An assay system for in vitro detection of permeability in human "endothelium".

The molecular mechanisms by which endothelial permeability occurs are often studied more readily in vitro, underscoring the importance of the use of systems that mimic human endothelium in vivo. We present an assay that accurately models human endothelium by use of primary human microvascular endothelial cells (hMVEC), because permeability primarily occurs at the microvascular level, and transwell filter units coated with Matrigel, extracellular matrix that mimics basal lamina, the matrix that is tightly associated with endothelium and is critical for its proper function. As a tracer molecule, we used 3-kDa dextran-FITC to detect leakage through the small gaps present in the early stages of permeability induction. The permeability-inducing agents IL-8 and VEGF were added to the lower chamber of the transwell units to mimic inflammatory conditions in vivo. After optimization, we were able to minimize basal permeability and to detect rapid changes in permeability stimulated by IL-8 and VEGF, similar to that observed in vivo. Furthermore, we have used this system to delineate the importance of the transactivation of VEGFR2 in IL-8-induced permeability and have confirmed the relevance of this signaling in vivo, suggesting that our permeability assay system adequately mimics the in vivo situation. Therefore, this system can be used to better understand the molecular mechanisms of human vascular permeability in a more in vivo-like setting and, thus, may be used to test effective therapeutics to prevent and treat diseases involving persistent permeability.