Lung Microvascular Endothelial Monolayers Research Articles

Quantification of Malignant Breast Cancer Cell MDA-MB-231 Transmigration Across Brain and Lung Microvascular Endothelium.

Tumor cell extravasation through the endothelial barrier forming the microvessel wall is a crucial step during tumor metastasis. However, where, how and how fast tumor cells transmigrate through endothelial barriers remain unclear. Using an in vitro transwell model, we performed a transmigration assay of malignant breast tumor cells (MDA-MB-231) through brain and lung microvascular endothelial monolayers under control and pathological conditions. The locations and rates of tumor cell transmigration as well as the changes in the structural components (integrity) of endothelial monolayers were quantified by confocal microscopy. Endothelial monolayer permeability to albumin P (albumin) was also quantified under the same conditions. We found that about 98% of transmigration occurred at the joints of endothelial cells instead of cell bodies; tumor cell adhesion and transmigration degraded endothelial surface glycocalyx and disrupted endothelial junction proteins, consequently increased P (albumin); more tumor cells adhered to and transmigrated through the endothelial monolayer with higher P (albumin); P (albumin) and tumor transmigration were increased by vascular endothelial growth factor, a representative of cytokines, and lipopolysaccharides, a typical systemic inflammatory factor, but reduced by adenosine 3',5'-cyclic monophosphate. These results suggest that reinforcing endothelial structural integrity is an effective approach for inhibiting tumor extravasation.

Reciprocal Regulation of Endothelial Substrate Adhesion and Barrier Function

ABSTRACTObjective: To examine how cell‐substrate adhesion is regulated during barrier changes produced by exposure to inflammatory mediators.Methods: Lung microvascular endothelial monolayers were treated with test agents ± blockers, and barrier was measured by transendothelial resistance; cell‐substrate adhesion was assessed by surface area conservation after trypsin treatment of monolayers. Protein phosphorylation and distribution were assayed by immunoblotting and fluorescent microscopy, respectively.Results: H2O2, histamine, bradykinin, and thrombin, decreased endothelial barrier function, and enhanced adhesion to the substratum. H2O2 enhanced cell adhesion to the substrate in a concentration (0–1 mM)‐ and time (0–60 minutes)‐ dependent fashion. This effect of H2O2 reversed within 120 minutes of removal of H2O2 and was blocked by the mean arterial pressure (MAP) kinase inhibitor, PD98059 and by chelating cytoplasmic Ca2+ but not PKC or PKG inhibition. H2O2 also stimulated tyrosine phosphorylation of several proteins and increased the association of the focal adhesive proteins paxillin, talin, and vinculin with the cytoskeleton and may promote localization of these proteins to junctions.Conclusions: Our data indicate that inflammatory mediators reduce cell‐cell contact, contributing to reduced solute barrier and simultaneously enhanced substrate binding, which may be reciprocal events in barrier regulation in vitro and in vivo.