The Adhesion of Choroidal Endothelial Cells to Selected Extracellular Matrix Components Depending on Culture Medium

Abstract

Abstract: : Purpose: The adhesion of choroidal endothelial cells (CEC) to extracellular matrix is an important stage of angiogenesis. Attachment of CEC to fibronectine, collagen type IV and laminine was analyzed in this study. Methods: Bovine CEC obtained by microdissection and Lycopersicon esculentum–coated paramagnetic beads were cultured in Endothelium Growth Medium containing 2% fetal calf serum (FCS). Second to fifth– passage cells grown to confluence were used. Fibronectine, collagen type IV and laminine were tested in different concentrations (from 0.5 µg/ml to 50 µg/ml). Attachment was measured by modified MTT assay. The morphological characteristic of cells attached to different matrix components was documented by confocal microscope Axiovent 200 M Zeiss. All experiments were repeated three times and compared using Student’s t–test with the level of confidence for statistical significance determined to be P<0.01. Results: All analyzed substrates (fibronectine, collagen and laminine) gave a dose–dependent kinetics on attachment. Fibronectine was the best substrate for adhesion with a maximum efficiency dose of 20 µg/ml at each time of the kinetics. On fibronectine, the maximal adhesion of 90% was reached after 24 hours. Collagen was less efficient with maximal efficiency dose at 5 µg/ml at every times studied and the level of adhesion after 24 hours was 65%. Laminine was the less efficient substrate for adhesion and even high dose (50 µg/ml ) gave only 58% of attachment after 24 hours. The influence of studied matrix components on CEC adhesion was more pronounced when the cells were seeded with the initial cell density of 5x10⁴ than 1x10⁴. The absence of FCS decreased the number of adhered cells almost by half irrespectively of matrix components. The morphological characteristic of CEC, documented by confocal microscope confirmed the prominent role of fibronectine as a strongest adhesion–promoting factor. Conclusions: The adhesion rate of CEC is dependent on initial cell density and matrix components. Fibronectine promotes cell attachment in the most important manner. We continue our study to evaluate the intracellular signaling pathways implicated in CEC adhesion, with special interest on MAP–kinase and on the PI3–kinase pathway, which as was already published, are involved in CEC proliferation.

Keywords: age-related macular degeneration • choroid: neovascularization • cell adhesions/cell junctions