Detection of endogenous 12-hydroxyeicosatrienoic acid in human tear film.

PURPOSE: Increased production of 12-hydroxyeicosatetraenoic acid [12(R)-HETE] and 12-hydroxyeicosatrienoic acid [12(R)-HETrE] positively correlates with the in vivo progression of ocular surface inflammation in rabbits. Tear film was collected from human subjects with inflamed eyes to determine whether these eicosanoids could be detected from endogenous sources. METHODS: Control and inflamed eyes were assessed and assigned a subjective inflammatory score. Tears were collected and extracted with an internal standard. Single-ion-monitoring gas chromatography-mass spectrometry (SIM-GC-MS) was performed to quantitate endogenous levels of 12-HETE and 12-HETrE. RESULTS: 12-HETrE was detected in the tear film of both control and inflamed eyes, with the mean level being seven times higher in inflamed tears. 12-HETE was not detected in control tears and was detected in only 6 of 38 inflamed-eye tear samples. CONCLUSIONS: The current findings demonstrate that the human eye produces detectable amounts of 12-HETrE, which is released into the tear flow. The increased levels of 12-HETrE associated with ocular surface inflammation suggest that this eicosanoid may contribute to inflammation of the ocular surface in humans.

Corneal epithelial VEGF and cytochrome P450 4B1 expression in a rabbit model of closed eye contact lens wear.

PURPOSE: The similar and overlapping activity of VEGF and the potent corneal-derived angiogenic eicosanoid 12(R)-HETrE calls for a study of the temporal relationship in the expression of these two autocoids. Since recent evidence suggests that hypoxia induces the expression of a CYP4B1 mRNA which might be involved in the conversion of arachidonic acid to 12(R)-HETrE, we determined its time-dependent expression and correlated it to that of VEGF mRNA in the rabbit model of closed eye contact lens-induced injury. METHODS: Rabbit eyes were fitted with contact lenses followed by a silk suture tarsorrhaphy. The anterior surface was analyzed at 2-, 4- and 7-days by slit lamp biomicroscopy, subjective inflammatory scoring and corneal pachymetry. Corneal epithelium was scraped and CYP4B1 and VEGF mRNA levels were measured by Southern hybridization of RT-PCR products amplified from a single cornea with specific primers. RESULTS: Corneal thickness and inflammatory scores increased in a time dependent manner in the model of closed eye contact lens induced hypoxic injury. Corneal epithelial CYP4B1 and VEGF mRNAs, as well as the production of the angiogenic eicosanoid, 12-HETrE, increased in a time-dependent manner and correlated with the in situ inflammatory response. CONCLUSIONS: The present study documents the increased expression of CYP4B1 isoform in the corneal epithelium during hypoxic injury in vivo. It also demonstrates the presence of VEGF mRNA in the corneal epithelium and its increased expression in this model of hypoxic injury. All together, the results of this study raise the possibility of interaction between these autocoids, VEGF and CYP4B1-12(R)-HETrE, in mediating the neovascularization response induced by the prolonged hypoxic state brought about by closed eye contact lens wear.

The effect of hypoxia on endogenous corneal epithelial eicosanoids.

PURPOSE: Injury to the corneal epithelium increases arachidonic acid (AA) metabolism through the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 pathways. The authors used the rabbit corneal organ culture model to demonstrate the effect of hypoxia on the endogenous formation of 12-hydroxy-5,8,11,14-eicosatetraenoic acid (12-HETE), 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), and prostaglandin (PG) E2 by the intact cornea in the absence of exogenously added cofactors or substrate. 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.

Functional human heme oxygenase has a neuroprotective effect on adult rat ganglion cells after pressure-induced ischemia.

