Immunohistochemical detection of Hsp90 and Ki-67 in pterygium

Roberto Sebasti 0 Marcelo Palis Ventura 0 Helena Parente Solari 0 Emilia Antecka 1 Maria Eugenia Orellana 1 Miguel N Burnier Jr 1 0 Department of Ophthalmology, Universitary Hospital Federal Fluminense University , Rio de Janeiro , Brazil 1 Henry C. Witelson Ocular Pathology Laboratory, McGill University , Montreal , Canada Background: To examine the immunohistochemical expression of heat shock protein 90 (Hsp90) and Ki-67 protein in human pterygium. Materials and methods: Tissues obtained during pterygium surgery of 15 patients who underwent the bare-sclera procedure and 10 normal conjunctivae were studied. All of these pterygia were primary ones. Recurrent pterygia were excluded. Normal bulbar conjunctivas (2 x 2 mm) were obtained from the nasal region close to the limbus from patients during their cataract and retina surgeries. Immunohistochemical detection of Hsp90 and Ki67 was done using the streptavidin-biotin method in paraffin embedded tissue sections. Results: The percentage of cells stained for Hsp90 was greater for pterygium epithelium (76 10.8) than for normal conjunctiva (1.4 0.8). In each pterigyum sample more than 60% of cells were positive. The differences in positive cells between normal and pterigyum epithelium were highly significant for Hsp90 (P < 0,001). Pterygium epithelium also showed a higher percentage of cells that stained for Ki67 (10.1 9.5) than for normal conjunctiva (2.1 1.9). The differences in positive cells were also statistically significant for Ki67 (P < 0.01). Although there were significant differences in the majority of samples observed. It was noted that in some samples there was no difference between normal and pterygium epithelium for Ki67. Conclusion: Our results indicate an abnormal expression of Hsp90 and ki-67 in pterygium samples when compared to normal conjunctiva.The finding of abnormal expression of levels of Hsp90 in pterygium samples can stimulate new research into pterygium and its recurrence. - Background The pterygium is an ocular surface disease characterized by the centripetal growth of fibrovascular tissue associated with inflammation and vascularization. It can reach and invade the corneal surface reducing visual acuity [1]. Lacrimal distribution may be altered by the irregularities of the ocular surface causing keratoconjunctivities [2]. Epidemiological studies have shown that pterygium is strongly related to sun exposure, with little evidence that exposure during any particular period of life is more important than in other periods. The implication for prevention of pterygium is that ocular protection is beneficial at all ages, as this disease is linked to excessive ultraviolet (UV) radiation [3]. Although the major cause of pterygia has been attributed to UV-B radiation [4] many others etiological theories have been suggested [5] such as: cell cycle regulation [6,7], inflammatory mediators [8,9], immunological mechanisms [10], growth factors [11,12], angiogenic stimulation [13], extracellular matrix modulation [14,15], cholesterol metabolism modification [16], viruses [17] and hereditary factors [18]. At the moment, the treatment of pterygium is eminently surgical. There is a great recidive incidence using conventional techniques [2]. Some authors have reported the recurrence in about 50% of cases after excision [19]. A lower recurrence of pterygium is observed using conjunctival [20] or oral mucosa autograft [21,22]. The use of mitomicin drops reduces the incidence of recurrence of pterygium [23]. Since there is a high incidence of failure in the treatment of pterygium, recent studies have aimed at a better understanding of the pathophysiology of the disease in order to improve the therapy. Although the pathogenesis of pterygium is yet undetermined, many features that suggest excessive or disordered growth have been found by different authors [7,24-26]. Tumorlike histologic characteristics, ranging from mild dysplasia to carcinoma in situ and local invasiveness, have been described [24]. The Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker [27] and HSPs in the epithelia may be important in the surveillance of epithelial cell integrity and may represent a first line of defense against the transformation of epithelial cells induced by stress agents [28]. The aim of this study was to examine the expression of Hsp90 and Ki67 and correlate their presence with the pterygium etiology. Methods Pterygium tissues obtained during pterygium surgery of 15 patients who underwent the bare-sclera procedure were included in this study. All of these pterygia were primary ones. Recurrent pterygia were excluded. Patients with previous ocular surgeries or with inflammatory or infectious disease were also excluded. None of these patients had an ophthalmic or systemic disease or used topical or systemic medication. Nine were men and six were women of ages that varied between 25 and 52 years (mean age 43 years). Except for local anesthetic no medicine or other chemical agent was used during pterygium excision. All cases were treated by the same surgeon (HPS) at the Universitary Hospital Universidade Federal Fluminense. Normal bulbar conjunctivas (2 2 mm) were obtained from the nasal region close to the limbus from patients during their cataract and retina surgeries. The tissues were then fixed using 10%paraformaldehyde and set in paraffin wax. Informed consent was obtained according to the Declaration of Helsinki. Local (CEP-UFF) and national (CONEP) ethical committees had approved this study. Immunohistochemistry The material was sent to the Department of Ophthalmology and Pathology at the McGill University Health Center and Henry C.Witelson Ocular Pathology Laboratory, Montreal, Quebec, Canada. Formalin-fixed, paraffin-embedded sections of the specimens were H&E stained for histopathologic assessment by three ocular pathologists. Immunohistochemistry was done using the Ventana benchmark machine according to the protocol (Ventana Medical Systems, Inc.). The fully automated processing of bar codelabeled slides included baking of the slides, solvent-free deparaffinization, and CC1 [Tris-EDTA buffer (pH 8.0)] antigen retrieval. Slides were incubated with the monoclonal mouse anti-Hsp90 (StressGen, Victoria, BC, Canada) at a dilution of 1:50, others slides were incubated with the monoclonal mouse anti-Ki67 (Abcam, Cambrige, MA, USA) at a dilution of 1:50 for30 min at 37C followed by application of biotinylated secondary antibody (8 min, 37C) and then an avidin/streptavidin enzyme conjugate complex (8 min, 37C). Finally, the antibody was detected in the presence of alkaline phosphatase enzyme by Liquid Fast-Red Substrate Kit (Abcam, Cambrige, MA, USA) and counterstained with hematoxylin. In the presence of alkaline phosphatase enzyme, Liquid Fast-Red produces a red reaction product that can be seen using microscopy. As a positive control, sections of colon ca (...truncated)