Abstract
purpose. Adenosine is increasingly released in metabolic stress conditions, like hypoxia or ischemia, and regulates many physiologic processes, such as aqueous humor secretion and intraocular pressure, via activation of four adenosine receptors. In the current study, the role of the adenosine system in the pathophysiology of pseudoexfoliation (PEX) syndrome, which is typically associated with anterior chamber hypoxia and elevated intraocular pressure, was examined.
methods. RT-PCR, Northern hybridization, in situ hybridization, and immunohistochemistry were applied to analyze the mRNA and protein expression of the adenosine receptor subtypes A1, A2A, A2B, and A3 in anterior segment tissues of PEX eyes, without and with glaucoma, in comparison to eyes with primary open-angle or angle-closure glaucoma and normal control eyes. Real-time PCR was used to study the effect of hypoxia and oxidative stress on adenosine receptor expression by nonpigmented ciliary epithelial cells in vitro. Levels of adenosine and its catabolites inosine, hypoxanthine, and xanthine were measured in cell culture supernatants and aqueous humor samples by HPLC.
results. All four adenosine receptor subtypes (A2A > A1 > A2B > A3) were coexpressed but differently distributed in the ciliary epithelium of control eyes, with the A3 receptor being localized to the basolateral membrane infoldings of the nonpigmented epithelial cells. A selective, approximately 10-fold upregulation of A3 receptor mRNA and protein was consistently found in the nonpigmented ciliary epithelium of all PEX eyes, with and without glaucoma, compared with the normal and glaucomatous control eyes. Significant upregulation of A3 receptor message in nonpigmented epithelial cells was induced by both hypoxia and oxidative stress in vitro, together with increased levels of inosine, hypoxanthine, and xanthine in the supernatants. Levels of adenosine and its catabolites, however, were not significantly elevated in the aqueous humor of patients with PEX.
conclusions. Considering the known role of the A3 adenosine receptor in modulating aqueous humor secretion, its selective, probably hypoxia-induced upregulation in the ciliary epithelium may not only confer cytoprotection but also influence aqueous humor dynamics and may be accessible to therapeutic intervention in patients with PEX.
The purine nucleoside adenosine is present in all tissues and body fluids and is known to function as a modulator of a variety of physiological processes, such as the regulation of cellular growth and differentiation, vasodilation and blood flow, inflammatory responses, central and peripheral neural function, neuroprotection, and apoptosis.
1 Adenosine levels in tissues change with activity and energy demand. In metabolic stress conditions, such as hypoxia or ischemia, the concentrations of adenosine and its metabolites inosine, hypoxanthine, and xanthine in the extracellular fluid increase dramatically, mainly through breakdown of adenosine triphosphate (ATP).
2 3 4 One of the primary roles of adenosine is cytoprotection against ischemia-induced cell damage, mainly in tissues such as the heart, brain, and kidney, which are especially prone to ischemic injury.
5 6 The cytoprotective role of adenosine is thought to be mediated by vasodilation, reduction of oxygen demand, suppression of formation of reactive oxygen species, increase in glucose uptake, decrease in the release of excitatory neurotransmitters, and inhibition of calcium influx.
4
These protective effects are mediated by activation of four pharmacologically and biochemically distinct adenosine receptors—A1, A2A, A2B, and A3—which belong to the family of G-protein-coupled receptors with different intracellular signaling pathways.
1 7 The A1 and A3 receptor subtypes couple to G
i-proteins, mediating the inhibition of adenylyl cyclase and a decrease in cAMP levels, whereas the A2A and A2B receptors activate adenylyl cyclase and increase cAMP levels via the stimulatory G
s-proteins.
Molecular and pharmacological studies have provided evidence that all adenosine receptor subtypes are expressed in ocular tissues.
8 9 10 11 12 Activation of these receptors has been shown to regulate retinal neurotransmission and neuroprotection,
8 13 retinal and choroidal blood flow,
14 15 16 17 18 19 photoreceptor phagocytosis,
20 and integrity of the blood–retinal barrier.
21 The adenosine system has also been shown to regulate ion transport in the corneal
22 and the ciliary epithelia
23 24 and to modulate aqueous humor in- and outflow.