Adult retina subjected to transient ischemia and reperfusion leads to controlled retinal ganglion cell (RGC) death over a period. Modification of intracellular mechanisms through a specific adenoviral vector containing the hemoxygenase gene (HO-1) provides avenues for RGC survival following HO-1 gene transfer and ischemia. RGC death rate was reduced by an average of 15% at 1, 2 and 3 weeks. A significant number of RGC transfected with functional HO-1 survived ischemic insults. Pharmacological stimulation of HO-1 may constitute a novel therapeutic approach to rescuing RGC experiencing ischemic/reperfusion injury.

Regulation of cyclooxygenase-2 by hypoxia and peroxisome proliferators in the corneal epithelium.

Hypoxic injury provokes inflammation of many tissues including the ocular surface. In rabbit corneal epithelial cells, both peroxisome proliferator-activated receptor (PPAR)-inducible cytochrome P450 4B1 and cyclooxygenase-2 (COX-2) mRNAs were increased by hypoxia. PPAR alpha and beta but not gamma mRNAs were detected in these cells. The PPAR activator, WY-14,643 increased COX-2 expression. Similarly, non-steroidal anti-inflammatory drugs with the ability to activate PPARs induced COX-2 independently of prostaglandin synthesis inhibition. COX-2 protein overexpression by hypoxia and PPAR activation was not associated with a parallel increase in prostaglandin E(2) accumulation. However, the enzyme regained full catalytic activity when: 1) hypoxic cells were re-exposed to normoxic conditions in the presence of heme and arachidonic acid, and 2) WY-14,643-treated cells were depleted of intracellular GSH. Consistent with previous observations showing that the corneal production of cytochrome P450-derived inflammatory eicosanoids is elevated by hypoxia and inflammation, the current data suggest that hypoxic injury is a model of inflammation in which molecules other than COX-derived arachidonic acid metabolites play a major proinflammatory role. This study also suggests that increased cellular GSH may be the mechanism responsible for the characteristic dissociation of PPAR-induced COX-2 expression and activity. Moreover, we provide new insights into the commonly observed lack of efficacy of classical non-steroidal anti-inflammatory drugs in the treatment of hypoxia-related ocular surface inflammation.

Retinal pigment epithelial cell-based gene therapy against hemoglobin toxicity.

To determine if overexpression of the human heme oxygenase (HO-1) protects retinal pigment (RPE) cells from hemoglobin toxicity, a human RPE cell line was infected by an adenoviral vector containing the HO-1 (Ad-HO-1) gene or transfected with a plasmid containing the cytomegalovirus promoter and HO-1 cDNA (pRc/CMV-HO-1) complexed to cationic liposomes. Phase contrast microscopy and acid phosphatase activity were examined to insure homogeneity of the cell line. Mitochondrial cytochrome and microsomal heme content were measured in both transduced and control cells. RPE cells were then challenged with hemoglobin and their viability estimated. We determined that cells transfected with Ad HO-1 overexpressed HO-1 compared to control cells: HO-1 mRNA levels were increased 3-fold within 3 days, decreasing in 7 days. In addition, we permanently transfected RPE cells with HO-1 gene. Transfected cell clones selected for neomycin resistance had elevated levels of HO activity 3-fold higher than control. Transfected cells exposed to hemoglobin had a survival rate of 93%; non-transfected cells had a 65-75% rate of survival. Transfected cells overexpressing HO-1 proved highly viable when challenged with hemoglobin. HO-1 appears to be an important component of the cellular anti-oxidant defense mechanisms against hemoglobin toxicity. However, the choice of transient or permanent expression of HO-1 against hemoglobin toxicity and hemorrhage needs to be further evaluated.

Alkali burn-induced synthesis of inflammatory eicosanoids in rabbit corneal epithelium.

