HDL and Apo A-I both enhance endothelial cell (EC) barrier protection by APC against thrombin-induced permeability. (A) The ability of the plasma lipoprotein HDL (1 mg/mL; red) to modulate protection against thrombin (5 nM) disruption of the EC barrier by APC (0.6-5 nM; blue) was measured. (B) Similarly, the ability of LDL (1 mg/mL) to mediate enhancement of APC barrier protection was tested under the same experimental conditions. (C) Evaluation of HDL and LDL enhancement of APC-mediated barrier protection was performed by titration of either HDL (blue) or LDL (red) in the presence of APC (5 nM) before thrombin (5 nM) exposure. (D) The ability of Apo A-I or Apo A-II (both 200 µg/mL) to enhance APC-mediated barrier protection was measured in EA.hy926 cells. (E) The stimulatory effect of Apo A-I on APC was confirmed by using primary human umbilical vein ECs. (F) Apo A-I (100-200 µg/mL) enhancement of APC barrier protection was titrated using the same assay. (G) To confirm that bioactive lipids potentially copurified with Apo A-I were not responsible for enhancement of APC barrier-protective function, the assay was repeated with either plasma-derived or recombinant Apo A-I (p-Apo A-I and r-Apo A-I, respectively; both 200 µg/mL). Experiments were performed in at least triplicate, and results are presented as the mean ± standard deviation. Statistical significance was determined by using the Student t test. *P < .05; **P < .01. n.s., not significant.