Purpose: Enhanced expression of transcription factor hypoxia inducible factor HIF-1 alpha is known to play a critical role in the modulation of cell metabolism and survival pathways as well as having stem-cell-like properties. Furthermore, accumulated data reveal the existence of cross-regulation between the oxygen-sensing and heat shock pathways contributing to the adaptation of cells under stressful conditions. Pterygium, a stem cell disorder with premalignant features, has been reported to demonstrate hypoxia. The purpose of this study was to investigate the co-expression patterns of transcription factor HIF-1 alpha and von Hippel Lindau protein (pVHL)-which normally acts to keep levels of HIF-1 alpha activity low under normoxic conditions-in pterygium and normal conjunctival human samples. Additionally, expression of HIF-1 alpha compared to the activation of heat shock proteins (Hsp90, Hsp70, and Hsp27) was studied. Emphasis was placed on the detection of HIF-1 alpha and Hsp90, which associates with and stabilizes HIF-1 alpha to promote its transcriptional activity. Methods: Semi-serial paraffin-embedded sections and tissue extracts from pterygium and normal conjunctival samples were studied by immunohistochemistry and western blot analysis, respectively, with the use of specific antibodies. Double labeling immunofluorescence studies on cryostat sections were also included. Results: Statistically significant increased expression of HIF-1 alpha 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-1 alpha immunoreactivity in all the epithelial layers of 23/32 (71.8%) pterygium tissues. Furthermore, all epithelial layers of the majority (75%) of pterygium samples showed strong cytoplasmic immunoreactivity for Hsp27 while Hsp27 expression was detected in all pterygia (100%) examined. Hsp27 expression was not observed in the superficial layer of goblet cells. In some samples, focal basal epithelial cells exhibited weak Hsp27 expression or were Hsp27 immunonegative. Immunoreactivity of phopsho-Hsp27 showed the same distribution pattern as Hsp27 did. Epithelium of all pterygia (100%) displayed moderate to strong Hsp90 cytoplasmic immunoreactivity. Furthermore, the majority 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 and suprabasal epithelial layers even though strong immunoreactivity in all epithelial layers was also observed in some pterygia. Stroma vessels were immunopositive for Hsps (Hsp90, Hsp70, and Hsp27) and pVHL. A statistically significant correlation between the expression of HIF-1 alpha and the activation status of Hsps (Hsp90, Hsp70, and Hsp27; p<0.05) was observed whereas HIF-1 alpha expression did not correlate with pVHL expression (p>0.05). Double labeling immunofluorescence studies showed nuclear HIF-1 alpha co-localization with cytoplasmic Hsp90 expression in cells distributed in the entire epithelium of pterygia, in contrast to, normal conjunctiva, which exhibited only a few scattered epithelial cells with cytoplasmic HIF-1 alpha expression and basal epithelial cells with Hsp90 expression. Conclusions: The upregulation of coordinated activation of HIF-1 alpha and Hsps in pterygium may represent an adaptive process for the survival of cells under stressful conditions. The significance of the association of HIF-1 alpha with Hsp90 with respect to the therapeutic approach of pterygium requires further evaluation.
