Fig 2: Graphical abstract showing the effects of CAR activation in endothelial cells. Activation of CAR by a ligand, promotes its translocation from the cytoplasm into the nucleus and heterodimerization with RXRa. This complex inhibits NF-?B activation induced by TNFa and consequently the transcription of the proinflammatory genes MCP-1, RANTES and VCAM-1 in endothelial cells. Black lines mean activation. Red line with rectangle means inhibition.

Fig 3: Knockdown of CAR by siRNA blocks the suppressive effect of CITCO on TNFa-induced leukocyte–endothelial interactions and VCAM-1 expression. Endothelial cells were transfected with control or CAR-specific siRNA. At 48 h post-transfection, cells were pretreated with CITCO (3 µM) for 24 h and then stimulated with TNFa (20 ng/mL, 24 h). (A) CAR protein expression in transfected HUVEC was analyzed by Western blot. Protein quantification was performed by densitometry and CAR protein levels were normalized to ß-actin. Values are expressed as mean ± SEM (n = 6). +p < 0.05 relative to control siRNA transfected cells. (B) After transfection HUVEC were treated with vehicle (0.1% DMSO) or CITCO (3 µM) for 24 h and then stimulated with TNFa (20 ng/mL, 24 h). Next, whole blood from healthy volunteers was perfused across the endothelial monolayers for 5 min at 0.5 dynes/cm2 and leukocyte adhesion was quantified. Data are mean ± SEM (n = 4 for each group). * p < 0.05 relative to the respective vehicle group, + p < 0.05 relative to the respective TNFa-stimulated cells. (C) Representative images showing VCAM-1 expression in transfected HUVEC. Immunoreactivity was visualized using an Alexa Fluor 488 secondary antibody (VCAM-1, green). Nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. (D) Fluorescence intensity of Alexa Fluor 488 (VCAM-1, green) was quantified using ImageJ. Values are expressed as mean ± SEM, n = 6. * p < 0.05 relative to the respective vehicle group, + p < 0.05 relative to the respective TNFa-stimulated cells.

Fig 4: Effect of CITCO on leukocyte–endothelial cell interactions under physiological flow conditions. (A) HUVEC were treated with vehicle (0.1% DMSO) or CITCO (0.1–3 µM) for 24 h and then stimulated with TNFa (20 ng/mL, 24 h). Next, whole blood from healthy volunteers was perfused across the endothelial monolayers for 5 min at 0.5 dynes/cm2, and leukocyte adhesion was quantified. Data are mean ± SEM (n = 8 for each group). * p < 0.05 relative to vehicle group, + p < 0.05 relative to TNFa stimulated cells. Representative images of leukocyte adherence to HUVEC monolayers under different treatment conditions are also shown. Arrows indicate adhered cells. (B) Effect of CITCO on VCAM-1 expression. Cells were pretreated with vehicle (0.1% DMSO) or CITCO (3 µM) 24 h before TNFa stimulation (20 ng/mL, 24 h). Representative images of VCAM-1 expression in HUVEC are shown. Immunoreactivity was visualized using an Alexa Fluor 488 secondary antibody (VCAM-1, green). Nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. Values are expressed as mean ± SEM, n = 3. * p < 0.05 relative to vehicle group, + p < 0.05 relative to TNFa-stimulated cells.