Generation and characterization of human soluble VE‐cadherin (sVE‐cadherin)
Authors: Knop Juna Lisa , Burkard Natalie , Ghoreishi Yalda , Flemming Sven , Schlegel Nicolas 
Published: April 2020

Endothelial barrier function is maintained by tight and adherens junctions. Adherens junction protein Vascular Endothelial (VE)‐cadherin plays a critical role for maintenance of endothelial barrier function by providing the mechanical strength between adjacent endothelial cells. Previously, it was shown that inflammation‐induced loss of endothelial barrier function is associated with the formation of soluble VE‐cadherin fragments (sVE‐cadherin) that comprise the extracellular domains (EC) 1‐5. Moreover, it was proposed that sVE‐cadherin could be involved in the loss of endothelial barrier function. Here, we generated a recombinant sVE‐cadherin EC1‐5 construct to test whether the presence of sVE‐cadherin alone and independent of pro‐inflammatory stimuli would affect the integrity of endothelial barrier function.DNA templates of extracellular domains EC1‐5 of human VE‐cadherin were amplified via PCR and ligated into different plasmids. Then, Chinese‐Hamster Ovary cells (CHO) were transfected with these vectors which resulted in CHO cells secreting functional active sVE‐cadherin. This sVE‐cadherin in CHO supernatants was purified and used for biochemical characterization. Thereafter, functional analysis was performed on human microvascular endothelial cells (HDMECs).First, sequencing of the purified products from CHO cells that had been transfected with the plasmids containing EC1‐5 confirmed the sequence of the extracelIular domains EC1‐5 of VE‐cadherin. In the cell culture supernatants of transfected CHO cells the presence of sVE‐cadherin was further verified using Western Blots where a 90 kDa protein in the cell culture supernatant was detected that proved to be sVE‐cadherin as revealed by an antibody directed against the extracellular domain of VE‐cadherin. In addition, by using an ELISA‐based system that specifically recognizes extracellular domains of VE‐cadherin, levels of 100–200 ng/ml of sVE‐cadherin were found in the cell culture supernatants of transfected CHO cells which in summary confirmed the successful generation of human sVE‐cadherin.To further test the hypothesis that sVE‐cadherin compromises endothelial barrier function HDMECs were incubated with different concentrations of sVE‐cadherin followed by measurements of transendothelial electrical resistance (TER) and immunostaining. In TER measurements sVE‐cadherin led to a significant loss of TER compared to controls after 2h hours of incubation beginning at concentrations of 75 ng/ml. In immunostaining sVE‐cadherin application on endothelial monolayers resulted in intercellular gap formation, loss of VE‐cadherin at the cell borders and led to the formation of stress fibers in the actin cytoskeleton. Preliminary experiments using immunoprecipitation assays showed that sVE‐cadherin binds to vascular endothelial protein tyrosine phosphatase (VE‐PTP) pointing towards a potential mechanism by which sVE‐cadherin induces loss of VE‐cadherin.In summary, we successfully generated human sVE‐cadherin. Our data in endothelial cells demonstrate that sVE‐cadherin disrupts endothelial barrier function in a dose‐dependent manner independent of the presence of pro‐inflammatory stimuli.Support or Funding InformationDFG SCHL1962/4‐2