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From the Department of Pharmacology, New York Medical College, Valhalla.
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Abstract |
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METHODS. Rabbit corneas were isolated and cultured for 24 hours in normoxia or hypoxia. After culture, PGE2 in media was quantitated by enzyme immunoassay. 12-HETE and 12-HETrE were extracted from culture media and corneal epithelium and quantitated by negative chemical ionization-gas chromatography-mass spectrometry. COX-1 and -2 protein expression in corneal epithelium was determined by Western blot. Acute (2 hours) COX activity in normoxia and hypoxia was determined as the conversion rate of [14C]AA to [14C]PGE2, quantitated through reverse-phase–high-performance liquid chromatography and radiodetection.
RESULTS. In the media of cultured rabbit corneas, both 12-HETE and 12-HETrE were detected, with 12-HETrE levels being four times higher. Hypoxia did not significantly increase extracellular 12-HETE or 12-HETrE; however, it caused more than 90% inhibition of PGE2 synthesis. Intracellular 12-HETE and 12-HETrE were undetectable in normal corneas but increased to 7.7 ± 1.3 and 2.2 ± 0.4 ng/mg protein, respectively, after 24 hours in culture. Culture in hypoxia further increased intracellular 12-HETE threefold but had no additional effect on 12-HETrE.
CONCLUSIONS. Hypoxia creates an environment in which epithelial COX activity is severely suppressed, whereas cytochrome P450-AA and/or 12-LOX metabolizing activity is maintained or enhanced. Additionally, the findings suggest that 12-HETE produced by the corneal epithelium acts intracellularly to promote corneal edema, whereas 12-HETrE acts in a paracrine manner to initiate an inflammatory cascade that can elicit neutrophil chemotaxis and neovascularization of the cornea.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Corneal surface injury increases the production of cyclooxygenase (COX)- and lipoxygenase (LOX)-derived eicosanoids by the corneal epithelium.3 4 5 After ocular surgery, the prostaglandin (PG) E2 level in tear film is elevated; however, no significant correlation is seen between PGE2 levels and clinical signs of inflammation, corneal thickness, polymorphonuclear (PMN) leukocyte levels, or tear secretion rate.6 Conversely, increased capacity of the corneal epithelium to synthesize cytochrome P450-derived 12(R)-hydroxy-5,8,11,14-eicosatetraenoic (HETE) and 12(R)-hydroxy-5,8,14-eicosatrienoic acid (HETrE) during closed-eye contact lens wear significantly correlates with the appearance and course of the ocular inflammatory response.7 12(R)-HETE is an Na+K+-adenosine triphosphatase (ATPase) inhibitor,8 9 vasoconstrictor and chemoattractant for PMN leukocytes, and can serve as a substrate for 12(R)-HETrE biosynthesis. 12(R)-HETrE can elicit conjunctival vasodilation and increase the permeability of the blood–aqueous barrier,10 stimulate limbal microvessel endothelial cell proliferation and angiogenesis,11 and serve as chemoattractant for PMN leukocytes.12 12(S)-HETE, reported to be the main LOX metabolite produced by the cornea in response to injury,13 can inhibit adenylyl cyclase,14 serve as chemoattractant for leukocytes, stimulate endothelial cell mitogenesis,15 and serve as a substrate for 12(R)-HETrE biosynthesis.
It is important to acknowledge that the formation of metabolites under optimized conditions in an assay does not ensure that the same reaction will occur or occurs to the same extent in vivo. Similarly, the ability of specific eicosanoids to elicit a biologic response when added exogenously in high doses does not ensure that they exhibit this activity (function in this regard) endogenously. Therefore, the present study was undertaken to characterize the endogenous production of corneal epithelial eicosanoids under both normoxic and hypoxic conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Corneal Organ Culture
Corneal organ culture was performed as described
previously.16
Briefly, on receipt, eyes were rinsed
with DMEM and antibiotics. The cornea was removed with the scleral rim,
and the iris ring was removed. Each cornea was then transferred to one
well of a 12-well culture plate containing 1 ml DMEM with 1x
pen/strep/amphotericin B and 0.2% LH (LH medium). Culture plates were
then placed into a 37°C incubator supplied with 5%
CO2-95% air (~20% O2;
normoxia), or into a modular tissue culture chamber
(Billups–Rothenburg, DelMar, CA) placed into a 37°C incubator and
supplied continuously with hydrated 5% CO2-95%
N2 (<0.05% O2; hypoxia,).
All cultures were incubated for 24 hours.
Endogenous PGE2 Quantitation
After 24 hours, culture medium from cornea-containing wells was
removed, diluted with enzyme immunoassay (EIA) buffer, and used for
quantitation of secreted PGE2 by a commercially
obtained EIA kit (Cayman).
