Figure 3.

The P2RY12 receptor promotes VSMC-derived foam cells by inhibiting cholesterol efflux. (A) Confocal images (upper) or optical images (bellow) of VSMCs labeled by BODIPY or ORO. Ox-LDL-stimulated VSMCs were treated with vehicle control, a P2RY12 receptor activator (ADPβs), a P2RY12 receptor inhibitor (2-MeSAMP), or a combination of the two and then imaged 24 h post-stimulation. The average body (left) or ORO (right) area per cell was assessed. Scale bar: 10 μm. (B) Total cholesterol levels in VSMCs were determined as described in A. (C) WB and PCR analysis of VSMCs transfected with a lentivirus containing the control vector (shctrl) or the P2RY12 receptor shRNA (shP2ry12). (D) ORO images of ox-LDL-loaded VSMCs in the indicated group. The graph shows the area positive for ORO per cell. The scale: 10 μm. (E) Total cholesterol levels in VSMCs were quantified as described in D. (F) Representative fluorescence images of VSMCs obtained in the indicated group and labeled with DIL-oxLDL at 37°C for 4 h. Cells were rinsed and incubated in complete medium without Dil-oxLDL. Scale bar: 10 μm. (G) Representative flow cytometry histogram of DIL-oxLDL-processed VSMCs obtained in the indicated group. Experiments were analyzed by flow cytometry to determine the mean DIL-oxLDL burden (mFI). (H) Representative fluorescent images of the NBD-cholesterol burden in VSMCs obtained in the indicated group after incubation with HDL or APOA1 for 4 h. The HDL and APOA1-mediated cholesterol efflux (%) were analyzed in these groups. Scale bar: 10 μm. (I) HDL- and APOA1-mediated cholesterol efflux (%) was measured in shctrl-infected or shP2ry12-infected VSMCs stimulated with ADPβs or vehicle control. All data are presented as the mean ± SEM from 3 to 5 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001