PURPOSE: Alkali burning of the rabbit cornea is a well-established model for the study of anterior surface inflammation, neovascularization, and wound-healing processes. 12-hydroxyeicosanoids have been implicated as mediators of such responses. 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) is a lipoxygenase-derived arachidonate metabolite and 12(R)-hydroxyeicosatetraenoic acid (12[R]-HETE) is formed by a cytochrome P450 monooxygenase; both give rise to the potent angiogenic factor 12(R)-hydroxyeicosatrienoic acid (12[R]-HETrE). In this study, the authors correlate the pattern of their synthesis in the corneal epithelium with the inflammatory response after alkali injury. METHODS: New Zealand albino rabbits were anesthetized and alkali burns created with 10-mm filter paper discs (1 N NaOH for 2 minutes). Corneas were then rinsed; 1 to 7 days later, corneal epithelium was scraped and used to assess 14C-arachidonic acid conversion to 12-HETE and 12-HETrE enantiomers in the presence of NADPH by chiral high-pressure liquid chromatography. The inflammatory response secondary to the alkali burn was quantified through area measurements of reepithelialization and neovascularization. RESULTS: Alkali burn induced a time-dependent production of corneal epithelial 12-HETE and 12-HETrE. A marked increase in 12-HETE and 12-HETrE synthesis was evident at day 2 (from 22 +/- 7 to 139 +/- 22 ng/hour) after injury, increasing to 800 +/- 68 ng/hour at day 7. Chiral analysis revealed a time-dependent synthesis of the R and S enantiomers of 12-HETE (24% R, 76% S) and 12-HETrE (72% R, 28% S). Total arachidonate metabolism, as well as the formation of 12(R)-HETrE, correlated with the area of neovascularization (P < 0.01 and P < 0.02, respectively). CONCLUSIONS: The results demonstrate that surviving and regenerating epithelium has an increased capacity of synthesizing 12(S)-HETE and 12(R)-HETE and that maximal production of 12(R)-HETrE, a known direct and indirect angiogenic factor, coincides with neovascularization in this model. Thus, the lipoxygenase and cytochrome P450-dependent activities increased in a time-dependent manner, indicating the potential involvement of both pathways in the inflammatory response to alkali burn. The formation of significant quantities of 12(R)-HETE and 12(R)-HETrE is a novel finding in this alkali injury model.

Heme oxygenase induction with attenuation of experimentally induced corneal inflammation.

Heme oxygenase (HO), by catabolizing heme to bile pigments, down-regulates cellular levels of heme and hemeproteins; certain of the latter, i.e. cytochrome P450s, generate pro-inflammatory products from endogenous substrates. Two HO isozymes, the products of distinct genes, have been described; HO-1 is the inducible one, whereas HO-2 is believed to be constitutively expressed. We studied the inducing effects of several metal compounds [CoCl2, SnCl2, ZnCl2, heme, and cobalt protoporphyrin (CoPP)] on HO-1 mRNA content and enzyme activity in cultures of rabbit corneal epithelial (RCE) cells; these metal compounds are known to induce HO in other tissues. Additionally, we studied HO-1 expression in an experimental model of ocular inflammation produced in rabbit corneas by extended contact lens wear, and the relation of HO expression to the induced inflammatory process. SnCl2 added to RCE cells in vitro produced marked time- and concentration-dependent increases in HO-1 mRNA and HO-1 enzyme activity; CoCl2, ZnCl2, and CoPP were inducers of HO as well, though to a lesser degree than SnCl2. Corneas treated for 6 days with contact lenses impregnated with SnCl2 displayed substantially less corneal inflammation, swelling, and new vessel invasion than did controls; attenuation of ocular inflammation was paralleled by SnCl2-induced increases in HO mRNA and HO activity in corneal epithelial cells from treated eyes. It is suggested that amelioration of the inflammatory response produced by extended contact lens wear is due, in part, to the induction of high levels of HO-1 activity by SnCl2, which results in diminished production of pro-inflammatory mediators generated through heme-dependent metabolic processes. Regulation of HO activity in this manner may have clinical applications.

Diminished heme oxygenase-1 mRNA expression in RPE cells from diabetic donors as quantitated by competitive RT/PCR.

