CHARACTERIZATION AND CLONING OF BOVINE RETINAL ENDOTHELIAL-CELLS (BREC) IN LONG-TERM CULTURE

The aim of this study was to generate a new experimental model of endothelial cells that continuously reproduce themselves in culture, in order to understand th biochemical modifications in the basement membrane of retinal endothelial cells in relation to diabetic retinopathy. bovine retina endothelial cells (BREC) have been obtained by mechanical dispersion after enzymatic digestion, and have been cultured long-term in a medium designed to select endothelial cells. The BREC 1.1 cell line has been characterized by electron microscopy analyses, immunohistochemical methods and by cellular cinetic methods. BREC 1.1 are polygonal cells showing a ''cobblestone'' pattern typical of endothelial cells. About 90% of cells in stable culture are positive for endothelial markers (Factor VIII related antigen, lectin I from Ulex europaeus); BREC 1.1 are also able to metabolize acetylated LDL. Population doubling time is 43 hours during the logarithmic phase of growth. The production of extracellular matrix components (ECM) (collagen type I, type III, type IV and fibronectin) has been evaluated using a specific and sensitive enzyme-linked immunoassay (ELISA). BREC 1.1 are able to secrete all the tested substances, but the main products are collagen type I and IV. The amount of the ECM component production varies with the cellular density in culture. Laminin secretion is detectable by the immunohistochemical method, but its production is lower than the detection limit of the ELISA assay. BREC 1.1 cells are also able to produce glycosaminoglycans in culture and their maximal production occurs in unstimulated, subconfluent cells. Cells have been cloned by the method of limiting dilutions, and all the 10 clones tested and studied are 100% positive fro the endothelial markers mentioned above. Individual clones are not homogenous for the production of different components of ECM: D4 being a maximal producer of collagen type I. G6 of type III, and A3 of type IV. On this basis BREC 1.1 may represent a good model for the study of the modification of ECM component production by endothelial cells in diabetic retinopathy.