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1 From the Academic Unit of Ophthalmology, Division of Immunity and Infection, and 2 Department of Endocrinology, Division of Medical Sciences, University of Birmingham, United Kingdom; and 3 Mass Spectrometry Facility, Children’s Hospital Oakland Research Institute, California.
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Abstract |
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METHODS. Immunohistochemical and reverse transcription–polymerase chain reaction (RT-PCR) studies were performed on sections of human ocular tissues, surgical trabecular meshwork (TM) specimens and a ciliary nonpigmented epithelial (NPE) cell-line. Free F and E concentrations in aqueous humor were determined by gas chromatography-mass spectrometry (GC/MS). IOP was measured in eight male volunteers before and after oral ingestion of carbenoxolone (CBX), a known inhibitor of 11ß-HSD.
RESULTS. 11ß-HSD1 was expressed in the basal cells of the corneal epithelium and the NPE. 11ß-HSD2 was restricted to the corneal endothelium. RT-PCR revealed mRNA for only the glucocorticoid receptor (GR) in the TM specimens, whereas GR, mineralocorticoid receptor and 11ß-HSD1 mRNAs were all present in the NPE cell line. The demonstration of free F in excess of E (F/E 14:1) in the aqueous humor suggested predominant 11ß-HSD1 activity. Compared with baseline (14.7 ± 1.06 mm Hg, mean ± SD), the IOP decreased significantly on both the third and seventh days of CBX ingestion (12.48 ± 1.11 mm Hg, P < 0.0001 and 11.78 ± 1.50 mm Hg, P < 0.0001, respectively).
CONCLUSIONS. These results suggest that the 11ß-HSD1 isozyme may modulate steroid-regulated sodium transport across the NPE, thereby influencing IOP.
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Introduction |
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In classic target tissues, such as the kidney, colon, and salivary gland, epithelial sodium transport is regulated, in part, by corticosteroids through stimulation of both the apical epithelial sodium channel and the basolateral Na+-K+-ATPase pump.2 3 4 At a prereceptor level, the activity of 11ß-hydroxysteroid dehydrogenase (11ß-HSD), responsible for the interconversion of hormonally active cortisol (F) and inactive cortisone (E), must be considered.5 Two isozymes have been characterized6 7 : an oxo-reductase (11ß-HSD1) that regulates F exposure to the glucocorticoid receptor (GR) at several sites, including liver8 and adipose tissue,9 and a dehydrogenase (11ß-HSD2) that protects the mineralocorticoid receptor (MR) from F by inactivating it to E.10 11 Deficiency of 11ß-HSD2 in the inherited form of hypertension, in the syndrome of apparent mineralocorticoid excess,12 13 or after liquorice or carbenoxolone ingestion14 15 16 results in cortisol-mediated, mineralocorticoid hypertension.
The eye represents an important target tissue for corticosteroids that express both the MR17 and GR.18 Corticosteroids have been implicated in the natural diurnal variation of IOP, and increased IOP may also occur in patients with endogenous or exogenous corticosteroid excess.19 20 21 22 23 Despite this, only a few studies have addressed the role of corticosteroids in the regulation of aqueous humor secretion and reabsorption. This may be of considerable relevance due to the widespread use of topical and systemic glucocorticoids in a variety of conditions in clinical ophthalmology, where one of the most important complications is corticosteroid-induced glaucoma. This condition is characterized by increased IOP secondary to increased outflow resistance19 20 21 22 23 and resembles the more common primary open-angle glaucoma (POAG), a leading cause of blindness due to irreversible optic neuropathy associated with uncontrolled IOP. Patients with POAG are at a higher risk of development of corticosteroid-induced glaucoma, and the two conditions appear to be linked by a common genetic defect in the trabecular meshwork-induced glucocorticoid response (TIGR)/myocilin gene, which is located on the long arm of chromosome 1 and mediates outflow resistance by the deposition of an extracellular protein.24 Other ocular complications associated with corticosteroid excess include posterior subcapsular lens opacities and florid ulceration of the cornea after injudicious use of topical steroids for the treatment of herpes simplex keratitis.
Our hypothesis was that corticosteroid regulation of aqueous humor production and drainage would be mediated at a prereceptor level through 11ß-HSD expression within ocular tissues. Furthermore, because of the established role of corticosteroids in other ocular tissues, we conducted a detailed analysis of the 11ß-HSD isozyme expression within the human eye.