PURPOSE: The RPE is essential in the maintenance of retinal vasculature homeostasis, since increased cellular expression of heme oxygenase-1 (HO-1) has been implicated as a defense mechanism against oxidative stress. This study was done in an effort to determine the levels of the stress protein (32 kD), HO-1, in retinal pigment epithelium (RPE) cells obtained from diabetic and normal eyes. METHODS: We measured the levels of HO-1 in the RPE from eight normal and six diabetic donor eyes. For comparison, HO-1 levels were assayed in RPE cells from four donor eyes with long-standing hypertension. Heme oxygenase-1 mRNA copy number was determined by competitive RT/PCR on various ex vivo samples and on RPE cultured from the same donors. Total RNA (1-200 ng) was reverse-transcribed and then amplified by PCR in the same tube as an internal standard obtained by deleting a 50 bp restriction site from the native HO-1 gene. RESULTS: Relative to the RPE obtained from control donor eyes, RPE from diabetic donors exhibited significantly decreased levels of HO-1 mRNA. In contrast, no significant difference in the levels of HO-1 mRNA was observed in RPE samples derived from hypertensive donor eyes. The diabetic group showed a range of 340-450 HO-1 mRNA copies/ng of total RNA, as compared to 425-8,000 HO-1 mRNA copies/ng of total RNA in RPE from normal donors and 460-7605 copies/ ng in hypertensive donor eyes. CONCLUSIONS: This study represents initial studies exploring the quantitative expression of heme oxygenase in the RPE human eyes. The decreased expression of HO-1, a stress/heat shock protein, may in the RPE contribute to the vulnerability of the neuroretina to significant metabolic alterations encountered in the diabetic state. This was a limited study; additional screening from different donor eyes will be done in order to establish the relationship between RPE, HO-1 expression and eye diseases in which oxidative stress is believed to be a determinant in the pathophysiology.

Hepatosplenic cat-scratch disease and abdominal pain.

BACKGROUND: There have been several recent reports that cat-scratch disease (CSD) causes a multiplicity of atypical clinical syndromes. We recently diagnosed hepatosplenic CSD in a child who was seen with fever and abdominal pain. We report this case and 10 other patients with hepatosplenic CSD and highlight the importance of abdominal pain in this clinical entity. METHODS: This was a retrospective review of charts of patients with a diagnosis of cat-scratch disease at Egleston Children's Hospital between January, 1985, and June, 1996. From these cases patients with hepatosplenic CSD were selected for study. RESULTS: Seven children (64%) had significant abdominal pain, and in three children abdominal pain was their chief complaint. All children in the study had pathologic evidence of CSD or elevated titers of antibodies to Bartonella henselae. Ultrasound examination showed that all children had microabscesses in the spleen, and eight had abscesses in the liver. CONCLUSIONS: One of the most remarkable findings in this large series of cases of hepatosplenic CSD was that 64% of the patients complained of abdominal pain. All children in this study received antibiotics. It was our clinical impression that once antibiotics had been started, the patients appeared to improve very quickly. With an increased index of suspicion, the use of B. henselae serology and an abdominal ultrasound examination, the diagnosis of this underrecognized disease might be more readily made.

Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues.

