Abstract
Abstract: :
Purpose: To determine the role of the adenosine system, which has been shown to be involved in the regulation of IOP, in eyes with pseudoexfoliation (PEX) syndrome and glaucoma. Methods: Differential gene expression analysis of anterior segment tissues from 3 eyes with PEX and 3 age–matched control eyes was performed by subtractive hybridization and differential screening as well as by cDNA macroarrays. Candidate genes were verified by Northern Blot analysis, RT–PCR, and in situ hybridization using 10 eyes with PEX syndrome, 10 eyes with PEX glaucoma, 8 eyes with primary open–angle glaucoma (POAG), 10 eyes with angle–closure glaucoma (ACG), and 10 normal donor eyes. Light and electron microscopic immunohistochemistry was performed using polyclonal antibodies against the adenosine receptor (AR) subtypes A1, A2a, A2b, and A3. Adenosine levels were measured in the aqueous humor of 20 patients with PEX syndrome, PEX glaucoma, POAG, and cataract by HPLC. Results: Both subtractive hybridization and macroarray analysis demonstrated overexpression of A3–AR mRNA in the ciliary processes of PEX eyes up to 40–fold. Northern Blotting and RT–PCR confirmed about 10–fold upregulation of A3–AR mRNA in all PEX eyes as compared to normal and glaucomatous control eyes, and in situ hybridization demonstrated increased signals for A3–AR mRNA particularly in the nonpigmented ciliary epithelium of PEX eyes. In contrast, the expression of A1–AR, A2a–AR, and A2b–AR was not significantly altered in PEX tissues. By immunohistochemistry, all AR subtypes could be immunolocalized to the ciliary epithelium, the trabecular meshwork, and other anterior segment tissues in a subtype–specific pattern in all eyes examined. However, only the A3–AR showed increased immunoreactivity in the iris vessels, the trabecular meshwork, and the nonpigmented ciliary epithelium, particularly at the tips of the ciliary processes, in PEX eyes as compared to normal and glaucomatous control eyes. The A3–AR was localized to the basolateral infoldings of the nonpigmented epithelial cells by immunoelectron microscopy. Mean aqueous adenosine levels were significantly increased in patients with PEX syndrome and PEX glaucoma as compared to cataract and POAG patients. Conclusions: Considering the known role of the A3–AR in modulation of aqueous humor secretion and regulation of IOP, its selective upregulation together with increased adenosine release may play an important role in glaucoma development in PEX patients. A3–AR antagonists may be useful in treating ocular hypertension in PEX patients.
Keywords: adenosine • receptors • candidate gene analysis