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Methods |
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Immunoperoxidase and immunofluorescence studies were performed using antisera raised in sheep against human 11ß-HSD1 (amino acids 18-33) and 11ß-HSD2 (amino acids 137-160 and 334-358), as previously reported.25 26 Antibody dilutions were 1:200 for 11ß-HSD1 and 1:100 for 11ß-HSD2. Control sections included the omission of primary antibody and use of antibody pretreated with the immunizing peptides. Secondary antibodies comprised donkey anti-sheep peroxidase conjugate (1:200) or donkey anti-sheep alkaline phosphatase conjugate (1:400; Binding Site, Birmingham, UK). Sections were developed with the peroxidase substrate 3,3'-diaminobenzidine, or an alkaline phosphatase substrate containing the alkaline phosphatase blocking agent levamisole (Vector Red; Vector Laboratories, Peterborough, UK).
Reverse Transcription–Polymerase Chain Reaction
TM specimens with associated drainage angle tissue, were
obtained after ethics committee approval, from eight patients
undergoing glaucoma filtration surgery. Specimens were snap frozen in
liquid nitrogen and then stored at -70°C until further analysis. The
presence of TM and the canal of Schlemm was confirmed histologically on
formalin-fixed tissue fragments. Human ciliary NPE cells
(ODM-2), were cultured as previously described.27
RNA was prepared from tissue and cultured cells using a single-step
extraction method (RNAzol B RNA isolation kit; AMS Biotechnology, Oxon,
UK) according to the manufacturer’s protocol.
Reverse transcription of RNA was performed using a commercial reverse transcription system (Promega, Southampton, UK). Briefly, 1 µg total RNA and 0.75 µg random hexamers were preannealed by incubation at 70°C for 5 minutes. Primer extension was then performed at 37°C for 60 minutes after the addition of reaction buffer, 1 mM of each dNTP, 80 U rRNasin RNase inhibitor, and 50 U avian myeloblastosis virus (AMV) reverse transcriptase. An aliquot of this reaction was taken for subsequent PCR reactions, by using a previously published method and primer pairs for 11ß-HSD1, 11ß-HSD2, GR, and MR,28 29 and transcript sizes were generated of 571, 477, 693, and 450 bp, respectively. Human hepatocyte cDNA was used as a positive control for 11ß-HSD1 and GR, whereas human placental cDNA was used for 11ß-HSD2 and MR. 18S ribosomal RT-PCR was performed to confirm the presence and integrity of RNA in all samples.
Aqueous Humor Cortisol and Cortisone Assays
Aqueous humor F and E concentrations were determined by gas
chromatography-mass spectrometry (GC/MS) by pooling aqueous humor
specimens from 16 patients who had given informed consent (seven males,
nine females; age 72.8 ± 12.8 years) undergoing routine
phakoemulsification of cataract at the Birmingham and Midland Eye
Centre. Patients with a history of glaucoma, diabetes, uveitis, or
underlying endocrine disease and those on topical or systemic
corticosteroids were excluded. GC/MS was performed, using an adaptation
of a previously reported method.30
Samples were pooled to
provide a total volume of 1.15 ml, diluted to 4-ml and 1-ml portions
(25%), for analysis. Isotope-labeled internal standards (24 ng
2H2-cortisone and 32 ng
2H4-cortisol) were added to
each 1-ml aliquot, diluted to 4 ml with water, and extracted by a
solid-phase extraction cartridge (Sep-Pak; Waters, Milford, MA). The
steroid extract was derivatized to make methyloxime-trimethylsilyl
ethers, which were then analyzed by selected-ion–monitoring GC/MS.
The ions—mass-to-charge ratio (m/z) 531 cortisone, m/z 533-labeled
cortisone, m/z 605 cortisol, and m/z 609-labeled cortisol—were
monitored, and the E and F concentrations determined from the 531:533
and 605:609 peak area ratios.
Clinical Study
A pilot observational clinical study was performed by recruiting
eight healthy male volunteers (age 21.5 ± 1.3 years) who were not
receiving any systemic or topical medications and had no family history
of glaucoma. The study protocol followed the tenets of the Declaration
of Helsinki and was approved by the local ethics committee. Informed
consent was obtained from all volunteers. Baseline IOP readings were
measured by a single observer using the same Goldmann applanation
tonometer at 8 AM and 12, 4, and 8 PM on two consecutive days. Using
this method, intraindividual variability in IOP was less than 0.5% at
any given time point. Systolic and diastolic blood pressures, recorded
with an automated digital blood pressure monitor, (HEM-705CP; Omron
Healthcare, Inc., Vernon Hills, IL) were measured at each time point.