PURPOSE: Heme oxygenase-1 (HO-1) is a stress protein induced up to 100-fold within a few hours after exposure to oxidative stress, and it has been shown to counteract oxidative injury induced by ultraviolet light or free radicals. The current study was undertaken to determine whether the HO-1 gene can be introduced into adult rabbit ocular tissues by microinjection of a recombinant replication-deficient adenovirus human HO-1 cDNA (Adv-HHO). METHODS: Human HO-1 gene was used for transfection studies to differentiate endogenous from transfected HO. The purified Adv-HHO construct (10(8) pfu/ml) was mixed with lipofectamine and microinjected into the anterior chamber, vitreous cavity, and subretinal space of New Zealand rabbit eyes. After 2 weeks, total RNA was extracted from different ocular tissues, reverse transcription-polymerase chain reaction was performed using specific human HO-1 primers, and amplification products were subjected to Southern hybridization. RESULTS: Transfection with the Adv-HHO construct into rabbit corneal epithelial cells in culture resulted in a functional expression of the human HO-1 gene; the human HO-1 mRNA was detected, and enzyme activity increased threefold. Human HO-1 mRNA was detected in the retina after microinjection of the Adv-HHO construct into the subretinal space. Microinjection into the vitreous resulted in HO-1 mRNA expression in the corneal endothelium, iris, lens, and retina; after intracameral injection of the Adv-HHO construct, human HO-1 mRNA was detected in corneal epithelium and endothelium, ciliary body, lens, and iris. Regardless of the injection site, transfected human HO-1 mRNA was undetectable in tissues outside the eye, that is, brain, liver, and kidney. CONCLUSIONS: These results demonstrated a tissue-selective functional transfer of the human HO-1 gene into rabbit ocular tissues in vivo. This technique may be a promising means for delivering HO-1 gene in vivo as a protective mechanism against oxidative stress that contributes to the pathogenesis of ocular diseases such as cataract, light-induced injury, age-related macular degeneration, and diabetic retinopathy.

A closed eye contact lens model of corneal inflammation. Part 1: Increased synthesis of cytochrome P450 arachidonic acid metabolites.

PURPOSE: To characterize a model of contact lens-induced corneal inflammation in the closed eye, with respect to inflammatory parameters and the metabolism of arachidonic acid by homogenates of the corneal epithelium. METHODS: Rabbit eyes were fitted with extended wear etafilcon A (58% water) hydrogel contact lenses in stacked fashion (two lenses per eye), followed by a silk suture tarsorrhaphy of approximately 90%. The anterior surface was analyzed over a 9-day period for inflammatory events through slit lamp biomicroscopy, subjective inflammatory scoring, corneal pachymetry, and corneal epithelial [1-(14)C]-arachidonic acid metabolism. RESULTS: Hydrogel contact lens wear in the closed eye resulted in a progressive anterior surface inflammatory response correlated over time (r = 0.999). Central corneal thickness progressively increased and was also correlated to the inflammatory score (r = 0.995). [1-(14)C]-arachidonic acid metabolism by homogenates of the corneal epithelium resulted in the time-dependent formation of two major products, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosatrienoic acid (12-HETrE). Correlations were established between the synthesis of 12-HETE and 12-HETrE, the subjective inflammatory score (r = 0.963) and the progressive increase in corneal thickness (r = 0.971), over 9 days. CONCLUSIONS: With this model of contact lens wear, eicosanoid synthesizing capacity of the corneal epithelium showed a time-dependent increase in the production of 12-HETE and 12-HETrE strongly correlating to the in situ inflammatory response. The relationship between 12-HETE and 12-HETrE synthesis and the degree of anterior surface inflammation implicate these eicosanoids, among others, as mediators of the inflammatory response to hydrogel contact lens wear in the closed eye.

A closed eye contact lens model of corneal inflammation. Part 2: Inhibition of cytochrome P450 arachidonic acid metabolism alleviates inflammatory sequelae.

