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Figure 7


Fig. 7. ICAM1-mediated VEC phosphorylation affects paracellular migration and coincides with increased EC permeability. (A) GPNT cells were grown to confluence, serum starved and then either left untreated (NT) or subjected to ICAM1 crosslinking (XL), 50 ng/ml VEGF, 10 µM lysophosphatidic acid (LPA), 10 µM bradykinin (BK), 100 µM histamine (HST) or 1 U/ml thrombin (TBN) for 15 minutes. Subsequently, cells were lysed and VEC immunoprecipitates analyzed by immunoblots using anti-phosphorylated tyrosine or anti-VEC antibodies. (B) The flux of 4 kDa or 140 kDa FITC-dextran across confluent GPNT cell monolayers was measured when ICAM1 was crosslinked (XL) or not (NT). In each case, the FITC-dextran flux was linear over 120 minutes. The values shown are mean permeability changes that occurred over the initial linear 50-minute period following crosslinking in three independent experiments. (C,D) Confluent GPNT cells were serum starved and ICAM1 crosslinked (XL). At the indicated times cells were lysed and subjected to immunoprecipitation of VEC (C) or {gamma}-catenin (D). Immunoprecipitates were then analyzed by immunoblotting using antibodies against phosphorylated tyrosine, {alpha}-, β-, {gamma}-catenins or VEC. Similar results were achieved when the order of the proteins for immunoprecipitates and immunoblots was inverted (data not shown). In all blots the position of size markers (in kDa) is indicated on the left. (E) GPNT cells were nucleofected with wt or Y to F mutants of pEGFP-N'-VEC as described in Fig. 3. They were then incubated with antigen-specific T cells, which were allowed to adhere and migrate for 1-4 h. Subsequently time-lapsed microscopy was performed over a period of 5-10 minutes to determine the fraction of T cells migrating in the paracellular area of the EC. Results are the mean ± s.e.m. of six replicates from at least three independent experiments. Significant differences were determined by Student's t-test (*P<0.05, **P=0.005, ***P<0.0001).





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