25 26 27 Recent studies in animal models have demonstrated that adenosine receptor agonists and antagonists can alter intraocular pressure (IOP) in vivo,
28 29 30 confirming that adenosine receptors play an important physiological role in IOP modulation. In ocular hypertensive individuals, the mean aqueous adenosine levels were significantly elevated when compared to normotensive subjects and correlated with IOP levels.
31 However, in eyes of healthy subjects, parenteral infusion of adenosine induced a small but significant decrease in IOP.
19
Apart from an involvement in IOP modulation, adenosine and its receptors have been implicated in many ocular and systemic ischemic diseases (e.g., retinal ischemia) and in conditions associated with oxidative stress.
2 18 32 Ischemia and anterior chamber hypoxia are well-recognized features of eyes with pseudoexfoliation (PEX) syndrome, a common age-related extracellular matrix disorder that often leads to the development of ocular hypertension and secondary open-angle glaucoma.
33 Marked alterations of the iris vasculature leading to a progressive obliterative vasculopathy have been well documented clinically, angiographically, and morphologically and account for significantly reduced oxygen partial pressure in the anterior chamber of PEX eyes.
34 In addition, increased concentrations of oxidative stress markers, such as 8-isoprostaglandin-F2α, have been measured in the aqueous humor of PEX eyes.
35
An involvement of the adenosine system in ischemia/hypoxia and IOP elevation in PEX eyes may therefore be hypothesized. This hypothesis has been substantiated by previous findings, in differential gene expression analyses, of a more than 30-fold overexpression of A3 adenosine receptor mRNA in the ciliary processes of PEX eyes compared with control eyes (Schlötzer-Schrehardt U, et al. IOVS 2004;45:ARVO E-Abstract 3535). We therefore further investigated the role of the adenosine system in eyes with PEX syndrome, without and with glaucoma, in comparison to eyes with primary open-angle (POAG) or angle-closure (ACG) glaucoma and normal donor eyes. In particular, we analyzed the expression of adenosine receptors in anterior segment tissues on the mRNA and protein level and measured the concentration of adenosine and its catabolites inosine, hypoxanthine, and xanthine in aqueous humor samples. In addition, we studied the effects of hypoxia and oxidative stress on adenosine synthesis and adenosine receptor expression by ciliary epithelial cells in vitro. The findings provided evidence of a selective upregulation of the A3 adenosine receptor in the nonpigmented ciliary epithelium of all PEX eyes, independent of the presence of glaucoma, that was also induced by hypoxia or oxidative stress in vitro.
Anterior segment tissues from 10 donor eyes with PEX syndrome (75.1 ± 7.9 years), 8 donor eyes with a history of POAG (78.8 ± 13.7 years), and 10 normal-appearing donor eyes (76.5 ± 6.2 years) were obtained at autopsy and fixed or processed within 10 hours after death. In addition, two pairs of donor eyes (79 and 81 years of age) with clinically and macroscopically unilateral PEX syndrome were used. The diagnosis of PEX syndrome was established by macroscopic observation of the presence of characteristic PEX deposits on ciliary processes, zonules, and lens and iris surfaces and confirmed by electron microscopy. Optic nerve cross sections were cut to exclude the presence of glaucomatous optic nerve atrophy. The diagnosis of POAG was taken from the patients’ files, and glaucomatous optic atrophy was confirmed by optic nerve cross sections. The normal donor eyes had no history or morphologic evidence of any known ocular disease.
We further included 5 eyes with PEX-associated open-angle glaucoma (80.2 ± 7.6 years), 5 eyes with PEX-associated closed-angle glaucoma (79.7 ± 5.1 years), 3 eyes with POAG (81.3 ± 1.5 years), and 10 eyes with secondary ACG due to rubeosis iridis (77.3 ± 9.8 years), all of which had to be surgically enucleated because of painful absolute glaucoma or associated malignant melanoma of the posterior choroid. These eyes were fixed or processed immediately after enucleation for optimal preservation of RNA.
Aqueous humor was aspirated intraoperatively from eyes of 10 patients with PEX syndrome without glaucoma (mean age, 79.8 ± 7.9 years), 10 patients with PEX glaucoma (mean age, 76.2 ± 8.4 years), 10 patients with POAG (mean age 75.4 ± 7.5 years), and 10 patients with cataract (mean age, 78.8 ± 8.0 years) during cataract or filtration surgery. One hundred microliters of aqueous humor were withdrawn through an abexterno limbal paracentesis site with a 27-gauge needle on a tuberculin syringe. The samples were immediately frozen in liquid nitrogen and stored at −80°C for up to 3 months. Patients with ophthalmic diseases other than glaucoma or cataract or with previous surgery were excluded from the study.