COX Activity
Total COX activity was determined using conversion of exogenously
added [14C]AA to
[14C]PGE2 in isolated
corneas. Control corneas (baseline activity) were isolated from rabbit
eyes and immediately incubated for 2 hours with 10 µM
[14C]AA. Incubations were performed at 37°C
in a humidified incubator under normoxic or hypoxic conditions. One
cornea per well was placed in a 12-well culture plate containing 1 ml
0.2% LH medium. After 2 hours, the medium was removed from each well,
acidified to pH 4.0 with formic acid, and extracted with ethyl acetate.
For cultured corneas, incubation was for 24 hours, as described, under
normoxic or hypoxic conditions. After 24 hours, the corneas were
removed from culture and rinsed once with fresh medium. These
conditioned corneas were subsequently incubated with
[14C]AA, as described, for 2 hours under
normoxic or hypoxic conditions. Samples were analyzed and
[14C]PGE2 quantitated
through reversed-phase high-performance liquid chromatography (RP-HPLC)
using radiodetection. Briefly, detection of
[14C]PGE2 was performed
in a chromatography system (model 1050; Hewlett Packard, Palo Alto, CA)
on a 5-µm ODS-Hypersil column (4.6 x 200 mm; Hewlett Packard)
using a solvent composed of 100% acetonitrile-water-acetic acid,
50:50:0.1 (vol/vol/vol) at a flow rate of 1 ml/min for 25 minutes.
Radioactivity was monitored by an online flow detector (Radiometric
Instruments, Tampa, FL).
Western Blot Analysis
After culture, the corneal epithelium was scraped from each cornea
and homogenized in lysis buffer (20 mM Tris-HCl [pH 7.5], 16 mM
CHAPS, 0.5 mM dithiothreitol, 1 mM EDTA, 1 mM benzamidine-HCl, 1
µg/ml leupeptin, and 10 µg/ml trypsin inhibitor). For basal COX-1
and COX-2, the corneal epithelium was scraped from eyes on receipt
without culture. Protein content was determined using the DC protein
assay (Bio-Rad, Hercules, CA). Sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS-PAGE) was performed using 10% and 3%
(wt/vol), acrylamide for the separating gel and stacking gel,
respectively. Protein transfer onto polyvinylidene fluoride membranes
(Amersham, Amersham, UK) was performed at 16 V for 1 hour. Membranes
were saturated in blocking buffer and incubated with a rabbit
anti-mouse COX-2 or goat anti-human COX-1 polyclonal antibody for 2
hours at room temperature. After washing, membranes were incubated with
anti-rabbit or anti-goat IgG conjugated with horseradish peroxidase
(Santa Cruz Biotechnology) for 45 minutes at room temperature. Positive
bands were developed through chemiluminescence and exposure to film
(Hyperfilm ECL; Amersham Pharmacia Biotech, Piscataway, NJ).
Densitometry was performed on individual bands by computer (Image;
National Institutes of Health, Bethesda, MD)
Negative Chemical Ionization–Gas Chromatography–Mass Spectrometry
12-HETE and 12-HETrE Quantitation from Culture Medium.
After 24 hours, culture medium from cornea-containing wells was
adjusted to 2 ng/ml
[2H3]-12(R)-HETrE
as an internal standard. The medium was then acidified to pH 4.0 with
formic acid and subsequently extracted with ethyl acetate. The extract
was subjected to RP-HPLC separation, and fractions containing 12-HETE
and 12-HETrE were collected. Briefly, purification of 12-HETE and
12-HETrE was performed (model 1050 chromatography system; Hewlett
Packard) on a 5-µm ODS-Hypersil column (4.6 x 200 mm; Hewlett
Packard) with a solvent composed of 80% acetonitrile-water-acetic
acid, 50:50:0.1 (vol/vol/vol) and 20% acetonitrile-acetic acid,
100:0.1 (vol/vol), at a flow rate of 1 ml/min for 25 minutes followed
by 100% acetonitrile-acetic acid, 100:0.1 (vol/vol) for 10 minutes.
Purified 12-HETE and 12-HETrE were derivatized to the pentafluorobenzyl
ester, trimethylsilyl ether. Negative chemical ionization–gas
chromatography–mass spectrometry (NCI-GC/MS) was performed on a mass
spectrometer (model HP5989A mass spectrometer; Hewlett Packard)
interfaced with a capillary gas chromatographic column (DB-1 fused
silica, 10 m, 0.25 mm internal diameter, 0.25 µm film thickness;
J&W Scientific, Rancho Cordova, CA) and programmed from 180
oC to 300 oC at 25°C/min
using helium as the carrier gas with a linear velocity of 0.4 m/sec.
