Members of the tetraspanin protein family are modulators of several fundamental cellular processes in various cell types. However, expression and function of these proteins have not been studied in microvascular endothelial cells despite their (patho-)physiological importance. Western blotting, FACS or RT-PCR analyses confirmed that CD9 and other tetraspanins are expressed in immortalized microvascular endothelial cells of the bovine retina (iBREC). In subconfluent cultures, most of the detected CD9 was located intracellularly as well as in the plasma membrane at cell-cell contact sites and in long spike-like extensions, whereas cells in confluent cultures predominantly showed plasma membrane staining. In wound healing assays, CD9 delocalized from the plasma membrane to its intracellular compartment in cells located at the gap border, and the gap closure was retarded by the addition of an anti-CD9 antibody. Migration of iBREC towards fibronectin and their adhesion to fibronectin were also strongly inhibited in the presence of an anti-CD9 antibody whereas other anti-tetraspanin antibodies had no effect. In summary, iBREC express members of the tetraspanin family of which CD9 was demonstrated to have a function in migration and adhesion of these cells.

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