Informed consent to tissue and aqueous humor donation was obtained from the patients or, in case of donor eyes obtained at autopsy, from the donors’ relatives. The protocol of the study was approved by the local Ethics Committee and adhered to the tenets of the Declaration of Helsinki for experiments involving human tissue and samples.
Total RNA was isolated from ciliary processes, iris tissue, lens epithelium, and trabecular meshwork specimens obtained from normal donor eyes or donor eyes with PEX syndrome and from surgically enucleated eyes with PEX-associated OAG or ACG or with POAG or ACG. RNA extraction was then performed (RNeasy kit; Qiagen, Hilden, Germany) and included an on-column DNase I digestion step, according to the manufacturer’s instructions.
First-strand cDNA synthesis was performed with 1 μg of total RNA, 200 U reverse transcriptase (Superscript II; Invitrogen, Karlsruhe, Germany), and 500 ng oligo dT primers (Roche Diagnostics, Mannheim, Germany) in a 20-μL reaction volume. Gene-specific primers (MWG Biotech, Anzing, Germany) were designed to anneal with sequences located in different exons by means of Primer 3 software
36 (Table 1) . The identity of PCR fragments was subsequently confirmed by sequence analysis with a sequence analyzer (Prism 3100; Applied Biosystems [ABI], Foster City, CA).
For semiquantitative RT-PCR, normalization of cDNA samples from different specimens was performed in 25-μL PCR reaction volumes with primers for glycerinealdehyde-3-phosphate dehydrogenase (GAPDH) and dilutions of the first-strand products. Dilutions resulting in the same band intensities were used for analytic amplifications. Amplification of each gene was performed at the exponential phase in 25-μL reaction volumes containing 0.5 μL of the normalized first-strand reaction product, 0.2 μM of 5′ and 3′ primers, 200 μM of each dNTP, 0.65 U Taq DNA polymerase (HotStar; Qiagen), in a program with an initial denaturation step of 95°C for 15 minutes, and 35 to 45 cycles of 95°C for 15 seconds, 56°C (58°C and 60°C, respectively) for 30 seconds, and 72°C for 1 minute. PCR products were analyzed in 1.2% agarose gels containing 250 ng/mL ethidium bromide. Images were captured, and band intensities were quantitated by densitometry (EagleEye II system; Stratagene, La Jolla, CA).
Quantitative real-time PCR was performed with a thermal cycler (iCycler IQ Thermal Cycler; Bio-Rad, Munich, Germany). A typical PCR reaction (25 μL) contained 0.5 μL of first-strand product (corresponding to 25 ng of total RNA), 0.4 μM of 5′ and 3′ primers, 3.5 mM MgCl2 (GAPDH, A3 adenosine receptor) or 4 mM MgCl2 (A1, A2A, and A2B receptors), respectively, and PCR mix (IQ SYBR Green Supermix; Bio-Rad) according to the manufacturer’s instructions. All samples were analyzed in triplicate, in a program with an initial denaturation step of 95°C for 3 minutes and 40 cycles of 95°C for 30 seconds, 65°C for 30 seconds, and 72°C for 30 seconds. For quantification, standard curves using serial dilutions (101 to 107 copies) of plasmid-cloned specific amplification products were run in parallel, and amplification specificity was checked by using melting-curve analysis. For standardization of levels of gene expression, mRNA ratios relative to the housekeeping gene GAPDH were calculated.
