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Immunohistochemical detection of heat shock protein 27 and Ki-67 in human pterygium
  1. NIKOLAOS PHARMAKAKIS
  1. Department of Ophthalmology, School of Medicine, University of Patras, Patras 26500, Greece
  2. Department of Anatomy, School of Medicine
  3. University of Patras
  1. MARTHA ASSIMAKOPOULOU
  1. Department of Ophthalmology, School of Medicine, University of Patras, Patras 26500, Greece
  2. Department of Anatomy, School of Medicine
  3. University of Patras
  1. Nikolaos Pharmakakis, MD, Department of Ophthalmology, School of Medicine, University of Patras, 26500 Greece npharmak{at}med.upatras.gr

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Editor,—Pterygium, a disorder of the ocular surface, consists of atrophic conjunctival epithelium and a highly vascularised hypertrophic and elastotic degenerated connective tissue. Ultraviolet irradiation is considered to be the principal environmental factor through an effect on the basal stem cells on the nasal limbus and activation of the fibroblasts.1-3 Previous studies have detected chromosomal allelic loss in slightly over 50% of pterygia and a low frequency of microsatellite instability.4 However, no differences in cellular proliferation between pterygial and normal conjunctival tissue have been detected with flow cytometry.5 On the other hand, a recent report suggests that pterygium may be the result of a failure of appropriate cellular apoptosis.6 We studied 17 pterygia and 12 normal conjunctiva tissues from the nasal conjunctiva for the expression of heat shock protein 27 (Hsp27) and cell proliferation associated nuclear antigen Ki-67.

Hsp27, a member of the small heat shock proteins family, is overexpressed in response to many environmental and pathophysiological stresses including ultraviolet radiation, hormones, growth factors, infection and anoxia, and may be important in surveillance of cell integrity acting as a “molecular chaperone.”7 Recently it has been found that Hsp27 constitutive overexpression in embryonic stem cells enhances the differentiation mediated decreased rate of cell proliferation and prevents these cells from undergoing apoptosis.8

Specimens from 17 patients (seven males and 10 females) (mean age 73.6) undergoing primary pterygium excision and 12 healthy people (three male and nine female) (mean age 77.7) undergoing cataract surgery were studied. None of these patients had an ophthalmic or systemic disease or used topical or systemic medication. Informed consent was obtained from patients participating in this study. Formalin fixed, paraffin embedded serial sections were immunostained using monoclonal antibodies against Hsp27 (clone G3.1) and Ki-67 (clone MIB-1). Morphological assessment of immunostained tissue preparations and manual cell counting of immunolabelled cells were performed. Hsp27 and Ki-67 labelled cell fractions were expressed in percentages.

Hsp27 cytoplasmic immunoreactivity was observed only in basal and suprabasal layers of normal conjunctival epithelium (mean 36.7) (Fig1A). On the other hand, strong Hsp27 immunopositivity in a high percentage of cells in all layers of epithelium was found in all pterygia examined in this study (mean 88.1) (Fig 1B, Table 1). Ki-67 immunoreactivity was confined in nuclei of scattered cells, located mostly in the basal layers of epithelium, in normal conjunctival epithelium (mean 5.2) as well as in pterygium (mean 9.4) (Table 1). No staining of Hsp27 and/or Ki-67 was observed in substantia propria in normal conjunctiva tissues and pterygia but in pterygia, Hsp27 strong immunoreactivity was observed in endothelial cells and smooth muscle cells of vessels. There was a statistically significant difference of Hsp27 immunoreactivity between normal conjunctival epithelium and pterygia (p<0.001) but no difference in Ki-67 immunoreactivity (p=0.1) although some pterygia contained large number of proliferative cells. There was no correlation between Hsp27 and Ki-67 labelling percentage in pterygia (p=0.7) and normal conjunctiva (p=0.9).

Figure 1

(A) Normal conjunctiva: Hsp27 cytoplasmic immunoreactivity in basal and subrabasal layers of epithelium (×400). (B) Pterygium: Hsp27 positive cells in all layers of epithelium. Cells of subepithelial connective tissue are Hsp27 negative while vessels are Hsp27 positive (arrows) (×400).

Table 1

Immunohistochemical staining of epithelial cells for Hsp27 and Ki-67 in pterygia and normal conjunctiva

Our findings concerning Ki-67 expression are consistent with previous results suggesting that pterygium may not be a disorder of cell proliferation.5 Overexpression of Hsp27 in all pterygia is interesting because this protein is known to be related to differentiation when expressed in other epithelial tissues—for example, skin,9 and in view of the recent report that Hsp27 transient expression seems essential for preventing embryonic stem cells from undergoing apoptosis.8 Furthermore, Tanet al 6 recently proposed that pterygium may be related to faulty apoptosis. The role of Hsp27 in pterygium remains to be elucidated since Hsp27 is expressed in basal epithelial cells of normal conjunctiva, where the cells are mainly differentiating stem cells and in all layers of epithelium in pterygia. Further studies would provide valuable information regarding the possible role of Hsp27, and the involvement of heat shock proteins generally in the pathogenesis of pterygium.

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