Entactin/nidogen: Synthesis by bovine corneal endothelial cells and distribution in the human cornea

Purpose. The purposes of this study were to determine whether entactin/nidogen (E/N) is synthesized and secreted by corneal endothelial cells and to characterize the distribution of E/N in the human cornea. Methods. Cultured bovine corneal endothelial cells were metabolically labeled with [³⁵S]methionine. Newly synthesized E/N was detected in cell lysates and culture medium by immunoprecipitation, using monoclonal anti-E/N antibodies, polyacrylamide gel electrophoresis, and autoradiography. The presence of E/N in the subendothelial extracellular matrix was demonstrated by Western blot analysis of solubilized extracellular matrix proteins. The distribution of E/N in normal human corneas was studied by indirect immunofluorescent staining of frozen sections, using monospecific anti-E/N antibodies. Results. E/N was detected in the basement membrane (BM)-like extracellular matrix deposited by corneal endothelial cells, as well as in cell lysates and culture medium. Immunofluorescence studies revealed the presence of E/N in both the epithelial and endothelial BM and to a much lower extent in the stroma. E/N was detected throughout the thickness of the epithelial BM, but its staining decreased in intensity toward the central part of the cornea. In the endothelial BM (Descemet's membrane), E/N fluorescence was limited to its most posterior portion, produced postnatally. Conclusions. Corneal endothelial cells synthesize and secrete E/N. E/N was found in both the epithelial and endothelial basement membranes but was primarily localized to the posterior portion of Descemet's membrane and the periphery of the epithelial BM. The authors suggest that E/N may be important in healing processes of corneal injuries and in the pathogenesis of diseases involving the postnatal region of Descemet's membrane.