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H. L. Deissler, E. Kuhn, G. E. Lang, H. W. Deissler; Expression of Tetraspanins in Endothelial Cells of the Bovine Retina: Involvement of CD9 in iBREC Migration. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1757.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferation and migration of endothelial cells are deregulated in the pathogenesis of diabetic retinopathy (DR). Since members of the tetraspanin protein family were shown to be involved in these processes in various cell types, we studied their expression and function in immortalized microvascular endothelial cells of the bovine retina (iBREC).
Expression of CD9, CD63 and CD151 in iBREC was analyzed by immunofluorescence staining, FACS and RT-PCR. In addition, change of the specific expression pattern was studied in a wound healing assay. Migration of iBREC towards fibronectin was measured in a modified Boyden-Chamber assay in the presence of antibodies against CD9 or CD151.
CD9 is localized mainly in the cytoplasm in non-confluent and in the plasma membrane in confluent iBREC as shown by FACS analyses and immunofluorescence staining. Interestingly, membrane-localized CD9 in non-confluent iBREC is detected at contact areas between cells and in long spike-like extensions, a distribution reminiscent of that of the EC-specific adhesion protein VECadherin (VECad). In a wound healing assays, CD9 like VECad, delocalizes from the plasma membrane to the cytoplasm in iBRECs located at the margin of the lesion within 4 hours after making the gap. Re-localization of CD9 and VECad to the membrane is seen after lesion closure. Migration of iBREC towards fibronectin is strongly (up to 90%) inhibited in the presence of an antibody against CD9. Interestingly, cytoplasmic staining seen for CD151 in non-confluent cells is not detected in confluent cells and reappears in iBREC located at the border of a gap. CD63 is mainly detected in the cytoplasm of the cell.
We showed for the first time that CD9, CD63 and CD151 are specifically expressed by microvascular iBREC and that CD9 is involved in migration and iBREC wound healing in an in vitro assay, suggesting an important role of this protein in DR and other pathogenic processes in the retina.
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