Endothelial ROS production induces VE-cadherin/VE-PTP dissociation. (A) Scavenging of ROS inhibits T-cell–induced VE-cadherin/VE-PTP dissociation. Preincubation of TNF-α–stimulated bEnd.5 cells with or without the ROS scavenger NAC (1 mM) (as indicated above) was followed by incubation with T cells and subsequent VE-PTP/VE-cadherin coimmunoprecipitation analysis. (B) T-cell adhesion induces ROS production in endothelial cells. TNF-α–stimulated bEnd.5 cells were loaded with the fluorescent ROS indicator DCF and were subsequently incubated either with H2O2 (as a positive control) or with T cells, followed by recording intracellular fluorescence. (C) Inhibition of ROS production prevents VE-cadherin/VE-PTP dissociation. TNF-α–stimulated bEnd.5 cells were pretreated with 2 μM of the flavoprotein inhibitor DPI, followed by incubation with or without T cells and subsequent VE-PTP/VE-cadherin coimmunoprecipitation analysis. (D) The same process was done as in panel C, but bEnd.5 cells were stimulated with anti-VCAM-1–loaded beads (VCAM XL) instead of T cells. (E) NOX inhibition prevents VE-cadherin/VE-PTP dissociation. TNF-α–stimulated bEnd.5 cells were preincubated with the specific NOX inhibitor VAS2870 (10 μM) and were subsequently analyzed as was done in panel C. (F) NOX inhibition prevents VE-cadherin/VE-PTP dissociation in murine primary lung endothelial cells. TNF-α–stimulated cells were pretreated with 10 μM of the VAS2870 NOX inhibitor, followed by stimulation with anti-VCAM-1–loaded beads (VCAM XL) and subsequent VE-PTP/VE-cadherin coimmunoprecipitation analysis.
