Figure 4. TRPM2 activation mediates VEGF-induced disassembly of adherens junctions and endothelial cell migration.

A) Immunofluorescent staining of p120-catenin (green) and β-catenin (red) in mouse lung ECs. VEGF stimulation induced multiple discontinuities in the expression of p120-catenin and β-catenin at the plasma membrabe in wild type ECs (arrows) whereas these changes were reduced in TRPM2^-/- mouse ECs and AJs appeared continuous. Scale bar=20μm.

B) Bar graph of mean fluorescent intensities at AJs in wild type and TRPM2^-/- ECs show significantly reduced intensity of p120-catenin and β-catenin in WT ECs compared to TRPM2^-/- ECs. Mean flluorescent intensity (MFI) was calculated from images taken from 5-6 different fields for each group. The mean response from two indpendent experiments is shown in the bar graph. *p<0.05, ns: non significant.

C) TER measurement of AJ disassembly (with SEM bars) in control and TRPM2-depleted human pulmonary artery ECs. siRNA depletion of TRPM2 suppressed AJ disassembly induced by VEGF. Representative tracings are shown. The mean response after VEGF stimulation is shown in bar graph from two independent experiments each consisting of 4 replicates.

D-E) EC migration measured using TER showing markedly reduced EC migration following VEGF stimulation in siRNA TRPM2-depleted or siRNA c-Src-depleted ECs compared to control ECs. Study was made after uniform injury of EC monolayers made at the arrow. Each trace is representative of 5 or more replicates. The mean response is shown for n=3 for TRPM2 siRNA and n=2 for c-Src siRNA.

F) Wound scratch healing motility assay carried out over 18 h showed that human ECs transfected with TRPM2 siRNA and c-Src siRNA exhibited impairment in wound closure as compared to control siRNA-treated human ECs. Experiment was repeated twice. Bar represent 50 μm.