered saline solution), 10% FCS, or 10 and 30 µM LPA (18:1), alkenyl-GP (18:1), and LPS (18:1), all complexed with 30 µM BSA. DNA synthesis was determined by measuring the incorporation of [methyl-3H]thymidine (Amersham Life Sciences, Arlington Heights, IL; sp act 185 GBq/mmol). After 21 h in the presence of the mitogens, 1 µCi [methyl-3H]thymidine was added per well, and cells were harvested 3 h later using a cell harvester (Brandel, Gaithersburg, MD). The incorporated acid-insoluble radioactivity was determined by liquid scintillation counting using a Beckman LS 5000TA counter.

Statistical evaluation. Values are means ± SE. Student's t-test was used to determine whether injury-induced changes in the LPA-like activity were statistically significant. P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

LGF and AH activate oo-ICl. LGF and AH were collected from uninjured eyes (control) as well as from eyes with injured corneas 24 and 90 h after injury. Aliquots were taken from the eyes separately; the collected fluids were rapidly frozen and stored at -80°C until further processing. LGF and AH sample aliquots were tested in the oocyte bioassay for LPA-like activity at 100-fold dilution in frog Ringer solution by a blinded investigator in a random sequence. The dose-response relationship for the LPA-evoked oo-ICl was calibrated for every oocyte, with oleoyl-LPA as a standard. The responses elicited by LGF and AH samples were determined for three oocytes and normalized to the mean response of 92 ± 11 nA (n = 15 oocytes from 5 frogs) elicited by 1 nM oleoyl-LPA, which was arbitrarily chosen to be 1 response unit (1 U = mean response to 1 nM oleoyl-LPA = 92 nA). Figure 1 shows the means of the normalized oo-ICl responses elicited by LGF and AH samples collected 24 and 90 h after injury compared with the specimens collected from the uninjured eye.


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Fig. 1.   Relative titers of 1-acyl-2-lyso-sn-glycero-3-phosphate (LPA)-like activity in lacrimal gland fluid (LGF) and aqueous humor (AH) from normal corneas (control, C) and injured corneas 24 and 90 h after injury. For clarity, responses were normalized to that activated by 1 nM oleoyl-LPA (1 U = 92 nA). Samples were tested on 3 oocytes at a 100-fold dilution in frog Ringer solution. * Significantly different (P < 0.01) from control.

Very little LPA-like activity was detected in control LGF specimens, which did not show any significant change 24 and 90 h after injury. In contrast, a high basal level of LPA-like activity, 0.48 ± 0.22 unit, was found in the AH control compared with 0.07 ± 0.02 unit in LGF. This elevated level of activity increased by ~13-fold to 6.35 ± 0.90 units after 24 h of wound healing and remained elevated by ~7-fold at 3.29 ± 0.47 activity units at 90 h of wound healing. All AH specimens contained significantly higher LPA-like activity than the corresponding LGF specimens when examined with a Student's t-test. Moreover, injury also caused a statistically significant rise in the LPA-like activity of the AH.

Lipid content of LGF and AH. To determine the active component(s) in AH and LGF, polar lipids were extracted with methanol. For control, 4.1 g of AH and 2.4 g of LGF (wet weight) were collected from eyes of rabbits (n = 12) with normal, uninjured corneas. These fluids were lyophilized before extraction with methanol. Because of the limited amount of AH and LGF, samples collected at 24 and 90 h after injury were pooled; the total yield of 0.48 g (wet weight) of AH and 0.84 g of LGF liquids was lyophilized. The freeze-dried specimens were weighed, and lipids were extracted three times with vigorous shaking for 10 min in 5 ml/g (dry wt) methanol containing 50 µg/ml butylated hydroxytoluene as antioxidant. After centrifugation for 10 min at 2,000 g, supernatants were collected, combined, dried under a stream of N2 at 40°C, and weighed.

