Figure 4. RvD1 inhibits cholesterol crystals-induced H₂O₂ production in suppressing Frk activation, AJ disruption and endothelial barrier dysfunction.

A. Quiescent HAEC were treated with and without CC (200 μg/ml) in presence and absence of RvD1 (200 ng/ml) for 10 min and H₂O₂ production was measured. B-E. Quiescent HAEC were treated with and without CC (200 μg/ml) in presence and absence of RvD1 (200 ng/ml) for the indicated time periods and cell extracts were prepared. Equal amounts of proteins from control and each treatment were immunoprecipitated with PY20, VE-cadherin or α-catenin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins. The same cell extracts were also analyzed by IB for total Frk, α-catenin, β-catenin, p120 catenin or VE-cadherin to show that the treatments do not affect their steady state levels. F-H. Quiescent HAEC were treated with and without CC (200 mg/ml) in presence and absence of RvD1 (200 ng/ml) for 2 hrs and examined for either AJ integrity by double immunofluorescence staining for VE-cadherin and α-catenin (DAPI for nucleus) or subjected to flux assay or for the indicated time periods and TER was measured. The bar graphs represent Mean ± SD values of three experiments. *, p < 0.05 vs control; **, p < 0.05 vs CC.