Five micrograms of total RNA was separated by denaturing agarose/formaldehyde gel electrophoresis, transferred to nylon membranes (ZetaProbeGT; Bio-Rad), and fixed by UV cross-linking. The membranes were prehybridized for 6 hours at 42°C in hybridization buffer containing 33% formamide, 5× SSC, 5× Denhardt’s solution, 0.5% SDS, 100 μg/mL of denatured salmon sperm DNA, and 100 mM potassium phosphate (pH 6.5). The hybridization probe against the human A3 adenosine receptor was labeled with [α-32P]dCTP (Prime-It II Random-Primer labeling kit; Stratagene), according to the manufacturer’s instructions, and column-purified (Mini Quick Spin columns; Roche Diagnostics). The specificity of the probe was confirmed by sequence analysis. The membranes were hybridized with the cDNA probe at a concentration of 2 × 106 cpm/mL overnight at 42°C. After hybridization, the membranes were washed in 2× SSC and 0.5% SDS and in 0.5× SSC and 0.1% SDS. Blots were exposed on imaging plates (Fuji; Düsseldorf, Germany) and analyzed with a phosphorescence imager (BAS-2000 Phosphor-Imager; Fuji). The band intensities were quantitated on computer (Tina 2.0 software; Raytest, Straubenhardt, Germany). For normalization, blots were hybridized with a cDNA probe for the housekeeping gene GAPDH.
The cDNA probe specific for the human A3 adenosine receptor was cloned into the pCR II vector (Invitrogen). In vitro transcription of the linearized constructs was performed with T7 and Sp6 RNA polymerase (Promega, Mannheim, Germany) in the presence of digoxigenin-11-uridine triphosphate (DIG-UTP) to produce DIG-labeled single-strand antisense or sense RNA probes with the DIG-RNA labeling kit (Roche Diagnostics) used according to the instructions of the manufacturer. The quality of the transcripts was controlled by using denaturing formaldehyde-agarose gels before and after DNase I digestion.
Whole eyes, with and without PEX syndrome, were fixed in freshly prepared 4% buffered paraformaldehyde overnight and embedded in paraffin. For in situ hybridization of 6-μm-thick paraffin sections, deparaffinized sections were rehydrated, pretreated with proteinase K (20 μg/mL), postfixed, acetylated in 0.25% acetic anhydride, and hybridized for 16 hours at 44°C in chemiluminescent gold hybridization buffer (ECL; Amersham Biosciences, Freiburg, Germany) containing 0.3 M NaCl, 5% blocking reagent (Roche Diagnostics) and 1 ng/μL DIG-labeled RNA probe. After hybridization, the sections were washed at 40°C in 1× SSC for 10 minutes and in 0.3× SSC for 1 hour; treated with RNase A (5 μg/mL) and RNase T1 (25 U/mL) in 2.5 M NaCl, 50 mM Tris-HCl, and 25 mM EDTA (pH 7.5) for 30 minutes at 40°C; and washed again in 1× SSC for 10 minutes at room temperature for 2 hours at 50°C in 0.1× SSC and for 30 minutes at room temperature in 0.5× SSC. For detection of hybridized probes, slides were incubated with a sheep anti-DIG antibody conjugated to alkaline phosphatase (Roche Diagnostics) diluted 1:2000 in 0.1 M maleate buffer (pH 7.5) for 30 minutes at room temperature, and the color reaction was processed with 0.5 mM nitroblue tetrazolium chloride (NBT) and 0.5 mM 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) for 3 days at room temperature.
Hybridization with sense strand riboprobes served as the negative control and with 18S rRNA probes served as the positive control.
To determine the concentration of adenosine and its catabolic reaction products inosine, hypoxanthine, and xanthine in aqueous humor, 10 aqueous samples from each of four groups of patients (PEX syndrome, PEX glaucoma, POAG, and cataract) were analyzed. Whereas the concentration of adenosine was below the limits of detection (<200 nM) in all aqueous humor samples, inosine, hypoxanthine, and xanthine were reliably measured in all samples. The aqueous concentrations of inosine were 1.13 ± 0.45 μM; of hypoxanthine, 0.88 ± 0.83 μM; and of xanthine, 9.43 ± 2.64 μM, in all groups of patients (n = 40). Although the hypoxanthine and xanthine levels were generally higher in both PEX groups (1.13 ± 0.70 and 10.07 ± 3.14 μM, respectively; n = 20) compared with the cataract group (0.66 ± 0.26 and 8.49 ± 2.46 μM, respectively; n = 10), the differences were not statistically significant (P = 0.07).
Effects of Hypoxia and Oxidative Stress on A3 Adenosine Receptor Expression and Adenosine Secretion
Expression and Functional Significance of Adenosine Receptors in Intra- and Extraocular Tissues