Data obtained for the dry material and polar lipid content of AH and LGF are summarized in Table 1. In control corneas the dry-to-wet weight ratio was 66 mg to 4,100 mg (1.6%) for AH and 98 mg to 2,400 mg (4.1%) for LGF. For injured eyes, these values were 8.4 mg to 480 mg (1.8%) and 42 mg to 840 mg (5.0%), respectively. The proportion of methanol-soluble lipids showed a substantial difference between AH and LGF. A total of 55 mg of the 66 mg of dry material (83%) was methanol-soluble lipid in control AH. In contrast, only 20 mg of the 98 mg of dry material (20%) were methanol-soluble lipid in control LGF. In samples taken from injured eyes, 7.4 mg of the 8.4 mg of dry material (88%) and 6.9 mg of the 42 mg of dry material (16%) were methanol-soluble lipid in AH in LGF, respectively.

                              
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Table 1.   Dry material and lipid content of LGF and AH

Fractionation of the methanol-soluble lipids by HPLC. To further characterize the polar lipids extracted by methanol, an HPLC method was developed for the separation of the major phospholipid classes. Lipids were dissolved in solvent A at a concentration of 10 mg/ml, and a total of 20 mg was injected into the column. Representative chromatograms of the extracts from normal corneas are shown in Fig. 2 for AH and LGF. Five-minute fractions were collected, dried under N2, and redissolved in 500 µl of methanol. LPA-like activity present in the fractions was tested using the oocyte bioassay. LPA-like activity was detected in the fractions that eluted from 5 to 15 min at a 100-fold dilution and in the region that eluted from 25 to 45 min at a 1,000-fold dilution (Fig. 2, bars). The active fractions of AH and LGF in the two regions were pooled separately and dried under a stream of N2. The active fractions are referred to as AH-(5---15), AH-(25---45), LGF-(5---15), and LGF-(25---45). The retention times for different known phospholipid standards under the conditions of this experiment are given in Table 2. The first peak of activity showed a retention time similar to that of cyclic PA (7 min), whereas the second peak of activity overlapped with the retention times of LPA, alkenyl-GP, PA, and LPS (Table 2).


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Fig. 2.   Fractionation of methanol-extractable lipids by HPLC. A and B: representative chromatograms (solid lines) for AH and LGF, respectively. LPA-like activity in 5-min fractions (bars) was tested using oocyte bioassay, diluting methanol-reconstituted fractions with frog Ringer solution. Active fractions were found between 5 and 15 min of retention at 100-fold dilution and between 25 and 45 min of retention at 1,000-fold dilution. ELSD, evaporative light-scattering detector.

                              
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Table 2.   HPLC retention times of different phospholipid standards under conditions used for separating the major phospholipid classes of LGF and AH

Two-dimensional TLC analysis of AH and LGF lipids. To identify the active phospholipid components in LGF and AH, two-dimensional TLC separations were carried out with the active HPLC fractions LGF-(25---45) and AH-(25---45) obtained from control corneas and the unseparated methanol extract of AH from injured corneas. Samples were reconstituted in a mixture of 1:2 (vol/vol) chloroform-methanol, and ~200 µg of lipid were spotted per plate. The plates were developed first in the basic and then in the acidic solvent system, stained with primulin, and viewed under ultraviolet illumination. Stained spots of lipids were marked with a pencil, scraped from the plate, and eluted with a large excess of methanol. Figure 3 illustrates representative TLC patterns, showing the lipid species present in LGF-(25---45), AH-(25---45), and AH from injured corneas. Individual lipid spots eluted from the TLC plate were reconstituted in 200 µl of methanol and tested for activation of oo-ICl using the bioassay.


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Fig. 3.   Two-dimensional TLC separation of polar lipid fractions of LGF and AH. A and B: active HPLC peaks LGF-(25---45) and AH-(25---45), respectively, in normal corneas. C: methanol-soluble polar lipids of AH from injured corneas. Position of primulin-stained components is shown. Retardation factors and activities in oocyte bioassay of active spots are given in Table 3.

