Figure 2: siRNA knockdown of EMCN leads to increased neutrophil–endothelial cell interactions.

HUVECs seeded at 50% confluence were transfected with siRNA oligonucleotides targeted against EMCN. Analysis was performed 48 h after transfection on confluent endothelial cell monolayers. (a) siRNA led to a more than 80% knockdown of EMCN as determined by western blot in HUVECs. (b) Flow chamber studies revealed that at shear stresses of 0.5–1.0 dynes per cm² there was >6-fold increase in adhesion of neutrophils compared to scramble-treated HUVECs. Representative images showing a freeze frame of neutrophils interacting with siScramble and siEMCN transfected HUVECs are shown. (c) Knockdown of EMCN did not alter the expression of pro-adhesive molecules, E-selectin, VCAM-1 or ICAM-1, as measured by flow cytometry. (d) Neutralizing antibody to LFA-1 reversed cell–cell interactions (including rolling and adherent cells) in siEMCN-treated HUVECs. Values for siRNA in (a) are expressed as mean±s.e.m., and results are representative of three independent experiments. Data in (c) represent one of the three independent experiments performed. In (b) and (d), results are representative of two to three human donors, and flow chamber experiments were performed in triplicate conditions, mean±s.e.m. Significance was determined for (a) using Student’s t-test or (b and d) using one-way ANOVA followed by Newman–Keuls post hoc test. *P<0.05, **P<0.01 and ***P<0.001; NS, nonsignificant. Scale bar, 50 μm.