Samples were injected through a splitless injector maintained at a
temperature of 250°C. Electron capture ionization was performed under
methane as a moderating gas at a flow resulting in ion source pressure
of 2.8 Torr. The other mass spectrometer parameters were as follows:
ion source temperature, 200°C; electron energy, 220 eV; and transfer
line temperature, 250°C. Single ions were monitored with m/z 391
corresponding to the derivatized 12-HETE, m/z 393 for the
derivatized 12-HETrE, and m/z 396 for the derivatized
[2H3]-12(R)-HETrE
internal standard. Total 12-HETE and 12-HETrE in each sample was
determined by comparison of the ratio of ion intensities (391:396 and
393:396, respectively) versus a standard curve of derivatized
12-HETrE/[2H3]-12(R)-HETrE
molar ratio obtained from NCI-GC/MS analysis.
Corneal Epithelial Tissue.
Basal tissue levels were obtained by scraping the corneal epithelium
(two eyes per sample) from anesthetized (intramuscular injection of 50
mg/kg ketamine and 20 mg/kg xylazine and application of 0.5% topical
proparacaine-HCl) healthy rabbits with no ocular inflammation. Cultured
corneas (24 hours) were rinsed once with ice-cold 0.1 M
KPi buffer [pH 7.5], and the corneal epithelium
from two corneas per sample was then scraped into a total volume of 1
ml KPi buffer. Each sample was homogenized in a
Teflon homogenizer and centrifuged for 2 minutes at maximum speed in a
centrifuge (Eppendorf, Freemont, CA) to remove particulate tissue.
Protein concentration in the supernatant was determined by the method
of Bradford (protein assay; Bio-Rad). Samples were adjusted to equal
protein concentration with deionized water, and 2 ng
[2H3]-12(R)-HETrE
was added per milligram protein as an internal standard. Total
tissue-associated fatty acids (free + esterified) were obtained by
Bligh and Dyer extraction.17
18
12-HETE and 12-HETrE were
then purified by RP-HPLC and quantitated by NCI-GC/MS, as described.
Data and Statistical Analyses
Student’s t-test was used to evaluate the significance
of differences between groups (control versus treatment).
P < 0.05 was considered significant. All data are
presented as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGE2
Hypoxia has been shown to induce the expression of COX-2 protein
in HUVEC cells19
; however, the catalytic activity of COX
under hypoxic conditions and the contribution of its major product in
the cornea, PGE2, to corneal inflammation are
uncertain. To investigate this activity, we measured the accumulation
of endogenous PGE2 in the culture media of rabbit
corneas cultured in either normoxia or hypoxia (Fig. 1)
. After 24 hours in culture, the amount of PGE2
detected in media from hypoxic cultures (36.3 ± 5.5 ng/ml,
n = 11) was less than 10% of that detected in media from
normoxic cultures (416 ± 39 ng/ml, n = 9).
PGE2 synthesis in both normoxia and hypoxia was
effectively blocked by the addition of 5 µM indomethacin (normoxia:
9.2 ± 1 ng/ml, n = 8; hypoxia: 11.2 ± 0.7 ng/ml,
n = 8) to the culture medium at the beginning of culture
confirming that PGE2 production was COX-dependent
under these conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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250 µM) but that under hypoxic conditions
(5–20 µM O2) the activity of these enzymes
becomes limited. The current findings suggest that oxygen availability
is the major limitation for COX catalysis under hypoxic conditions.
This hypothesis is supported by the observations that the accumulation
of endogenous PGE2 in the medium of corneas
cultured in hypoxia is only 10% of that produced by corneas cultured
under atmospheric oxygen tension; the acute ability of COX to
metabolize exogenously added [14C]AA is rapidly
inhibited by hypoxia; there are equivalent amounts of COX protein under
normoxic and hypoxic conditions, and these proteins exhibit nearly
equivalent catalytic activity when normal oxygen conditions are
restored; and phospholipase activity, and thus free AA, is elevated
under hypoxic conditions,22
23
which should serve to
increase COX activity. The enhanced oxygenation of AA to 12-HETE appears paradoxical in the setting of hypoxia-induced inhibition of COX activity; however, it should be noted that COX requires two molecules of O2 per molecule of product, whereas P450 and LOX require only one. Furthermore, even while suppressed by hypoxia, COX is still producing more than 30 ng/ml PGE2 versus 0.38 ng/ml 12-HETE (in medium). Moreover, the enhanced induction of 12-HETE synthesis stimulated by hypoxia above that stimulated by stress or injury alone suggests that these are separate stimuli that appear to have an additive effect. It should be noted, however, that the substantial inhibition of COX activity by hypoxia may contribute to the increase in 12-HETE synthesis, this resulting from a shunting of free AA from COX to the cytochrome P450 and 12-LOX pathways. Srinivasan and Kulkarni24 have hypothesized that this phenomenon may also occur during COX inhibition with NSAIDs. Nonetheless, the shunting of AA cannot account for the increase in metabolic capacity of homogenates of hypoxic corneal epithelium in vitro when exogenous [14C]AA is used.7 16 25 We recently reported the hypoxia-stimulated induction of a cytochrome P450 4B1 isoform in rabbit corneal epithelium26 that was associated with increased metabolism of [14C]AA to 12-HETE by corneal epithelial homogenates. Taken together, these results suggest a multifaceted amplification of 12-HETE production during hypoxic ocular inflammation.
