An anti vimentin antibody promotes tube formation

Abstract

In recent years, there has been an increasing appreciation of the importance of secreted and extracellular proteins that traditionally have been considered as intracellular components. Vimentin is a highly abundant intermediate filament protein, and its intracellular functions have been investigated in a large number of studies. Recently, however, vimentin has been shown to take part in significant processes outside the cell. Our understanding of the functions of extracellular vimentin is, however, limited. In this study we demonstrate that a vimentin specific antibody, obtained by phage antibody technology, promotes tube formation of endothelial cells in a 2D matrigel assay. By binding vimentin, the antibody increases the tube formation by 21% after 5 hours of incubation. Addition of the antibody directly to cultured endothelial cells does not influence endothelial cell migration or proliferation. The enhanced tube formation can be seen for up to 10 hours where after the effect decreases. It is shown that the antibody-binding site is located on the coil 2 domain of vimentin. To our knowledge this is the first study that demonstrates an enhanced tube formation by binding vimentin in a 2D matrigel assay under normoxic conditions.

Figure 1

Characterisation of LOB7. Top panel: (a) The commercial mouse anti-vimentin V9 was used to identify vimentin in the sample of immuno-precipitated proteins. (b) LOB7 presented on phage was tested against vimentin in ELISA. A dilution series of vimentin was coated in the wells of an ELISA plate and detected using the same amount of LOB7 presented on phage. (c) Western blot using the commercial mouse anti-vimentin V9 and LOB7 respectively on cytoplasmatic extracts from HUVEC cells and extracts from the extracellular matrix (Matrigel).

Figure 2

Testing specificity of LOB7 and V9 by western blotting. To test the specificity of LOB7 and the mouse monoclonal anti-vimentin antibody V9, seven fragments of vimentin were produced as fusions to the pagP proteins all carrying a His-tag. To the left, the fragments F1-F7 are schematically shown. In the right top panel, the seven fragments are detected by an anti-His antibody. In the lower right panel, LOB7 is seen to bind specifically to the F6 fragment. As there is no recognition of the F5 and F7 fragments, the epitope of LOB7 is between amino acid 268 and 396 of vimentin. The mouse monoclonal antibody V9 specifically bind to the F7 fragments, therefor the epitope for V9 is between amino acid 405 and 466.

Figure 3

Tube formation using growth factor reduced matrigel in IBIDI angiogenesis slides. The tube length was measured as number of pixels using a MathWorks MatLab program. To the right, pictures are shown for 5 hours of incubation, from the top: LOB7, isotype control (anti HNE-α7), and medium only. Error bars: Standard error of the mean (SEM).