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Endothelial injury is a central finding in vascular disease and is accompanied by disruption of the endothelial barrier function leading to tissue edema and inflammation. Endothelial barrier dysfunction is caused by loss of transmembrane proteins such as Occludin and VE-cadherin, and by loss of cell-cell contacts and cytoskeletal contraction and reorganisation leading to paracellular gaps and penetration of protein-rich fluid and inflammatory cells. Bone morphogenetic proteins (BMPs) are important in endothelial inflammation, but their effects on endothelial permeability have not been investigated until now. The intention of this thesis is to determine, why and by which effects specific BMPs and their antagonists influence the endothelial permeability.
In this thesis, in vitro and in vivo methods were established to detect and to measure permeability. In vitro, a transwell was applied where epithelial or endothelial cells were seeded on the upper insert. As permeability indicators, fluorescent dye (FITC-dextran) or isolated immune cells (PBMCs) were inserted. The amount of diffused FITC-dextran or transmigrated PBMCs through the cell layer was measured after the stimulation of the endothelial cells by adding the BMPs or the BMP antagonists. For analyzing the endothelial permeability in vivo, mice were stimulated with bleomycin or BMP4 and subsequently injected with Evans blue (EVB) into the tail vein as permeability indicator. By using a spectrophotometer, the amount of diffused Evans blue in heart, lung, kidney, bronchioalveolar fluid (BAL), and ascites fluid was quantified.
After bleomycin induced injury of endothelial and epithelial cells, the content of diffused FITC-dextran was higher, compared to the untreated cells. In vivo, the content of EVB in lungs, kidneys, and BAL fluid was significantly higher after bleomycin stimulation.
In vitro, endothelial cells were seeded on transwell chambers and were cultured in the presence of BMPs. Stimulation with BMP2, 4, 7, and 9 enhanced the endothelial permeability. In vivo, mice were stimulated with recombinant murine BMP4. After three days, EVB was injected intravenously. In lung, kidneys, heart, and ascites the EVB content after BMP4 injection was significantly higher.
Inflammation was induced in vitro with TNF-α and in vivo with thioglycolate. After three days of additional BMP4 stimulation endothelial permeability was increased. In vitro, more PBMCs were able to transmigrate through the endothelial cell layer. In vivo, leukocytes in blood and leukocytes in ascites were significantly higher.
Bone morphogenetic proteins regulate protein expression of tight and adherens junctions in endothelial cells. Endothelial cells were exposed to BMP2, 4, and 7 and were lysed afterwards. The lysates were used for western blotting with anti-occludin, anti-VE-cadherin and anti-PECAM1 antibody. Using western blotting BMP2 and BMP4 decreased expression of occludin. After stimulation with BMP2, 4, and 7, the expression of VE-cadherin was downregulated. Furthermore, the VE-cadherin expression in endothelial cells was shown by immunohistofluorecence and was down regulated after injection of BMP4. The VE-cadherin expression was increased after BMP4 knock down. BMP9 decreased the expression of PECAM1, depending on the BMP9 concentration, this was shown by western blotting and immunohistofluorescence. Bone morphogenetic proteins increase actin stress fiber formation and endothelial cell contraction. Therefore, endothelial cells were exposed to BMP2, 4, 7 and 9. The cells were fixed and actin cytoskeleton was stained with phalloidine.
To find a protein to protect the endothelial barrier function, endothelial cells were cultured in the presence of BMP-modulators Gremlin, Noggin, TSG, BMPER, LDN and Chordin, with the result that Gremlin decreased endothelial permeability due to upregulation of occludin.
In conclusion BMP4 and the BMP-antagonist Gremlin were identified as novel regulators of endothelial barrier function with opposing effects. While BMP4 increased endothelial leakage due to downregulation of occludin and VE-cadherin, antagonist of BMP activity Gremlin increased occludin expression and reduced endothelial permeability. Inhibition of BMP activity may open new therapeutic options in the treatment of edema and transmigration of inflammatory cells.