The detection of 12-HETrE in rabbit corneal epithelium and culture medium demonstrates the endogenous production of this eicosanoid. In contrast to 12-HETE, the intracellular level of 12-HETrE was not significantly increased by hypoxia. This may indicate that the synthesis of 12-HETrE is already maximal as a result of the culture conditions (as with COX-2 induction), and no further induction can be elicited by hypoxia. These findings differ from our previous observation that 12-HETrE synthesizing activity was increased in homogenates from hypoxic tissue in vitro.16 This discrepancy may indicate that the synthesis of 12-HETrE is subject to regulatory mechanisms in vivo. Unlike the tear film that is continuously produced and washed away, the culture medium creates a static environment in which metabolites accumulate over the 24-hour culture period. In this environment, the synthesis of 12-HETrE may diminish once sufficient concentration is achieved. In fact, the detected extracellular concentration was approximately 4 nM, which is 40 times higher than that required for 12(R)-HETrE to elicit a maximal biologic response in neutrophil chemotaxis, vasodilation, and angiogenesis when applied exogenously.10 In preliminary experiments using human tear film, which more accurately reflects the in vivo environment of the cornea, we have detected increased amounts of 12-HETrE in inflamed eyes (unpublished data, 1999). These observations are consistent with a correlation between 12-HETrE production and ocular surface inflammation as previously noted in rabbits.7 27
Comparison of the relative concentrations of 12-HETE and 12-HETrE in corneal epithelium and culture medium indicates a difference in the likely site of action of each. In corneal epithelium, the amount (nanograms per milligram protein) of 12-HETE was three and eight times higher than 12-HETrE (in normoxia and hypoxia, respectively), whereas in culture medium, 12-HETrE was approximately four times (nanograms per milligram protein) higher than 12-HETE. This represents up to a 32-fold difference (in hypoxia) in the ratio of 12-HETE to 12-HETrE between cells and culture medium, respectively. Although a much smaller amount of 12-HETrE is present inside corneal epithelium (versus 12-HETE), a substantially greater proportion of it appears to reach the extracellular environment. This phenomenon was confirmed in human tears where 12-HETrE was found in greater amounts than 12-HETE (data not shown). This difference in localization is not predicted by in vitro metabolism studies using homogenates or microsomes. Such studies have consistently demonstrated that 12-HETE is produced in greater quantities than 12-HETrE. However, although the pattern of increased 12-HETE production in inflammation and hypoxia observed previously16 27 is paralleled by the current findings in corneal epithelial cells, the concentration of 12-HETE detected in culture medium does not reflect these increases. The dissociation of intracellular increases in 12-HETE from changes in its extracellular concentration implies an intracellular site of action for 12-HETE and is supported by the observed ability of 12(S)-HETE to inhibit adenylyl cyclase14 and of 12(R)-HETE to inhibit Na+K+-ATPase.8 In binding studies using [3H]12(R)-HETrE, Stoltz and Schwartzman28 found that less than 10% of cell-associated 12-HETrE was esterified into membrane phospholipids, whereas specific binding indicated a possible receptor site with an apparent kd of 43 pM. In contrast, Hurst et al.29 found that [3H]12(S)-HETE injected intracamerally into rabbits was mainly incorporated into the membrane phospholipids with free 12(S)-HETE representing less than 1% of the total label. Taken together, the current data and previous observations suggest that the intracellular synthesis of both 12-HETE and 12-HETrE is increased by inflammatory stimuli, but 12-HETrE is preferentially secreted to act in a paracrine manner on neighboring cells. The effects of 12(R)-HETrE as a chemoattractant of PMN leukocytes12 and a stimulator of microvessel endothelial cell proliferation and angiogenesis11 further support this hypothesis.
In summary, the observed suppression of corneal epithelial PGE2 synthesis by hypoxia further challenges the role of PGE2 in mediating ocular surface inflammation and provides a possible explanation for the inefficacy of NSAIDs in alleviating such diseases. Figure 5 represents the proposed scheme for the intracrine and paracrine roles of corneal epithelium–derived eicosanoids in hypoxia-induced ocular inflammation.
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Acknowledgements |
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Footnotes |
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Submitted for publication August 18, 1999; revised November 30, 1999, and January 21, 2000; accepted February 10, 2000.
Commercial relationships policy: N.
Corresponding author: Michal Laniado Schwartzman, Department of Pharmacology, New York Medical College, Valhalla, NY 10595. michal_schwartzman{at}nymc.edu
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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