Vascular Endothelial Growth Factor VEGF and p53 Expression in Tunisian Patients with Pterygium: Immunohistochemical Analysis O Beltaief, Kh Errais, I Ammous, A Meddeb-Ouertani Charles Nicolle University Hospital- Tunis- Tunisia Authors have no financial interest

INTRODUCTION Pterygium is characterized by abnormal fibrovascular corneo-conjunctival tissue Recurrence probability after surgery incompletely understanding of its pathogenesis Vascular Endothelial Growth Factor (VEGF) and P53 are known to participate in tumor vascularization Purpose: to study VEGF and p53 expression in active primary and recurrent pterygium in Tunisian patients.

PATIENTS AND METHODS Prospective case-control study in Charles Nicolle University Hospital- Tunis- Tunisia 15 cases of active primary pterygium (hyperemic vascularized tissue ) 5 cases of recurrent pterygium in Tunisia 10 Normal human conjunctiva as control (negative control) Immunohistochemical study Antibodies against VEGF and p53 were used to analyze the distribution and expression of these markers in pterygia and normal human conjunctiva Tissue sections were examined in brightfield microscopy Positive cells was counted with a monocular grid

RESULTS Active primary and recurrent pterygium have different patterns of expression Active pterygium: VEGF and p53 proteins were found in all primary pterygia in epithelial cells, vascular endothelial cells, and fibroblasts VEGF expression was moderate to strong P53 expression was strong Recurrent pterygium: p53 immunoreactivity was weak to moderate VEGF immunoreactivity was strong Normal human conjunctiva: VEGF and p53 expression was weak to negative.

Table 1: Immunohistochemical expression of VEGF and p53 on histological tissue sections of conjunctiva with primary and recurrent pterygia   VEGF p53 patients Age (years) Gender Type Ep Vx Fb 1 71 F P 4+ 3+ 2+ 2 58 M 3 52 1+ 4 65 5 55 6 30 7 40 8 51 9 39 10 42 11 61 12 63 13 24 14 20 15 16 R 17 36 18 62 19 44 P: primary pterygia ; R: recurrent pterygia ; Ep: Epithelial cells; Vx: Vascular endothelial cells; Fb: Fibroblasts

Comparative expression of VEGF and p53 in primary pterygium (scale bar is 30 µm) VEGF and p53 expression in primary pterygium in a 52-year-old patient (patient 3) with stage III pterygium: moderate epithelial (Ep) staining for VEGF (A) and low cytoplasmic and nuclear staining for p53, mainly detected in epithelial cells (Ep), were observed (B). VEGF and p53 expression in primary pterygium in a 58-year-old patient (patient 2) with stage II pterygium: very strong epithelial (Ep) staining and strong staining in fibroblasts (Fb) and vascular endothelial cells (Vx) were observed both for VEGF (C) and p53 (D).

Comparative expression of VEGF and p53 in primary pterygium (scale bar 30 µm) Weak immunoreactivity of VEGF was mainly seen in the epithelial cells (Ep) of a 65-year-old patient (patient 4) (A), whereas strong expression was observed in all pterygial cells (particularly in epithelial (Ep) and vascular endothelial cells (Vx)) in a 63-year-old patient (patient 12) (B). Weak p53 immunoreactivity was mainly seen in basal epithelial (Ep) and vascular endothelial cells (Vx) in a 39-year-old woman (patient 9) (C), whereas strong expression was found in all pterygial cells in the 63-year-old man (patient 12) (D).

Expression of VEGF and p53 expression in recurrent pterygium Expression of VEGF and p53 expression in recurrent pterygium. Very strong and diffuse expression of VEGF (A) observed in all pterygial cells of a 36-year-old patient (patient 17) and weak expression of p53 (B) in epithelial (Ep) and fibroblast cells (Fb). Very strong and diffuse, and strong expression of VEGF (C) in epithelial cells (Ep) and in fibroblasts (Fb) and vascular endothelial (Vx) cells, respectively, and slight or no expression of p53 (D) in epithelial (Ep) and fibroblast (Fb) cells, in a 62-year-old patient (patient 18).

CONCLUSION We found that VEGF and p53 expression was correlated in primary pterygium, suggesting relationships between angiogenesis triggered by VEGF and the level of p53 overexpression. different patterns of VEGF and p53 expression in primary and recurrent pterygium not only contribute identifying etiology of this disease underline the substantial role played by neovascularization and cellular proliferation in the pathogenesis of pterygia

CONCLUSION Overexpression of VEGF in active primary and recurrent pterygium suggests that angiogenesis may play a role in their pathogenesis Expression of p53 in primary pterygium supports the hypothesis that actinic radiation may be involved in its genesis. VEGF and p53 may be useful biomarkers for understanding the physiopathology of pterygium.