A new, rapid and reproducible method to obtain high quality endothelium in vitro.

Human umbilical vein endothelial cells (HUVECs) cultured in vitro are a commonly used experimental system. When properly differentiated they acquire the so-called cobblestone phenotype; thereby mimicking an endothelium in vivo that can be used to shed light on multiple endothelial-related processes. In the present paper we report a simple, flexible, fast and reproducible method for an efficient isolation of viable HUVECs. The isolation is performed by sequential short trypsinization steps at room temperature. As umbilical cords are often damaged during labor, it is noteworthy that this new method can be applied even to short pieces of cord with success. In addition, we describe how to culture HUVECs as valid cobblestone cells in vitro on different types of extracellular matrix (basement membrane matrix, fibronectin and gelatin). We also show how to recognize mature cobblestone HUVECs by ordinary phase contrast microscopy. Our HUVEC model is validated as a system that retains important features inherent to the human umbilical vein endothelium in vivo. Phase contrast microscopy, immuno-fluorescence and electron microscopy reveal a tight cobblestone monolayer. Therein cells show Weibel-Palade bodies, caveolae and junctional complexes (comparable to the in vivo situation, as also shown in this study) and can internalize human low density lipoprotein. Isolation and culture of HUVECs as reported in this paper will result in an endothelium-mimicking experimental model convenient for multiple research goals.

Endothelial cell senescence is associated with disrupted cell-cell junctions and increased monolayer permeability.

BACKGROUND: Cellular senescence is associated with cellular dysfunction and has been shown to occur in vivo in age-related cardiovascular diseases such as atherosclerosis. Atherogenesis is accompanied by intimal accumulation of LDL and increased extravasation of monocytes towards accumulated and oxidized LDL, suggesting an affected barrier function of vascular endothelial cells. Our objective was to study the effect of cellular senescence on the barrier function of non-senescent endothelial cells. METHODS: Human umbilical vein endothelial cells were cultured until senescence. Senescent cells were compared with non-senescent cells and with co-cultures of non-senescent and senescent cells. Adherens junctions and tight junctions were studied. To assess the barrier function of various monolayers, assays to measure permeability for Lucifer Yellow (LY) and horseradish peroxidase (PO) were performed. RESULTS: The barrier function of monolayers comprising of senescent cells was compromised and coincided with a change in the distribution of junction proteins and a down-regulation of occludin and claudin-5 expression. Furthermore, a decreased expression of occludin and claudin-5 was observed in co-cultures of non-senescent and senescent cells, not only between senescent cells but also along the entire periphery of non-senescent cells lining a senescent cell. CONCLUSIONS: Our findings show that the presence of senescent endothelial cells in a non-senescent monolayer disrupts tight junction morphology of surrounding young cells and increases the permeability of the monolayer for LY and PO.