Our study involved tissue samples (pterygium and conjunctiva) obtained from surgery, and tears from patients, with and without pterygium. All patients who underwent surgery had active pterygium. Conversely, outpatients with pterygium were classified as active if they presented with vascularization, edema and active hyperaemia, or as inactive if they showed a fibrotic component. Using RT-PCR we found that HCC was expressed in both normal conjunctiva and pterygium, supporting the findings of Chang et al., who found HCC in another mucosa [11
]. Abrahamson et al., found HCC in several autopsy tissue specimens (heart, kidney, lung, liver, uterus and seminal vesicles) and in several fluids (tears, saliva, cerebrospinal fluid and seminal plasma) [18
]. Wasselius et al., studied cystatin C in the anterior segment of rat and mouse eyes and found that expression of the protein was localized to the ciliary body, cornea and retina [19
]. In that study, cystatin C was not detected in rat and mouse conjunctiva. Our results presented here contradict those presented by Wasselius et al.; this may be because of the anatomical and histological variations between rodent and human eyes. Other authors have noted that HCC plays an important role in the regulation of proteolysis and inflammatory diseases, controlling the activity of cathepsin B in tissues and fluids, including tears [20
Using immunohistochemistry, we found that normal conjunctiva contained HCC in the most superficial epithelial layers. In contrast, HCC was expressed in all epithelial tissues of pterygia, from basal epithelial cells to the most superficial layer of the epithelium. It is interesting to note that this compartment has a high proliferatives rate in comparison with a normal conjunctiva, such as morphological changes we observed in a picture from two tissues stained with H&E technique (Figures
A and B). Other authors have studied HCC by immunohistochemistry but in different tissues. Chang et al., showed light immunostaining intensity corresponding to the presence of HCC in normal sinus mucosa, and intense staining in an inflamed sinus mucosa [11
]. Based on the anti-inflammatory function(s) of HCC, Chang et al., suggest that this protein may play an important role in the protection of normal sinus mucosa, and also in the prevention of aggravation of inflammatory conditions that occur in chronic sinusitis [11
]. Hung et al., compared cystatin C expression in normal human oral mucosa and oral submucous fibrosis (OSF). He demonstrated that OSF was expressed at levels significantly higher than HCC in normal oral mucosa at the RNA and protein level [21
]. They hypothesized that cystatin C had synergistic effects with other antimicrobial substances such as lysozyme, lactoferrin, and defensins, that make it more potent against infectious pathogens. It contrast, it has been shown that the balance between cystatin C and C1 cathepsin is of major importance in the regulation of proteolytic activity under normal physiological conditions, and for pathological degradation that occurs in inflammatory diseases. Our findings may be explained in the context of the protective role(s) of HCC; the protein may be trying to maintain homeostatic redox at the anterior surface of the eye.
Our ELISA and western blot results indicate low levels of HCC in the tears and tissues of patients with a normal conjunctiva. The amount and concentration of HCC were increased in the tears and tissues from patients with pterygium, with the highest levels observed in patients with active recurrent pterygium. Sharman et al., assess the concentration of HCC in gingival crevicular fluid (GCF) and serum from patients with various periodontal diseases, the mean HCC concentration in GCF and serum was observed to be highest in patients with periodontitis, and lowest in healthy individuals, they suggested that HCC levels increased with disease progression to prevent further periodontal degeneration; HCC levels then decrease after treatment to maintain bone metabolic homeostasis [22
]. Lertnawapan et al., found that concentrations of cystatin C were significantly higher in the sera of patients with Systemic Lupus Erythematosus (SLE) compared with controls. These increased cystatin C levels correlated with increases in erythrocyte sedimentation rate, Tumor Necrosis Factor-alpha, and Interleukin 6, suggesting that cystatin C concentrations in SLE may be affected by inflammatory mechanisms [23
]. Henskens et al., found that levels of cystatin C were increased in the saliva of patients with inflammatory periodontal disease in comparison with healthy people [24
]. Consider these findings and combined with our results presented here, point to a possible role for HCC in fluids, as some sort of acute-phase protein during inflammatory diseases.
We found that HCC was present to a great extent in epithelial cells of pterygium samples. Underlying cells in CT did not exhibit strong staining for HCC, suggesting that the production of HCC is confined to epithelial cells. This further indicates that pterygium does not involve a clear inflammatory process. Based on these findings, we can deduce that HCC acts as an epithelial proliferatives stimulant, and at the same time as a modulator of inflammation in underlying CT. This is similar to what was outlined by Koroloenko et al., (2008) [25
] and Tavera et al., (1990) [26