Thymosin Beta 4 (Tß4) Expression in Vernal Keratoconjunctivitis (VKC)

Abstract

Abstract: : Purpose: Vernal Keratoconjunctivitis (VKC) is a chronic inflammatory allergic disease leading to conjunctival scarring, fibrosis and tissue remodelling. Thymosin beta 4 (Tß4) is the major actin–sequestring peptide in the mammalian cells involved in wound–healing having also anti–inflammatory properties. It is reasonable to explore if Tß4 may play a role in modulating the progression of VKC. Here we evaluated the expression of Tß4 in the conjunctiva of patients with VKC. Methods: Conjunctival biopies obtained from VKC patients (n=4) and healthy subjects (n=5) were evaluated for molecular (relative real time–PCR) and biochemical (immunohistochemistry, Confocal Laser Scanning Microscopy (CLSM) and western blot technique) expression of Tß4. The Tß4 expression in lymphocytes (CD4+), mast cells (AA1), eosinophils (EG2) and fibroblasts/myofibroblasts (vimentin/a–SMA) in VKC conjunctiva was further evaluated by double–labelled immunofluorescence. All data were elaborated using the ANOVA program followed by Tukey–Kramer post–hoc. Results: The real time–PCR analysis showed that Tß4mRNA was up–regulated in VKC conjunctival biopsies (500 fold increase, p<0.05) when compared to controls. Immunohistochemical staining showed an intense localization of Tß4 in the conjunctival epithelium of healthy subjects, meanwhile a weak staining was observed in the stroma. In VKC conjunctiva Tß4 expression increased in both epithelium and stroma, as confirmed by CLSM, consistently with the molecular data. CLSM technique also demonstrating localization of Tß4 in fibroblasts and in inflammatory cells infiltrating the conjunctiva. Interestingly, some cells co–expressed Tß4 and α–SMA, a typical marker of smooth muscle cells and myofibroblasts. Conclusions: Our data show that Tß4 is over–expressed in the conjunctival in both epithelium and stroma of VKC patients. This observation is suggestive for a role of Tb4 in the progression of VKC and encourages further studies to fully understand the underlying mechanism.

Keywords: conjunctivitis • inflammation • protein structure/function