TRPV2 and TRPM2 channels facilitate pulmonary endothelial barrier recovery after ROS-induced permeability

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), are known signaling molecules that increase endothelial barrier permeability. In this study, we investigated the roles of two redox-sensitive transient receptor potential (TRP) ion channels, TRPM2 and TRPV2, in H2O2- induced endothelial barrier dysfunction. Using primary human pulmonary microvascular endothelial cells (HPMEC), we employed impedance-based resistance measurements, Western blot, and immunofluorescence staining to assess the effects of H2O2 on the endothelial barrier. Exposure to sublytic concentrations of H2O2 caused an acute loss of endothelial barrier integrity, accompanied by the cleavage of vascular endothelial cadherin (VE-cadherin), which was also apparent after application of the TRPV2 activator cannabidiol. The inhibition of either TRPV2 with tranilast or a disintegrin and metalloprotease domain-containing protein 10 (ADAM10) with GI254023X significantly reduced H2O2-induced VE-cadherin cleavage, while TRPM2 inhibition by econazole significantly increased H2O2-driven VE-cadherin cleavage. Although inhibition of either TRP channel did not prevent the initial loss of barrier resistance upon H2O2 exposure, both were essential for the subsequent recovery of barrier integrity. Time-course immunofluorescence stainings revealed that HPMEC barrier recovery involved a transient localization of N-cadherin proteins at cell-cell junctions, which were replaced by VE-cadherin within 90 minutes. This process of cadherin-switching did not occur upon inhibition of TRPV2 or ADAM10. Our results highlight complementary roles for TRPM2 and TRPV2 as redox sensitive ion channels in the microvascular endothelium and provide insight into the mechanisms underlying pulmonary microvascular endothelial barrier recovery. Graphical Abstract: See text for more details O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/638083v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@5dbdacorg.highwire.dtl.DTLVardef@4f9bb5org.highwire.dtl.DTLVardef@77927dorg.highwire.dtl.DTLVardef@32894e_HPS_FORMAT_FIGEXP M_FIG C_FIG