Figure 2.

Primary HCAECs in culture retain endothelial characteristics and internalize LDL in a polarized fashion. (A) Coronary artery endothelial cells exhibit cobblestone morphology under phase contrast microscopy (left panel), as well as continuous rings of occludin (middle) and VE-cadherin (right panel) as determined by immunostaining. Images are z-stack projections and are representative of >5 independent experiments; nuclei are stained with DAPI (blue). Size bar is 19 µm. (B) Confluent HCAECs were pulsed with DiI-conjugated LDL and then rinsed to remove unbound ligand. After incubation at 37°C, monolayers were fixed at progressive time points and imaged by confocal microscopy. The apical membrane is labelled with a fluorescein-tagged lectin (green). Long images are side views (yz-axis) of separate cells with corresponding xy images shown at left for reference. xy images were taken 5 slices from the top of the cell in both cases, and white arrows denote vesicles containing LDL. Note the apical localization of LDL (red) at time zero (indicated by colocalization with the green lectin) followed by its movement towards the basal membrane (dotted line) 30 min later. Quantification is shown at right. Scale bar represents 3 µm, nuclei are stained with DAPI (blue); n = 4 independent experiments; *P < 0.001 by Student's t-test for 0 vs. 30 min.