The relationship between in-vitro endothelial permeability and molecules of the intercellular junction
Changes in endothelial permeability are known to contribute to many pathologies, including inflammation seen in skin irritation. The regulation of permeability has been linked to inter-endothelial junctions, specifically the tight and adherens junctions (TJ and AJ). The functional state of the junction is thought to be associated with numerous factors including cytoskeletal changes and the interactions between junctional components. How these factors interact and respond to inflammatory stimuli is still not fully understood. This project examined two human endothelial cell-lines for use in in-vitro permeability studies, ECV304 and HMEC-1. Changes in permeability along with arrangement of the F-actin cytoskeleton and the adherens junction molecule VE-cadherin were studied in response to a variety of compounds. Macromolecular permeability was assessed by measuring the leakage of fluorescently labelled dextrans of varying molecular weights. The F-actin and VE-cadherin were visualised using immunocytochemical techniques. TEM was undertaken to examine the ultrastructure of the junctions The ECV304 cells, whilst showing an increased permeability to a variety to vaso-active mediators, did not express some of the pertinent junctional molecules of the endothelium and thus are not recommended for use. The basal permeability of the HMEC-l cell-line was shown to act in predictable fashion, giving comparable permeability coefficients to other endothelial cells. The increase in permeability following exposure to A23187, CAPB, EGTA and PMA was shown to correlate to an altered expression of VE-cadherin and F-actin. These observations were furthered using histamine, where a quantifiable change in the levels of continuous and stitch VE-cadherin staining was demonstrated. The permeability response to histamine occurred much later than the VE-cadherin and F-actin changes. This could be due to methodology and/or the cells lack of TJs. The apparent lack of mature AJs was approached by exposing the cultures to cAMP-raising media. This significantly reduced the basal permeability and increased the expression of AJ components, apart from β-catenin, at the cell-cell contacts. Indeed α-catenin was redistributed from the triton-soluble fraction of the cells to the triton-insoluble fraction, which is proposed to contain the junctional components. These results demonstrate additional information on the role that the adherens junction molecules play in endothelial permeability and characterise the HMEC-1 cell-line for further use in this field.