Alpha-B-Crystallin in the Limbal and Corneal Epithelium

Abstract

Abstract: : Purpose:The proliferative compartment of the corneal epithelium consists of stem cells(SC) and transient amplifying cells which are exclusively located in the basal limbal and basal corneal epithelium respectively. SC are supported and protected by a unique stromal environment called the SC niche. SCs by definition continuously produce progenies throughout a life span and need adequate protection against various insults. Heat shock proteins (HSP) are a family of inducible and constitutively expressed intracellular proteins that play a major role in protecting cells from environmental stress by preventing aggregation and unfolding of proteins. a-B-crystallin (aBc), a member of the small hear shock protein family has been shown to have antiapoptotic capacity in response to cellular stress in retinal pigment epithelium. In this study we wanted to investigate the constitutive and inducible expression of aBc in human limbo-corneal epithelium. Methods:In a first step we determine the constitutive expression pattern of aBc in the limbal and corneal epithelium of human eyes. Immunostaining was performed in 4 human donor corneas. The distribution of aBc in the limbo-corneal epithelium was analyzed by fluorescence microscopy. In a second step we analyzed the expression level under 4°C and 37°C storage conditions, comparable to the two most common ways of cornea storage. Six human donor cornea pairs were stored at either 4°C in conservation medium (Optisol GS) or 37°C under cell culture conditions for 7 days. The amount of aBc expression was investigated using western blot analysis. Results:The epithelium of the limbo-corneal epithelium showed strong aBc expression. The strongest signal was found in the basal layer with significant lower levels in the suprabasal and superficial layers. After a storage period of seven days, the epithelium of corneas stored at 4°C showed significant lower protein levels of aBc compared to those stored at 37°C under cell culture conditions as measured by western blot analysis (RDI 1:2). Conclusions: The epithelium of the limbo-corneal epithelium does constitutively express aBc with higher levels in the proliferative compartment. The expression of aBc may represent the need for intrinsic protection against various insults of these cell populations. The increase of aBc expression under culture conditions at 37°C compared to cold preservation at 4°C could be either temperature related or reflect a higher stress level during tissue culture at 37°C. Whether the higher level of aBc expression may protect the SC containing limbal epithelium for later keratolimbal transplantation needs to be further investigated.

Keywords: cornea: epithelium • chaperones • cornea: storage