The platelet-derived Wnt antagonist Dkk1 is implicated in the stimulation of TNFα-mediated ICAM-1/VCAM-1 expression in AECs. E10 cells were incubated with 50% Con_CM or Wnt3a_CM together with 200 μM Dkk1 for 12 hours and then stimulated with 20 ng/mL TNFα for 2 hours (A-B) or 24 hours (C). Platelets were isolated from LM-challenged mice, resuspended in Tyrode-HEPES buffer, and treated with 0.1 U/mL thrombin for 90 minutes. Platelet releasate (PR) was collected and added to cultured primary mouse AECs together with isotype-matched control IgG or a function-blocking monoclonal antibody against Dkk1 (10 μg/mL) for 24 hours. After that, mouse AECs were stimulated with TNFα (20 ng/mL) for 2 hours (D-E). The mRNA levels of ICAM-1 (A,D) and VCAM-1 (B,E) were measured by real-time PCR using mouse 18S rRNA as a reference gene and normalized to control untreated cells. (C) Protein levels of ICAM-1, VCAM-1, and ABC were determined by western blotting with β-actin as an internal control. Data are expressed as the mean ± SEM from 3 independent preparations and statistical significance was tested with ANOVA analysis followed by post hoc Tukey test. *, **, ***, and # represent P < .05.