PURPOSE: The authors have previously shown a marked increase in corneal epithelial arachidonic acid metabolism to 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosatrienoic acid (12-HETrE) in a model of closed eye-contact lens wear. Their formation was predominantly cytochrome P450-dependent and significantly correlated with inflammatory score and corneal thickness. In the current study, the authors used stannous chloride to inhibit the epithelial cytochrome P450-dependent synthesis of 12-HETE and 12-HETrE to assess the role of these eicosanoids as mediators of the inflammatory response to contact lens wear in the closed eye. METHODS: Hydrogel contact lenses were soaked in stannous chloride (100 micrograms/ml) or vehicle and fitted to the rabbit eye in stacked fashion (two lenses/eye), followed by a silk suture tarsorrhaphy of approximately 90%. Eyes were analyzed over a 7-day period for inflammatory responses through slit lamp biomicroscopy, subjective inflammatory scoring, ultrasonic pachymetry, and corneal epithelial [1-14C]-arachidonic acid metabolism. RESULTS: Closed eye-hydrogel contact lens wear resulted in a progressive anterior surface inflammatory response. Coinciding with these events was a time-dependent increase in corneal thickness and 12-HETE and 12-HETrE production rates by corneal epithelial homogenates. Treatment of the lenses with stannous chloride (100 micrograms/ml) significantly attenuated by day 7 the inflammatory score (56% decrease), corneal thickness (17% decrease), and 12-HETE and 12-HETrE synthesis (77% and 71% decrease, respectively). CONCLUSIONS: This study further substantiates the involvement of cytochrome P450, through the synthesis of 12-HETE and 12-HETrE, in the inflammatory response associated with hydrogel contact lens wear in the closed eye. Thus, inhibition of cytochrome P450, with subsequent decreases in 12-HETE and 12-HETrE, may attenuate the pathophysiologic response to contact lens wear in the closed eye.

Modulation of corneal heme oxygenase expression by oxidative stress agents.

Heme oxygenase, the rate-limiting enzyme in the degradation of heme to bile-pigments and carbon monoxide, is induced in response to increased oxidative stress and is believed to provide a cytoprotective effect. We investigated the role of heme oxygenase in cultured rabbit corneal epithelial cells (RCE), and its potential to alleviate oxidative stress-induced cell damage. Heme oxygenase in RCE was effectively and potently induced by most metals tested, including tin, silver, and gold, and cytokines such as IL-6, and TGF beta. Stannous chloride and heme-induced heme oxygenase mRNA by 40 and 100 fold within 1-3 hours and increased enzyme activity by 9.2- and 10-fold, respectively, over a 24 hour period. IL-6, TGF beta and H2O2 induced heme oxygenase by 2-3 fold. Zinc protoporphyrins were effective inhibitors of heme oxygenase activity in vitro. However, when incubated with cells for 24 h they induced heme oxygenase mRNA but decreased or had no effect on its activity. Administration of heme, SnCl2, and H2O2 resulted in some degree of glutathione perturbation (GSH/GSSG). However, in all cases, depletion of glutathione was exacerbated if heme oxygenase was simultaneously inhibited. Conversely, perturbation of glutathione levels was minimized if heme oxygenase was induced by heme or stannous chloride. These results demonstrate that RCE cells exhibit functional heme oxygenase activity which is inducible in response to inflammatory cytokines and oxidative stress agents and suggest a cytoprotective role for heme oxygenase against cell injury.

Effect of metabolic inhibitors on arachidonic acid metabolism in the corneal epithelium: evidence for cytochrome P450-mediated reactions.

The corneal epithelium of several species, has the capacity to metabolize arachidonic acid (arachidonic acid) via an NADPH-dependent cytochrome P450 mechanism. The major metabolites are 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), both of which exist in stereoisomeric configurations. However, the R enantiomers are predominantly produced by this enzyme system and exhibit potent biological activities. 12(R)-HETE inhibits Na-K-ATPase, increases corneal thickness and reduces intraocular pressure. 12(R)-HETrE causes vasodilation, neutrophil chemoattraction and angiogenesis. The formation of these metabolites is unaffected by cyclooxygenase and lipoxygenase inhibitors (indomethacin, diclofenac and BW755C) but inhibited by cytochrome P450 enzyme inhibitors such as carbon monoxide, SKF-525A and clotrimazole. The capacity of the normal corneal epithelium to metabolize arachidonic acid via cytochrome P450 is very low although under certain conditions this enzymatic pathway may become greatly induced. Corneal epithelial hypoxia in response to contact lens wear results in the time-dependent formation of NADPH-cytochrome P450-dependent arachidonate metabolites, 12(R)-HETE and 12(R)-HETrE. Under this condition, metabolite production correlates strongly with the in situ inflammatory response and inhibition of their formation significantly attenuates inflammation. It is evident that the cytochrome P450 arachidonate metabolites should be added to the realm of cyclooxygenase and lipoxygenase-derived eicosanoids as possible inflammatory mediators. Therefore, studies to evaluate eicosanoid involvement in inflammation should examine inhibitors of this pathway in addition to the classically studied non-steroidal antiinflammatory drugs (NSAIDs).

