Purchase this article with an account.
M. Singh, Z. Zhang, F. Grossi, S. Dick, P. Olson; In–vitro and in–vivo Evidence Suggesting Abundant Expression of MFG–E8 by Human Retinal Pigmented Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4882.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
MFG–E8, or lactadherin, is a glycoprotein that participates in several important biological processes. Among these are tissue remodeling, phagocytic removal of apoptotic cells, oocyte–gamete interactions and neovascularization. Accordingly, various cell types in mammals have been documented to synthesize this molecule. However, little is known about its production by ocular cells. We studied the expression and localization of MFG–E8 in cultured APRE–19 cells, as well as in the normal human eye sections.
First, ARPE–19 cells were grown in tissue culture. Cell lysates and supernatants were processed separately to confirm MFG–E8 expression using specific antibodies in western blot assays. Second, total RNA was extracted from cultured ARPE–19 cells and subjected to RT–PCR to determine the levels of MFG–E8 transcription. Third, sub–cellular localization of MFG–E8 in APRE–19 cultured cells was visualized by indirect immunofluorescence microscopy. Finally, the presence of MFG–E8 was demonstrated in total cell lysates and in tissue sections prepared from normal human donor eyes using immunoblotting and immunohistochemical methods, respectively.
Immunoblot analysis yielded a prominent band from the supernatant of ARPE–19 cells in comparison to the ARPE–19 cell lysate and cell lysate from the normal donor eyes tissue. The RT–PCR analysis of total RNA from ARPE–19 exhibited a band of the appropriate size thus confirming the immunoblot data. Furthermore, immunolocalization showed MFG–E8–specific staining of ARPE–19 cells. Immunohistochemistry demonstrated MFG–E8–specific reactivity mainly in the retinal pigmented cell layer of sections prepared from normal human donor eyes.
For the first time, we have demonstrated that MFG–E8 is expressed in human ocular tissue. The synthesis of MFG–E8 by retinal cells and its putative abundance in ocular tissue reflects the possible importance of this glycoprotein in retinal homeostasis and overall ocular health.
This PDF is available to Subscribers Only