Figure 4: Fasudil treatment prevents histamine- or PAF-stimulated vascular leakage and anaphylactic response.

(a) HUVECs were treated with histamine for 10 min, immunostained with anti-VE-cadherin, anti-vinculin antibodies, and stained with rhodamine-phalloidin. VE-cadherin, F-actin, vinculin and merged images are shown, boxed areas are enlarged (right). (b) The percentage of cells forming focal adherens junctions was quantified after treatment with Fasudil (n=30–40 cells per group). (c) Endothelial monolayers were treated with Fasudil (10 μM) 30 min prior to histamine treatment (orange) and challenged for permeability in vitro. Quantification of at least three independent experiments shown (n=5 per group). (d) Mice were treated with normal saline or Fasudil (20 mg kg⁻¹) by i.p. injection 2 h prior to in vivo permeability experiments. Evans blue extravasation was determined after subcutaneous injection of 20 μl of the indicated doses of histamine. Quantification of two independent experiments is shown (n=8 per group). (e) Schematic representation of the passive cutaneous anaphylaxis model. (f) Quantifications of Evans blue extravasation in ear biopsies correlate to the anaphylactic response in tamoxifen-treated RhoA^f/f Tom-GFP^f/f (control) versus RhoA^f/f Tom-GFP^f/f Chd5Cre-ERT² (RhoA ECKO) littermates. Quantification of two independent experiments is shown (n=8 per group). (g,h) C57 mice were treated with PBS or Fasudil 2 h before the experiment. Evans blue extravasation was determined in ear biopsies and representative images are shown. Quantification of two independent experiments is shown (n=8 per group). (i) Survival of mice sensitized with BSA and challenged 14 days later with BSA was monitored for 2 h. Fasudil treatment took place 2 h before the challenge (n=5 per group). (j) Mice were treated with Fasudil (20 mg kg⁻¹) by i.p. injection 2 h prior to in vivo permeability experiment. Evans blue extravasation was determined after subcutaneous injection of 20 μl of the indicated doses of PAF. Quantification of two independent experiments is shown (n=6 per group). (k) Survival of mice challenged with PAF i.v. injections was monitored for 2 h. Fasudil treatment took place 2 h before the challenge (n=7 per group). Statistical significance was determined by an unpaired Student’s t-test or by the Mantel–Cox test for survival experiments. Data represented as mean±s.e.m. NS, not significant; *P<0.05; **P<0.01; ***P<0.001.