Induction of corneal epithelial cytochrome P-450 arachidonate metabolism by contact lens wear.

Two biologically active cytochrome P-450 arachidonate metabolites previously were characterized: 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(R)-HETE) and 12(R)-hydroxy-5,8,14-eicosatrienoic acid (12(R)-DH-HETE), which are endogenously formed in the corneal epithelium. The functional activity of these novel metabolites mimics changes observed in hypoxic corneas. Therefore, the effect of hypoxic stress was examined on metabolite formation in rabbits fitted with polymethylmethacrylate contact lenses. Although applied lenses fit tightly to the rabbit cornea, mechanical irritation also may contribute to the ocular response. Contact lens-induced hypoxic stress stimulated endogenous formation of both 12(R)-HETE (a sodium, potassium adenosine triphosphatase inhibitor) and 12(R)-DH-HETE (a vasodilatory, chemotactic, and angiogenic factor) in a time-dependent manner. After 4 hr of contact lens wear, a 21-fold increase in endogenous 12(R)-HETE formation concomitant with an increase in corneal thickness was observed. After prolonged contact lens wear (144 hr), a 23-fold increase in endogenous 12(R)-DH-HETE formation was found, corresponding with the appearance of a marked conjunctival inflammation characterized by corneal neovascularization. The increased formation of these compounds was associated with time-dependent changes in corneal endothelial morphology. The ability of 12(R)-HETE and 12(R)-DH-HETE to mediate the clinical signs of corneal hypoxia suggest these metabolites may be potential mediators of contact lens complications that followed conditions of hypoxic stress and possibly mechanical irritation in this model.

12(R)-hydroxyeicosatrienoic acid, a potent chemotactic and angiogenic factor produced by the cornea.

Human and bovine corneal epithelial cytochrome P450 convert arachidonic acid to compound D [12(R)-hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid], a metabolite with inflammatory properties including vasodilatation and breakdown of the blood-aqueous barrier. Angiogenic properties of the endogenous compound D and the synthetic enantiomers DR and DS were examined using the corneal micropocket technique. The synthetic compound DR was as active as the endogenously formed compound D. Neovascularization of the cornea was found in all the implants containing as little as 0.5 micrograms of compound DR. In contrast, the stereoisomer DS at the same concentration (0.5 micrograms) was inactive. Since angiogenesis can be secondary to a local inflammatory response, we evaluated the effects of compound DR and its stereoisomer DS on human neutrophil chemotaxis by using a modified Boyden chamber technique. DR, but not DS, was found to be a potent chemotactic factor, exhibiting dose-dependent neutrophil chemotaxis with significant responses observed at doses as low as 10(-11) M, a concentration at which leukotriene B4 does not exhibit significant chemotactic activity. Therefore, compound D produced by the cornea may qualify as an intrinsic corneal angiogenic factor which, in association with other inflammatory mechanisms, account for the growth of new vessels in the cornea that appear in chronic inflammation or in the reparative stages of an acute process.

Hormonal stimulation of 12(R)-HETE, a cytochrome P450 arachidonic acid metabolite in the rabbit cornea.

