Effect of APC on the nuclear cytokine-mediated proinflammatory responses in the absence and presence of polyP-70 in cellular and animal models. (A) Confluent endothelial cells were treated with APC (20-40 nM) before measuring cell permeability in response to polyP-70 (2.5 µM) + HMGB1 (10 nM). (B) The same as (A), except that the effect of APC on the polyP-70-mediated permeability in response to H4 (0.44 µM) was measured. (C) The effect of APC (10-40 nM) on the H4 (3.5 µM)-mediated expression of either VCAM-1 (gray bar) or E-selectin (black bar) was studied. (D) The same as (C), except that the effect of APC on the polyP-70 (2.5 μM)-mediated expression of ICAM-1 (white bar), VCAM-1 (gray bar), and E-selectin (black bar) by H4 (0.44 µM) was monitored. (E) The same as (D), except that the effect of APC on the polyP-70 (2.5 μM)-mediated expression of ICAM-1 (white bar), VCAM-1 (gray bar), and E-selectin (black bar) by HMGB1 (10 nM) was monitored. (F-H) In vivo analysis of the effect of HMGB1, H4, and polyP-70 on vascular leakage. Mice (n = 3 for every experiment) were intravenously injected with 1% bovine serum albumin-bound Evans blue dye followed by an immediate intraperitoneal injection of HMGB1 (1-5 µg/g body weight), H4 (2.5-10 µg/g body weight), or polyP-70 (35-150 µg/g body weight) with 0.7% acetic acid as a positive control. Vascular permeability was determined from the extent of extravasation of Evans blue to the peritoneal cavity as described in the Materials and methods section. (I) The same as above (F-H), except that the effect of APC (0.2 µg/g body weight) on in vivo permeability was monitored in response to polyP-70 (35 µg/g body weight) plus either HMGB1 (1 µg/g body weight) or H4 (2.5 µg/g body weight). (J-M) The same as (F-I), except that the in vivo analysis of the effect of polyP-70, HMGB1, and H4 on the migration of leukocytes to peritoneal cavity in the absence and presence of APC treatment was studied. *P < .05; **P < .01; ***P < .001.