Figure 6.

Rac1 deactivation results in barrier breakdown that is not aggravated by lack of IQ domain GTPase-activating protein 1 (IQGAP1). A, Immunocytochemistry for VE-cadherin and 4,6-diamidino-2-phenylindole (DAPI) from confluent human microvascular endothelial cells (HMVECs) 2 hours after treatment with NSC 23677 (25 mmol/L) or vehicle. Red arrows highlight severe paracellular gap formation similar to IQGAP1-silenced cells (representative of 3 experiments, scale bars=10 μm). B, Normalized transendothelial electrical resistance (TER) was measured in real time after treatment with NSC 23677 or control vehicle. After 2 hours—when TER was noted to drop continuously—medium was carefully changed to test for reversibility of NSC-induced permeability (n=4 per condition). TER normalization refers to the start point of the experiment for each condition (ie, 1 hour before NSC treatment). C, TER was recorded in real time similarly to the method described in B. At the start of the experiment, HMVECs were treated with angiopoietin-1 (Angpt-1) (300 ng/mL, orange and red) or control vehicle (green and blue). One hour later, NSC 23677 (blue and red) or control vehicle was added, and TER followed for another 6 hours. NSC-induced permeability was notable after 2 hours and was also not reversed by pretreatment with Angpt-1 (n=2 per condition). TER normalization refers to the start point of the experiment for each condition (ie, 30 minutes before Angpt-1 treatment). D, TER 2 hours after treatment with NSC 23677 or control vehicle in control or IQGAP1-silenced (48 hours) HMVECs. NSC-induced permeability was not aggravated by silencing of IQGAP1 (n=2 per condition). TER normalization refers to the start point of the experiment for each condition (ie, 36 hours after small interfering RNA [lsqb]siRNA[rsqb] transfection). ns indicates not significant.