12(R)-HETE [12(R)-hydroxy-5, 8, 10, 14 eicosatetraenoic acid] is one of the major arachidonic acid metabolites produced by microsomal cytochrome P450 of the corneal epithelium. This metabolite is a potent inhibitor of Na(+)-K(+)-ATPase activity in several tissues. We investigated endogenous production of 12(R)-HETE in the rabbit corneal epithelium. Incubation of corneal epithelial sheets (prelabeled with 14C-arachidonic acid) with arginine vasopressin resulted in the production of radioactive 12(R)-HETE suggesting its formation from endogenously labeled-arachidonic acid. The maximal response was obtained with 1 microM arginine vasopressin and represents a 15-fold increase in 12(R)-HETE formation compared with that of control tissues. Stimulation of 14C-arachidonic acid release with a detergent, digitonin, also resulted in endogenous 12(R)-HETE formation. Analysis of the incubation media following digitonin treatment of prelabeled corneal epithelial sheets revealed that 12(R)-HETE production was maximal at 20 microM digitonin, a 17-fold increase over control values. This study is the first to describe hormonal and traumatic stimulation of 12(R)-HETE formation from endogenously labeled arachidonic acid in intact corneal tissues. This study demonstrates that the formation of this Na(+)-K(+)-ATPase inhibitor can be modulated by physiological and pathophysiological regulation.

12(R)-hydroxyeicosatetraenoic acid, an endogenous corneal arachidonate metabolite, lowers intraocular pressure in rabbits.

12(R)-hydroxyeicosatetraenoic acid (12(R)HETE) is an endogenous corneal epithelial arachidonic acid metabolite formed by the cytochrome P450 system and a potent inhibitor of Na(+)-K(+)-ATPase activity. We studied the effect of topically applied 12(R)HETE, either derived endogenously from corneal epithelium or synthetically prepared, on the IOP of the rabbit eye and compared it to its stereoisomer 12(S)HETE. Topical application of 1 microgram of biologically derived 12(R)HETE to both eyes of rabbits resulted in a marked reduction in IOP: a reduction of 4-7 mmHg occurred within 30-120 min. The IOP reduction effect of a single application of 12(R)HETE was long-lasting (9 days), whereas no effect on IOP was found for the vehicle control. Using synthetic compound, we demonstrated that the effect of 12(R)HETE on IOP is dose-dependent. Single topical application of 1, 10, and 50 micrograms of 12(R)HETE caused a reduction in IOP of 4, 6, and 12 mmHg, respectively. The stereoisomer, 12(S)HETE, did not have any effect on IOP at doses up to 5 micrograms. The IOP reduction effect of 12(R)HETE was not associated with hyperemia, appearance of flare, miotic response, or increased protein concentration of the aqueous humor. This study was the first to demonstrate that an endogenous inhibitor of Na(+)-K(+)-ATPase generated by the corneal epithelium potently and specifically lowers IOP in rabbits. Further studies are needed to elucidate the mechanism by which 12(R)HETE lowers IOP.

Ocular effects of a novel cytochrome P-450-dependent arachidonic acid metabolite.

Compound D is a novel arachidonic acid metabolite formed by a cytochrome P-450-dependent mono-oxygenase in bovine corneal epithelium. The structure of compound D was recently identified as 12-hydroxy-5,8,14-eicosatrienoic acid. In the current study, we described the biological properties and the ocular effects of this compound. Compound D is a potent vasodilator, and caused a dose-dependent relaxation of the rat tail artery preconstricted with phenylephrine with an EC50 of 1.5 microM, 4-5-fold more potent than acetylcholine. Topical application of as little as 10 ng of compound D onto the rabbit cornea produced a vasodilation of the conjunctival blood vessels. Intracameral injection of compound D (1-10 ng) caused a dose-dependent increase of up to 30-fold of the aqueous humor protein, indicating a breakdown of the blood-aqueous barrier. Compound D was also found to be a potent angiogenic factor, ie, it induced the appearance of new vessels in the cornea. The effects of 12-(R)-hydroxyeicosatrienoic acid (compound D) on the rabbit eye mimics the response of the eye to an inflammatory stimulus. We hypothesize, therefore, that ocular inflammation that occurs following injury to the cornea is mediated, at least in part, by the production of compound D by the corneal epithelium.