Pterygium is a chronic condition, characterized by the invasion of an altered ocular surface tissue, into the normal cornea
Histologically, actively growing pterygium exhibit both degenerative and hyperplastic changes as well as proliferative and inflammatory disorders. They consist of an overlying conjunctival epithelium, which can appear normal or mildly hyperplastic. The underlying fibrovascular tissue usually presents a chronic inflammatory cellular infiltration and rich vasculature
The pathogenesis of pterygium is still controversial. Many causative environmental factors may induce ocular inflammation, and various types of cytokines may cause abnormal cell changes of the ocular surface. UV-B exposure causes oxidative stress, leading to an excess of many potential mediators of pterygium growth
]. The noxious effects of UV irradiation are caused either directly by UV phototoxic effects or indirectly by formation of radical oxygen species (ROS). ROS are very harmful to cells, because they injury cellular DNA, proteins, and lipids, called oxidative stress. ROS produced by UVB and UVA radiation through photosensitized oxidation can target DNA base guanine, giving rise to 8-hydroxydeoxyguanosine (8-OHdG) in DNA molecules. 8-OHdG has been shown to be a sensitive and stable biomarker for evaluating the degree of oxidative DNA damage
Although pterygium is considered an oxidative-degenerative disease many authors believe that pterygium is a cellular proliferative disease probably induced by UV radiation. Growth factors have been studied for their role in pterygium development. They have been shown to be elevated in pterygium, playing an important role in cell proliferation, inflammation, connective tissue remodelling and angiogenesis
A study of growth factors in cultured pterygium shows that active primary and recurrent pterygium fibroblasts immunoreact strongly with platelet-derived growth factor (PDGF) and basic fibroblast growth factor (b-FGF), respectively
Incubation of pterygium-derived epithelium cells (PECs) and pterygium fibroblasts (PFs) with HB-EGF resulted in a significant increase in [3H] thymidine incorporation compared with that of control cells. HB-EGF stimulated chemotaxis of both PECs and PFs. These effects were abolished by the addition of a neutralizing antibody to HB-EGF
]. HB-EGF also induces cell migration of both pterygium epithelium and fibroblasts. Previous studies found that b-FGF, VEGF and HB-EGF are upregulated after UV-B exposure
The mRNA levels were evaluated for 5 peptide growth factors: transforming growth factor (TGFB1), vascular endothelial growth factor (VEGFA), basic fibroblast growth factor (FGF2), epidermal growth factor (EGF) and insulin-like growth factor (IGF1) in pterygium and phenotypically normal conjunctiva by employing qRTPCR which detected significantly higher levels of expression of FGF2 and VEGFA in the former, compared with the latter. Furthermore, FGF2 and VEGFA expression was significantly higher in more advanced pterygium, compared with less advanced ones, whereas VEGFA was also significantly correlated with postoperative recurrence. Results support the involvement of FGF2 and VEGFA in pterygium pathogenesis and imply that pharmacological modification of the clinical behavior of pterygium may be possible with agents directed against specific growth factors, such as VEGFA
The cell cycle, specifically defective regulation of apoptosis, possibly plays a significant role in pterygium formation and growth
]. The p53 tumor suppressor has been extensively investigated for its role in pterygium development. Increased p53 expression in the limbal epithelia of pingueculae, pterygium, and limbal tumors indicates the probable existence of p53 mutations in these cells at an early stage in their development, which is consistent with UV irradiation causation
]. p53 acts as a checkpoint by preventing proliferation and subsequently inducing apoptosis if DNA mutations exist.
Disorganization in the extracellular matrix modulation has also been attributed to the pterygium formation
].Pterygium epithelial cells (PEC) and limbal epithelial cells (LEC), but not normal conjunctival epithelial cells (CEC), respond to UVB exposure by an enhanced production of metalloproteinase-1 (MMP-1), an enzyme involved in the turnover of extracellular matrix, supporting the concept of UV-induced genetic trauma to LEC as a pathogenetic mechanism for pterygium
Hsp90 (heat shock protein 90) is a molecular chaperone and as its name implies, heat shock proteins protect cells when stressed by elevated temperatures
]. It is an abundant and highly conserved molecular chaperone that is essential for viability in eukaryotes
It has been estimated that Hsp90 accounts for 1% of the total soluble cytosolic protein in unstressed cells, making it one of the most abundant proteins
Hsp90 promotes protein folding by preventing unfolded proteins from aggregating
]. Similarly, it can prevent unfolding and aggregation of folded proteins, with which it is more or less stably associated. The first hints that Hsp90 may help stabilize metastable protein domains came from studies on steroid receptor complexes in the70s and 80s, and even preceded the formal identification of the 90-kDa component as Hsp90
It also stabilizes a number of proteins involved in tumor growth. Cancerous cells overexpress a number of proteins, including growth factor receptors, such as EGFR. Disregulation of these growth factor receptor pathways by over-expression or constitutive activation can promote tumor processes including angiogenesis and metastasis and is associated with poor prognosis in many human malignancies
It was demonstrated that 17-allylamino-17-demetho-xygeldanamycin (17-AAG), the Hsp90 inhibitor can induce the degradation of mutant EGFR.