Table 3 demonstrates the migration of the active components present in LGF-(25---45) and AH-(25---45), respectively. Table 3 also shows the retention factors of the active spots of AH from injured corneas and phospholipid standards. On the basis of their matching retention factors, the following four compounds could be tentatively identified by their comigration with known lipid standards in the two-dimensional TLC system: 1) injured AH spot 6 comigrates with the alkenyl-GP control, 2) LGF-(25---45) spot 4 and AH-(25---45) spot 4 comigrate with the dioleoyl-PA control, 3) AH-(25---45) spot 5 comigrates with the LPS control, and 4) LGF-(25---45) spot 1, LGF-(25---45) spot 2, and AH-(25---45) spot 3 comigrate with the LPA control.

                              
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Table 3.   Two-dimensional TLC retention factors of LPA-like phospholipids in AH and LGF

The structural formulas of LPA analogs tentatively identified in this manner in the ocular fluids are shown in Fig. 4. The slight variations observed between the retention factors of LGF-(25---45) spot 1, LGF-(25---45) spot 2, AH-(25---45) spot 3, and the oleoyl-LPA standard (Table 3) are likely due to the fact that endogenous LPAs with different acyl chain lengths have slightly different mobilities in the acidic solvent system. Estimated concentrations based on the oocyte bioassay for the identified bioactive lipids present in the LGF and AH are shown in Table 4.


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Fig. 4.   Structural formulas of growth factor-like phospholipids detected in ocular fluids.

                              
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Table 4.   Estimated concentrations of growth factor-like phospholipids in ocular fluids

FAB-MS spectra of the active TLC spots. After activity tests using the oocyte bioassay, the active components were concentrated to a volume of ~20 µl under a stream of N2 and subjected to MS analysis using FAB-MS in the positive and negative mode. MS analysis of the active spots corroborated, via the (m-H)- ion, the identity of these naturally occurring molecules with those of the comigrating TLC standards; those data reveal the presence of components with molecular masses corresponding to LPA, PA, and LPS, with different hydrocarbon chains (data not shown). LPA, PA, and LPS have long been known as physiological constituents of biologic fluids (8, 36). In contrast, alkenyl-GP has not previously been identified as an endogenous constituent of a physiological fluid. The negative-ion FAB-MS spectra of the lipid recovered from spot 6 and that of an alkenyl-GP standard are shown in Fig. 5, A and B, respectively. Because of its relative abundance, the high signal-to-noise ratio that was obtained allowed the identification of the major molecular species present in this spot. The compound with an m/z of 421 corresponds to an 18:0 aliphatic chain-substituted alkenyl-GP (m422/z-H+ = 421). In addition, C18-1 aliphatic chain-substituted alkenyl-GP was detectable with an m/z of 419. 


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Fig. 5.   Negative ion fast atom bombardment-MS spectra of AH spot 6 and alkenyl-GP standard. A: MS spectrum of highly active AH TLC spot 6 from injured corneas (Fig. 3C and Table 3) B: MS for an alkenyl-GP standard dissolved in methanol at a concentration of 2 mg/ml. Molecular ions for 18:0 and 18:1 alkenyl-GP are identified as (m-H)- at mass-to-charge ratios (m/z) of 421 and 419.

LPA-induced DNA synthesis in freshly dissociated keratocyte cultures. Cultures of freshly dissociated corneal keratocytes from uninjured rabbits (n = 3) were treated with 10 and 30 µM LPA, alkenyl-GP, or LPS immediately after isolation or with 10% FCS to establish whether these lipids induce cell proliferation in corneal keratocytes (Fig. 6). All three lipids, at both concentrations, caused a significant increase in [3H]thymidine incorporation. Therefore, it appears that corneal cells acquire mitogenic responsiveness to LPA-like lipids within the first 24 h of their isolation.


