Μελέτη των μοριακών μηχανισμών ανάπτυξης του οφθαλμικού πτερυγίου στον άνθρωπο: ο ρόλος του μεταγραφικού παράγοντα HIF-1α, της πρωτεΐνης pVHL και των πρωτεϊνών θερμικού σοκ

Abstract

Enhanced expression of transcription factor hypoxia inducible factor HIF-1α isknown to play a critical role in the modulation of cell metabolism and survivalpathways as well as having stem-cell–like properties. Furthermore, accumulateddata reveal the existence of cross-regulation between the oxygen-sensing andheat shock pathways contributing to the adaptation of cells under stressfulconditions. Pterygium, a stem cell disorder with premalignant features, has beenreported to demonstrate hypoxia. The purpose of this study was to investigatethe co-expression patterns of transcription factor HIF-1α and von Hippel Lindauprotein (pVHL)—which normally acts to keep levels of HIF-1α activity low undernormoxic conditions—in pterygium and normal conjunctival human samples.Additionally, expression of HIF-1α compared to the activation of heat shockproteins (Hsp90, Hsp70, and Hsp27) was studied. Emphasis was placed on thedetection of HIF-1α and Hsp90, which associates with and stabilizes HIF-1α topromote its transcriptional activity.Semi-serial paraffin-embedded sections and tissue extracts from pterygium andnormal conjunctival samples were studied by immunohistochemistry andwestern blot analysis, respectively, with the use of specific antibodies. Doublelabeling immunofluorescence studies on cryostat sections were also included.Statistically significant increased expression of HIF-1α and Hsps (Hsp90, Hsp70,and Hsp27) in pterygia compared to normal conjunctiva was demonstrated(p<0.05). In contrast, no significant difference was detected for pVHL expression(p≥0.05). Immunohistochemical findings revealed nuclear HIF-1αimmunoreactivity in all the epithelial layers of 23/32 (71.8%) pterygium tissues.Furthermore, all epithelial layers of the majority (75%) of pterygium samplesshowed strong cytoplasmic immunoreactivity for Hsp27 while Hsp27 expressionwas detected in all pterygia (100%) examined. Hsp27 expression was notobserved in the superficial layer of goblet cells. In some samples, focal basalepithelial cells exhibited weak Hsp27 expression or were Hsp27immunonegative. Ιmmunoreactivity of phopsho-Hsp27 showed the same distribution pattern as Hsp27 did. Epithelium of all pterygia (100%) displayedmoderate to strong Hsp90 cytoplasmic immunoreactivity. Furthermore, themajority of pterygia, specifically, 30/32 (93.7%) and 27/32 (84.3%) demonstrated, respectively, Hsp70 and pVHL cytoplasmic immunoreactivity.Hsp90, Hsp70, and pVHL immunoreactivity was mainly detected in basal andsuprabasal epithelial layers even though strong immunoreactivity in all epitheliallayers was also observed in some pterygia. Stroma vessels were immunopositivefor Hsps (Hsp90, Hsp70, and Hsp27) and pVHL. A statistically significantcorrelation between the expression of HIF-1α and the activation status of Hsps(Hsp90, Hsp70, and Hsp27; p<0.05) was observed whereas HIF-1α expressiondid not correlate with pVHL expression (p≥0.05). Double labelingimmunofluorescence studies showed nuclear HIF-1α co-localization withcytoplasmic Hsp90 expression in cells distributed in the entire epithelium ofpterygia, in contrast to, normal conjunctiva, which exhibited only a few scatteredepithelial cells with cytoplasmic HIF-1α expression and basal epithelial cells withHsp90 expression.The upregulation of coordinated activation of HIF-1α and Hsps in pterygium mayrepresent an adaptive process for the survival of cells under stressful conditions. The significance of the association of HIF-1α with Hsp90 with respect to thetherapeutic approach of pterygium requires further evaluation.