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E.-M. Kuhn, H. Deissler, G. E. Lang, H. L. Deissler; CD9 is Important for Migration of Retinal Endothelial Cells - A Novel Target for Therapeutic Intervention?. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5884.
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© ARVO (1962-2015); The Authors (2016-present)
Cellular processes that are deregulated in endothelial cells in retinal diseases like diabetic retinopathy, including migration and adhesion to components of the extracellular matrix, are the same for which members of the tetraspanin protein family are known modulators. Therefore, a contribution of these proteins to the genesis of such diseases can be assumed. Recently we have shown that the tetraspanin CD9 indeed is involved in migration and adhesion of endothelial retinal cells (iBREC). To elucidate its function, putative partners of CD9 in protein complexes were studied.
Complex partners of CD9 were identified by co-immunoprecipitation with a CD9-specific antibody followed by Western blotting. Their co-localization was verified by double-immunofluorescence staining. Involvement of these proteins in migration of iBREC towards fibronectin was confirmed in a modified Boyden-Chamber assay in the presence of specific antibodies. In addition, effects of glucose on the expression of CD9 was investigated.
CD9 was mainly localized at the plasma membrane in confluent iBREC but considerable intracellular staining was also seen. However, in non-confluent or migrating cells, CD9 was usually found in intracellular compartments. Integrin beta-1 (CD29) and integrin alpha-5 (CD49e) which form the classical fibronectin receptor, were identified as complex partners of CD9 in iBREC by double-immunofluorescence staining and/or immunoprecipitation. A portion of the co-localized CD29 and CD49e was also co-localized at the plasma membrane with CD9, whereas intracellular CD9 was not associated with these proteins. Antibodies against CD29 or CD49e both inhibited migration of iBREC sightly but their effect was much stronger in the presence of an anti-CD9 antibody. The amount of plasma membrane-localized CD9 was strongly increased in iBREC cultivated in medium with elevated glucose levels (> 5 g/l) which also stimulated CD9-dependent migration of these cells.
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