Abstract
Abstract: :
Purpose:There is increasing evidence that cellular stress conditions (e.g. free radical formation and hypoxia) constitute major mechanisms involved in the pathobiology of pseudoexfoliation (PEX) syndrome. To determine whether PEX syndrome/glaucoma is associated with an altered or faulty cellular stress response, we investigated the expression of stress–related genes in PEX and control tissues. Methods:Anterior segment tissues were obtained from three eyes with PEX syndrome with or without glaucoma and three age–matched control eyes. After RNA extraction and reverse transcription, cDNA from ciliary processes, which are mainly involved in the PEX process, was amplified by means of SMART cDNA amplification. Radioactively labeled cDNA from PEX and control patients was then hybridized to the "Atlas human Stress–Array" (Clontech) and the "Hypoxia Signaling Pathway Array" (Superarray). Differentially expressed genes were identified by different–intensity hybridization signals and tested by means of RT–PCR and Northern blot analysis for further verification. To localize the differentially expressed genes or gene products in the whole anterior segment, in situ hybridizations and immunohistochemistry were performed. Results:A set of 15 stress–related genes showed consistent differential expression in ciliary processes of PEX eyes with and without glaucoma. The expression of several heat shock proteins (HSP 27, 40, 60), Mn+ superoxide dismutase, and protein phosphatase PP2A was up to 4–fold enhanced in PEX specimens. In contrast, a larger set of antioxidant defense enzymes (e.g. glutaredoxin and glutathion–S–transferases), components of the ubiquitin proteasome pathway (ubiquitin conjugating enzyme E2A and E2B), DNA repair proteins (ERCC1, hMLH1, GADD153), and apolipoprotein J were found to be downregulated in PEX specimens. The differential expression of these genes was partially confirmed by RT–PCR, Northern blot analysis, in situ hybridization, and immunohistochemistry. Conclusions:Our data provide evidence for the reduced expression of a large set of protective gene products in PEX syndrome suggesting an impaired cellular stress response in PEX eyes. The resulting enhanced cellular stress conditions leading to chronic stimuli may be implicated in the abnormal extracellular matrix metabolism characteristic of PEX syndrome.
Keywords: gene microarray • candidate gene analysis • ciliary body