Urine was collected for cortisol (tetrahydrocortisol [THF], alloTHF,
urinary free F [UFF]) and cortisone (tetrahydrocortisone [THE],
urinary free E [UFE]) metabolites.
The UFF-to-UFE ratio was used as an index of renal 11ß-HSD2 activity, and the THF+alloTHF-to-THE ratio as an index of global 11ß-HSD activity (i.e., 11ß-HSD1 and -2), as previously validated by our group.30 Subjects then received carbenoxolone (CBX; 100 mg) treatment three times a day for seven consecutive days. IOP measurements and blood pressure recordings were repeated at each time point on the third and seventh days of CBX ingestion. A further 24-hour urine collection was performed on the last day of CBX ingestion. Statistical analysis was performed by computer (Minitab 13.1 for Windows; University Park, PA). A combination of multiple linear regression and balanced analysis of variance was used to analyze IOP, and a paired t-test was used to evaluate the urinary steroid metabolites before and after CBX treatment. The association between changes in IOP and urinary steroid metabolites was assessed by linear regression and Spearman rank correlation.
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Results |
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Clinical Study
There was no significant difference in measured IOP between either
eye of each subject before or after the ingestion of CBX. The mean IOPs
measured at baseline on days 1 and 2 were similar at 15.05 ± 1.19
and 14.31 ± 1.04 mm Hg, respectively. Compared with mean daily
baseline levels on days 1 or 2, IOP was lower on the third (12.48 ± 1.11 mm Hg, P < 0.001) and seventh (11.78 ±
1.50 mm Hg, P < 0.001) days of CBX ingestion (Fig. 4)
. The difference between days 3 and 7 were not significant
(P = 0.14). There was a small reduction in IOP during
the course of the day (i.e., from 8 AM to 8 PM; baseline reduction of
0.38 ± 0.58 mm Hg [2.55%], P = 0.30), which
became more marked on days 3 and 7 of CBX ingestion (reduction of
0.87 ± 0.74 mm Hg [6.5%], P = 0.01 and
1.69 ± 1.73 mm Hg [13.3%], P = 0.03,
respectively).
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The UFF-to-UFE ratio increased significantly after CBX administration (0.50 ± 0.19 vs. 1.14 ± 0.38, P < 0.01) indicating inhibition of 11ß-HSD2. Despite this, the urinary THF+alloTHF-to-THE ratio decreased significantly (0.92 ± 0.23 vs. 0.70 ± 0.19, P = 0.001), reflecting concomitant inhibition of 11ß-HSD1 activity. There was a significant positive correlation between the reductions in IOP and urinary THF+alloTHF-to-THE ratio (r = 0.83, P = 0.01), but no correlation was seen with the UFF-to-UFE ratio.
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Discussion |
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Much more is known about endogenous or exogenous corticosteroids and their effect on reducing aqueous outflow.36 37 This occurs in approximately 30% of patients taking glucocorticoids, increasing to more than 90% in patients with established POAG.21 38 This ocular hypertensive effect of corticosteroids is thought to have a hereditary component and may be a marker for the subsequent development of glaucoma.38 39 Susceptible individuals may have an increase in IOP within a few hours or as long as months to years after the administration of corticosteroids. Both the acute and chronic forms of corticosteroid-induced glaucoma appear to respond to the cessation of corticosteroid therapy. The underlying pathogenesis is unclear but is thought to be mediated by deposition of an extracellular protein in the trabecular meshwork that is likely to be the product of the TIGR gene, also known as myocilin.24 37
Our study demonstrated the expression of predominantly 11ß-HSD1 within human ocular tissue, principally the NPE and the corneal epithelium. 11ß-HSD2 expression was restricted to the corneal endothelium. Although the presence of the 11ß-HSD isozymes in the corneal tissues could imply a role in stromal dehydration and the preservation of corneal transparency, the intense 11ß-HSD1 expression seen in the NPE and the ODM-2 cell line and the absence of expression of either 11ß-HSD isozyme in the TM suggest that 11ß-HSD1 may have a role in aqueous production rather than drainage. These data are supported by a recent in situ hybridization study demonstrating the expression of mRNA for 11ß-HSD1 in ciliary epithelial cells, for both MR and GR in the NPE, and for GR in the TM.40 Contrary to our findings, mRNAs for both isozymes were also demonstrated in the TM.