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Fig. 6.   LPA induces DNA synthesis in freshly isolated keratocytes. Keratocytes were exposed to solvent (30 µM BSA), 10 or 30 µM LPA, alkenyl-GP, and LPS (18:1) solubilized in 30 µM BSA, or 10% FCS for 21 h, then 1 µCi of [3H]thymidine was added. All 3 lipids at both concentrations significantly (P < 0.05) increased DNA synthesis, as indicated by increased [3H]thymidine incorporation. Data represent mean disintegrations per minute obtained for triplicate wells and are representative of data obtained with keratocytes from 3 rabbits.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Keratocytes residing within the collagen network of the corneal stroma play an essential role in repair after injury. Keratocytes normally are quiescent but, when activated, migrate to the site of injury and proliferate, constituting the wound-healing process. Activated keratocytes generate proteases (15, 16); phagocytose foreign bodies and injured tissue (22); produce a variety of mediators, including interferon, prostaglandin, and complement factor I (23, 30, 31); secrete components of the extracellular matrix, including fibronectin, collagen, and proteoglycans (1, 18); and show an increase in thymidine incorporation, indicating their proliferation (42). Epithelial growth factor (28), fibroblast growth factor (11), interleukin-1, transforming growth factor-beta (9), insulin (42), retinoic acid, and serum (14) have been shown to cause activation of keratocytes. Serum initially captured our attention as a keratocyte activator because of the abundance of compounds present in serum that have been found to enhance growth. A major group of growth-promoting agents in serum, however, does not belong to the polypeptide growth factor family but are phospholipids. Before this report, there were no reports available concerning the presence of growth factor-like phospholipids in the biologic fluids bathing the cornea.

LPA is the best-characterized member of a family of lipid mediators capable of positive or negative regulation of cell proliferation (35, 34). We and others have shown that, along with LPA, alkyl-GP, cyclic PA, LPS, and PA can activate LPA receptors and elicit LPA-like responses (20). We have also demonstrated the expression of at least two distinct receptors for glycerophosphatidates, which are pharmacologically distinguished through cyclic PA responsiveness. The subtype-specific LPA receptor agonist cyclic PA has been shown to be a stable intermediate produced by phospholipase D (6), an enzyme that is under tight regulation by hormones and growth factors. After our initial report that LPS also elicited LPA-like effects in Xenopus oocytes and PC-12 cells, Xu and co-workers (45) demonstrated a complete heterologous desensitization between LPA and LPS, indicating that LPS acts through LPA receptors. More recently, we isolated and synthesized a novel LPA analog, alkenyl-GP (29), that until now had not been shown to be a naturally occurring component of any biologic fluid.

LPA has been shown to activate a Cl- conductance in keratocytes from injured, but not from control, corneas (39). Twenty-four hours after injury was sufficient for the occurrence of LPA responsiveness, which was maintained for at least 4 days. Here, we applied the Xenopus oocyte bioassay to detect and monitor the changes in LPA-like activity of LGF and AH samples collected at 24 and 90 h after injury and in control eyes with uninjured corneas. LGF and AH showed characteristic changes during wound healing (Fig. 1). The LPA-like activity in LGF was low and did not change significantly after 24 and 90 h of healing. In contrast, a significantly higher basal level of LPA-like activity was found in control AH, and this activity increased rapidly, by as much as ~13-fold, 24 h after injury and remained high for up to 90 h. It is important to point out the corresponding occurrence of LPA responsiveness in keratocytes and the increase in the LPA-like activity in AH. These data indicate that the LPA mediator family and its receptors could play a physiological role in corneal wound healing. This hypothesis is supported by the inducible nature of some of the recently identified putative LPA receptor genes (21). Further experiments are necessary to establish the expression of the different LPA receptor subtypes and their regulation in resting and activated keratocytes.

The role of lipid mediators in the physiological maintenance of corneal integrity and regeneration is underlined by the unexpectedly high methanol-soluble phospholipid content of AH (Table 1). Despite the similar overall lipid content of AH (1.4%) and LGF (0.8%), the dry materials of AH consisted of ~90% polar lipids in contrast to the 20% lipid content of LGF. The high lipid content of AH suggests that it is almost like a lipid suspension, whereas LGF is a solution with a smaller, but still significant, lipid content. In the context of the relatively high lipid content of these ocular fluids, the lipid-soluble LPA-like mediators could play a role in the normal cellular function of the cornea, where there are intact cellular barriers, as well as in injured corneas, where the epithelial and endothelial cell barriers are disrupted. Moreover, given that AH and LGF bathe ocular tissues other than the cornea, including the iris, lens, ciliary body, conjunctiva, and sclera, it is possible that these lipid mediators play a physiological role throughout the anterior portion of the eye.