Inhibition of Hsp90 may induce apoptosis through inhibition of the PI3K/AKT signaling pathway and growth factor signaling generally
The heat shock proteins (HSPs) induced by cell stress are expressed at high levels in a wide range of tumors and are closely associated with a poor prognosis and resistance to therapy. The increased transcription of HSPs in tumor cells is due to loss of p53 function and to higher expression of the proto-oncogenes HER2
, and is crucial to tumorigenesis. The HSP family members play overlapping, essential roles in tumor growth both by promoting autonomous cell proliferation and by inhibiting death pathways. The HSPs have thus become targets for rational anti-cancer drug design: HSP90 inhibitors are currently showing much promise in clinical trials, whereas the increased expression of HSPs in tumors is forming the basis of chaperone-based immunotherapy
Hsp90 is also required for induction of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS).Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. It was demonstrated that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation
]. They are important for angiogenesis that is required for tumor growth.
In our study we found Hsp90 overexpressed in all pterygium samples (Mean: 76 ± 10.8). We observed statistical significance (p<0,001) if compared to normal conjunctiva (Mean: 1.4 ± 0.8). In some tumors, the same factors are involved in their etiopathogenesis
] as well as in pterygium
]. It was demonstrated that Hsp90 stabilizes many of these factors in tumors.
As far as we know this is the first report of Hsp90 expression in pterygium samples. Hsp90 is a protein involved in stress response and in normal homeostatic control mechanisms. Tumor cells require higher Hsp90 activity than normal cells to maintain their malignancy, so Hsp90 is a promising target for a pterygium treatment development
]. We believe that searching for Hsp90 inhibitors (39, 40. 43) is of great importance and may represent a new therapeutic modality.
The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0), makes it an excellent marker for determining the so-called growth fraction of a given cell population
It was demonstrated that despite the strong downregulation of pKi-67 expression in non-proliferating cells, the protein can nevertheless be detected at sites linked to ribosomal RNA (rRNA) synthesis
]. Although this finding does not argue against the use of pKi-67 as a proliferation marker, it has wide ranging implications for the elucidation of pKi-67 function.
Inactivation of antigen KI-67 leads to inhibition of ribosomal RNA synthesis
The usefulness of the Ki-67 labeling index has been well established for various types of malignant neoplasms
]. In multivariate analysis, it was found that the Ki-67 labeling index is an independent and significant prognostic factor for disease-specific survival if all stage and grade categories are included
In our study we found Ki67 overexpressed in 60% of pterygium samples (Mean: (10.1 ± 9.5). We observed statistical significance (p<0.01) if compared to normal conjunctiva (Mean: 2.1 ± 1.9). Although we observed negative results in 3 samples (30%) and 1 sample (10%) weakly stained. Garfias et al.
] previously demonstrated expression of ki67 in epithelium of pterygium samples. The immunostaining was not observed in healthy conjunctivas. The results were in accordance with those found by Kase et al.
]. It is interesting to note that we demonstrated great variance in Ki67 staining among the studied samples. This probably can indicate proliferative cell variability and should be related to recurrence rate. We also observed some samples of normal conjunctiva that showed positive staining and it can indicate that those samples could present some pathology not detected in biomicroscopy examination.
It was demonstrated that Ki67 is associated with cell proliferation.
Overexpression in malignant tumors means a great celular activity and poor disease prognostic
]. Our finds proved the increase of cell proliferation in pterygium. The negative and weak results represent a minor activity and may be a better prognostic to recurrence of pterygium. We believe that new studies into the expression of Ki67 in pterygium are necessary for better understanding of the physiopathology of pterygium.
In this study abnormal Hsp90 and ki67 expression was found in pterygium epithelium, suggesting that the disease could be a result of uncontrolled cell proliferation. The staining pattern showed variations that could help understand the disease progress thus strategies in cell proliferation may be appropriate in the management of pterygium or its recurrence.