An anti vimentin antibody promotes tube formation.

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.

Confronting cellular heterogeneity in studies of protein metabolism and homeostasis in aging research.

In this chapter we review the different technologies that can be applied in the analysis of protein homeostasis and metabolism in aging research. Special focus will be on technologies with a potential to circumvent the problems associated with cell heterogeneity in biomarker discovery. Often studies aimed at increasing our understanding of cellular senescence take advantage of model systems. This can be in the form of cell culture, where specific celllines are cultivated, thus undergoing cellular senescence according to the Hayflick phenomenon. Alternatively, model organisms can be included, such as yeast, nematodes and zebra fish. Even though such model systems allow the researcher to control many parameters of the system, it is well established that even in a simple cell culture system the individual cells are morphologically and functionally different. The heterogeneity observed even within the least complex systems, makes it a difficult task to identify biomarkers of cellular senescence and to fully understand the various molecular networks. The complexity is further increased when taking the step from model systems of aging to human aging.

Evaluation of endothelial cell culture as a model system of vascular ageing.

The purpose of this study was to evaluate the relevance of long-term endothelial cell culture as a model system of vascular ageing. Micro- and macrovascular endothelial cells were serially passaged until replicative senescence and their ability to form tube-like structures when cultured on Matrigel was assessed throughout their lifespan. For both cell types low passage cultures adopted a homogeneous cobblestone morphology, while senescent cultures were extremely heterogeneous. Furthermore, both cell types showed a reduction in tube formation ability with in vitro ageing, which is in accordance with the reduction in angiogenic potential observed with ageing in vivo. Examination of senescence associated ß-galactosidase activity revealed an increased activity in cells forming tubes as compared to cells cultured on plastic, which could be attributed to an increased lysosomal content of cells undergoing tube formation. As this increased senescence associated ß-galactosidase activity was unrelated to the replicative age of the cells, senescence associated ß-galactosidase activity may not be a relevant senescence marker for differentiating endothelial cells. The age-related reduction in tube formation ability suggested that long-term culture of endothelial cells may be a valid model system of vascular ageing, which makes it an ideal platform for high throughput screening of compounds influencing angiogenesis.