Ηypoxia-inducible factor-1α, von Hippel-Lindau protein, and heat shock protein expression in ophthalmic pterygium and normal conjunctiva.
OBJECTIVE
Εnhanced expression of transcription factor hypoxia inducible factor HIF-1α 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-1α and von Hippel Lindau protein (pVHL)--which normally acts to keep levels of HIF-1α activity low under normoxic conditions--in pterygium and normal conjunctival human samples. Additionally, expression of HIF-1α compared to the activation of heat shock proteins (Hsp90, Hsp70, and Hsp27) was studied. Emphasis was placed on the detection of HIF-1α and Hsp90, which associates with and stabilizes HIF-1α 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-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-1α 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. Ιmmunoreactivity 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-1α and the activation status of Hsps (Hsp90, Hsp70, and Hsp27; p<0.05) was observed whereas HIF-1α expression did not correlate with pVHL expression (p>0.05). Double labeling immunofluorescence studies showed nuclear HIF-1α 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-1α expression and basal epithelial cells with Hsp90 expression.


CONCLUSIONS
The upregulation of coordinated activation of HIF-1α and Hsps in pterygium may represent an adaptive process for the survival of cells under stressful conditions. The significance of the association of HIF-1α with Hsp90 with respect to the therapeutic approach of pterygium requires further evaluation.
Department of Anatomy-Histology and Embryology, School of Medicine, University of Patras, Rion, Greece.
Dionysios
D
Pagoulatos
Department of Ophthalmology, School of Medicine, University of Patras, Rion, Greece.
Nikolaos
N
Pharmakakis
Department of Medical Physics, School of Medicine, University of Patras, Rion, Greece.
John
J
Lakoumentas
Department of Anatomy-Histology and Embryology, School of Medicine, University of Patras, Rion, Greece.
Martha
M
Assimakopoulou
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D003228:Conjunctiva / Q000378:metabolism* / Q000473:pathology*; D003710:Demography; D004848:Epithelium / Q000378:metabolism / Q000473:pathology; D005260:Female; D005455:Fluorescent Antibody Technique; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D015151:Immunoblotting; D008297:Male; D008875:Middle Aged; D021381:Protein Transport; D011625:Pterygium / Q000378:metabolism* / Q000473:pathology*; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000378:metabolism*
D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human