HPLC fractionation of the methanol-extractable lipids of LGF and AH from normal eyes showed two major fractions with LPA-like active lipids (Fig. 2). The first active peak eluted with a retention time in good agreement with that of cyclic PA (7 min). Consequently, cyclic PA is the most likely LPA-like compound present in the first active peak in LGF-(5---15) and AH-(5---15). The second active broad peak eluted near the LPA standard, between 25 and 45 min. The retention time of this second group of activities overlaps that of LPA analogs, including LPA, alkyl- and alkenyl-GPs, and LPS. Two-dimensional TLC and MS have corroborated the presence of different members of the LPA family in LGF and AH (Fig. 3, Table 3). LGF and AH from control corneas contain PA and LPA. Using negative-ion FAB-MS, we have, for the first time, been able to positively identify the presence of alkenyl-GP molecules in AH from injured corneas, establishing that this LPA analog also occurs endogenously. The level of LPS in LGF, if any, was below the limit of detectability of the assay systems used in the present study. In contrast, we found LPS to be abundant in AH from control corneas. LPS has a very high (20 µM) EC50 in oocytes (unpublished observations), whereas LPA, alkenyl-GP, and alkyl-GP have EC50 values in the low nanomolar range. This difference places LPS as a low-potency agonist in the LPA-like mediator family, which can potentially synergize with other mitogens. The relatively high activity and apparent abundance of spot 5 in AH-(25---45), which comigrates with LPS, suggest the presence of a high amount of LPS in the AH in the normal eye (Table 4). In addition, we have detected a few additional active minor compounds in LGF and AH, the identities of which are yet to be determined (Table 3). The observation that LPA and LPS were not among the detectable active components in injured AH is an interesting one. We do not know whether there is a true decrease in the relative abundance of these two lipids while other AH components, like alkenyl-GP, increase after injury. Nonetheless, more research is necessary to establish the biosynthetic and breakdown pathways for each of these lipids in the blood-free environment of the ocular anterior chamber.

We have also provided evidence that LPA-like lipids, after a 24-h incubation, induce keratocyte proliferation, as revealed by increased thymidine incorporation into freshly dissociated keratocytes from uninjured eyes. This observation, together with the lack of LPA-elicited Cl- currents in freshly dissociated keratocytes from uninjured eyes (39), might suggest a rapid induction of LPA responsiveness within the first 24 h after cell dissociation and placement into culture. One possible explanation for the development of LPA responsiveness would be de novo LPA receptor expression, given that the signaling pathway leading to Cl- channel opening could be activated by an osmotic stimulus in freshly isolated keratocytes that were unresponsive to LPA stimulation (39). The induced expression of LPA receptor subtypes during keratocyte activation is under investigation in our laboratories.

The present study establishes that growth factor-like glycerophosphate mediators are present and are generated in increased amounts after injury in AH and LGF. Furthermore, we provide evidence that analogs of the mitogenic lipid mediator LPA, including cyclic PA, LPA, LPS, PA, and, for the first time in any biologic system, alkenyl-GP, are physiological constituents of the biologic fluids bathing the cornea. We also show that LPA is mitogenic in freshly dissociated cells from the uninjured cornea, suggesting that LPA and its homologues are involved in maintaining the integrity of the control cornea and in promoting the regeneration of the injured cornea.

In summary, we propose that the novel group of LPA-like mediators that are abundant in the lipid-rich fluids bathing the cornea can potentially be important physiological regulators of ocular integrity and regeneration.

    ACKNOWLEDGEMENTS

We thank Dr. John Ubels for demonstrating the LGF collection technique.

    FOOTNOTES

This work was supported by National Science Foundation Grant IBN-9321940 (G. Tigyi) and National Eye Institute Grant EY-10178 (M. A. Watsky). G. Tigyi is an Established Investigator of the American Heart Association.

Address for reprint requests: G. Tigyi, Dept. of Physiology and Biophysics, College of Medicine, University of Tennessee, 894 Union Ave., Memphis, TN 38163.

Received 16 September 1997; accepted in final form 16 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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