Fig 1.
OxLDL-dependent release of TNF-α and other inflammatory factors from THP-1 macrophages.
TNF-α release from (A) THP-1 macrophages following 48 hours or (B) primary human macrophages following 24 hours of oxLDL (25 and 50 μg/mL) stimulation was determined by ELISA. LPS (100 ng/mL) was used as positive control. *P<0.05, **P<0.01, ***P<0.001 vs. control, n = 4–6 replicated experiments. (C) Conditioned medium from THP-1 macrophages following 48 hours of oxLDL (25 μg/mL) was analyzed by protein array. Conditioned medium from unstimulated THP-1 macrophages were used as control. Bar graph showing densitometric analysis of array membranes specific for inflammation-related factors. Factors with more than 2-fold increase after stimulation are indicated in the graph.
Fig 2.
Effect of conditioned media from oxLDL-stimulated THP-1 macrophages on endothelial adhesion molecules.
mRNA expression of the adhesion molecules (A) VCAM-1, (B) ICAM-1, (C) E-selectin and (D) P-selectin in HUVECs following stimulation with conditioned media from oxLDL stimulated THP-1 macrophages (oxLDL CM) was determined by real time PCR at the indicated time points. Conditioned medium from unstimulated THP-1 macrophages were used as control (control CM). *P<0.05, **P<0.01, ***P<0.001 vs. control CM, n = 4–5 replicated experiments.
Fig 3.
Effect of TNF-α inhibition with adalimumab on endothelial adhesion molecules.
mRNA expression of the adhesion molecules (A) VCAM-1, (B) ICAM-1 and (C) E-selectin in HUVECs following 6 hours of incubation with conditioned media from oxLDL-stimulated THP-1 cells (oxLDL CM) with or without adalimumab (ada, 1 μg/mL) was determined by real time PCR. Conditioned medium from unstimulated THP-1 macrophages were used as control (control CM). Protein expression of the adhesion molecules (D) VCAM-1, (E) ICAM-1 and (F) E-selectin in HUVECs following 6 hours of incubation with conditioned media from oxLDL-stimulated THP-1 cells (oxLDL CM) with or without adalimumab was determined by western blot. (G) Representative blots are shown. Conditioned medium from unstimulated THP-1 macrophages (control CM) and IgG isotype (1 μg/mL) were used as control. GAPDH was used as loading control for normalization. *P<0.05, **P<0.01, ***P<0.001 vs. control CM, #P<0.05, ##P<0.01 vs. oxLDL CM, n = 6–8 replicated experiments.
Fig 4.
Effect of TNF-α inhibition with adalimumab on adhesion of THP-1 monocytes to endothelial cells.
Adhesion assay under static conditions. (A) Fluorescence images depicting CellTracker green-labelled THP-1 monocytes on a HUVECs monolayer after incubation with conditioned media from oxLDL-stimulated THP-1 macrophages (oxLDL CM) with or without adalimumab (ada) for 4 hours followed by the addition of CellTracker green-labelled THP-1 monocytes. Pictures before and after washing with basal medium are shown. (B) Adherent cells per high power field were quantified after washing. (C) HUVECs monolayer was stimulated with TNF-α (10 ng/mL) for 4 hours followed by the addition of CellTracker green-labelled THP-1 monocytes. Adherent cells per high power field were quantified after washing. Adhesion assay under flow conditions. (D) Phase contrast images of THP-1 monocytes on a HUVECs monolayer after incubation with conditioned media from oxLDL-stimulated THP-1 macrophages (oxLDL CM) with or without adalimumab (ada) for 4 hours followed by the addition of THP-1 monocytes with a flow rate of 0.53 mL/min (0.5 dyne/cm2). (E) Adherent cells per high power field were quantified after washing. (F) HUVECs monolayer was stimulated with TNF-α (10 ng/mL) for 4 hours followed by the addition of THP-1 monocytes with a flow rate of 0.53 mL/min (0.5 dyne/cm2). Adherent cells per high power field were quantified after washing. Conditioned medium from unstimulated THP-1 macrophages (control CM), IgG isotype (1 μg/mL) and medium were used as control. Scale bar = 200 μm. Representative pictures are shown. ***P<0.001 vs. control CM or medium, ##P<0.01, ###P<0.001 vs. oxLDL CM, n = 3–4 replicated experiments.
Fig 5.
Effect of TNF-α inhibition with adalimumab on migration of THP-1 monocytes.
(A) Images depicting crystal violet stained THP-1 cells which migrated across the membrane of transwell inserts (pore size 8 μm) to the lower side after incubation with conditioned media from oxLDL stimulated THP-1 macrophages (oxLDL CM) with or without adalimumab (ada) for 4 hours. Representative pictures are shown. (B) Migrated cells per high power field were quantified. (C) Crystal violet stained THP-1 monocytes which migrated in response to TNF-α (10 ng/mL) across the membrane of transwell inserts (pore size 8 μm) to the lower side were quantified per high power field. Conditioned medium from unstimulated THP-1 macrophages (control CM), IgG isotype (1 μg/mL) and medium were used as control. Scale bar = 200 μm. Representative pictures are shown. ***P<0.001 vs. control CM. n = 3–4 replicated experiments.
Fig 6.
Effect of TNF-α inhibition with adalimumab on endothelial permeability.
A confluent monolayer of HUVECs on the membrane of transwell inserts (pore size 0.4 μm) was incubation with (A) conditioned media from oxLDL-stimulated THP-1 macrophages (oxLDL CM) with or without adalimumab (ada) or (B) with TNF-α (10 ng/mL) for 4 hours. Diffusion of Evan´s blue bound BSA across the endothelial monolayer from the luminal to the abluminal chamber was quantified by measuring absorbance at 620 nm of the abluminal chamber after additional 3 hours. (C) VE-cadherin immunofluorescence staining visualizes endothelial cell-to-cell adherens junctions. Conditioned medium from unstimulated THP-1 macrophages (control CM), IgG isotype (1 μg/mL) and medium were used as control. Scale bar = 25 μm. Representative pictures are shown. *P<0.05 vs. control CM or medium, #P<0.05 vs. oxLDL CM, n = 3–4 replicated experiments.
Fig 7.
Deposition of adalimumab in aorta, liver and spleen.
(A) Aortas of hypercholesterolemic Ldlr‒/‒ mice were washed and prepared en face 12 hours after injection of DyLight-549-labelled adalimumab (8.0 mg/kg, i.p.). Fluorescence images (bottom panels) from the aorta were captured before Oil Red O staining (top panels). Lesion area and vascular fluorescence accumulation are indicated by white arrows. Scale bars = 2.5 mm (top panel) and 200 μm (bottom panels). One representative experiment is shown. (B) Immunohistochemical analysis of adalimumab deposition with anti-human Fc or IgG control antibodies in liver and spleen of hypercholesterolemic Ldlr‒/‒ mice 12 hours after injection of adalimumab (8.0 mg/kg, i.p.). Scale bar = 100 μm. Representative pictures are shown.