Our data demonstrating the exclusive presence of 11ßHSD1, detected by
both RT-PCR and immunohistochemistry of the NPE, is surprising in view
of the established autocrine role of 11ß-HSD2, but not 11ß-HSD1, in
modulating corticosteroid-regulated sodium transport within other
epithelial cells, notably kidney, colon, and salivary
gland.5
7
10
11
Nevertheless, the novel analyses of F and
E within aqueous humor samples and our clinical study appear to support
the expression of a functional 11ß-HSD1 enzyme within the NPE. In
urine and saliva, free concentrations of E exceed those of F, giving
F-to-E ratios of 0.8 and 0.2, respectively.29
30
This has
been attributed to the predominant expression of 11ß-HSD2 in the
kidney and salivary glands. Conversely, in our study aqueous humor F
concentrations exceeded those of E yielding an F-to-E ratio in excess
of 14:1. In contrast to urine and saliva, E concentrations were very
low in kidney and salivary gland, indicating functional expression of
the 11ß-HSD1 isozyme. Earlier studies provide evidence of cortisol
metabolism within both human and rabbit ocular tissues, but these data
mainly refer to the cortisol A-ring metabolism (5
/ß-reductase,
tetrahydrocortisol) and not the interconversion of F and E by
11ß-HSD.41
42
In addition, the systemic administration of CBX to healthy volunteers provided further evidence for a potential role of 11ß-HSD1 within the eye. Previous in vitro and clinical studies have demonstrated that CBX inhibits both 11ß-HSD2 and 11ß-HSD1 activities.7 15 Our data supported this concept: The increase in the UFF-to-UFE ratio was indicative of inhibition of 11ß-HSD2 activity, whereas the concomitant decrease in the THF+alloTHF-to-THE ratio indicated inhibition of 11ß-HSD1 activity. Although our clinical study was single-blind and observational, our finding of a 17.5% decrease in IOP from baseline after 3 to 7 days, suggested an inhibition of 11ß-HSD1 activity within the NPE, a reduction in local F concentrations with consequent decreased aqueous production, and a decrease in IOP (Fig. 5) .
Under normal physiological conditions, activity of 11ß-HSD1 may mediate exposure of the GR within the TM to F, which could contribute to aqueous humor outflow resistance and increased IOP. This may account for the acute and chronic changes in IOP observed in steroid-induced glaucoma and certain patients with POAG. The positive correlation between the reduction in IOP after systemic administration of CBX and decrease in the THF+alloTHF-to-THE ratio potentially supported our hypothesis. If, as we had anticipated at the initiation of this study, 11ß-HSD2 was the predominant isozyme in human ocular tissues including the NPE, then the reverse would have been observed (i.e., an increase in IOP after CBX-induced enzyme inhibition). Although the type 1 isozyme may exhibit both reductase and dehydrogenase activities, in intact cells, 11ß-HSD1 is principally a reductase. Preliminary activity data in ODM-2 cells have confirmed that this is indeed the case (data not shown), consistent with the results from our novel analysis of free F and E concentrations in aqueous humor. Nevertheless, double-blind, placebo-controlled trials incorporating aqueous humor dynamics and outflow facility studies are now required to further evaluate this observation.
We conclude that by mediating local intraocular cortisol levels, 11ß-HSD1 may have a twofold role within the human eye: first, a short-term physiological role, centered around the sodium transporting NPE and the secretion of aqueous humor, maintaining a normotensive, intraocular environment; second, a more long-term pathologic role, through interactions with the GR and TM, contributing to outflow resistance in susceptible individuals. Relative expression and activity of this isozyme, could therefore represent one of the underlying pathogenic mechanisms of POAG, one of the most common causes of visual loss in the Western world.30 The future pharmacologic manipulation of 11ß-HSD activity with topical or systemic derivatives of CBX or more selective 11ß-HSD1 inhibitors may provide a novel treatment option for patients with glaucoma.
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Acknowledgements |
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Footnotes |
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This work forms the basis of an International Patent Application (No. 9914648.2), "Glaucoma Treatment."
Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2000.
Supported by the West Midlands National Health Service Executive (Locally Organised Research Scheme) and the Medical Research Council, United Kingdom. PMS is a Medical Research Council Senior Clinical Fellow.
Submitted for publication December 18, 2000; revised March 14, 2001 accepted April 6, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U. S. C. 
1734 solely to indicate this fact.
Corresponding author: Paul M. Stewart, Division of Medical Sciences, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK. p.m.stewart{at}bham.ac.uk
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