A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis

Aims Retention of low-density lipoprotein (LDL) cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosisdifficult. We developed a novel assay for measuring LDL transcytosisin real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. Methods and results Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosisoccurs in intact vessels. We then confirmed that LDL transcytosisoccurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosisof DiI-LDL in real time using total internal reflectionfluorescence microscopy. DiI-LDL transcytosiswas inhibited by excess unlabelled LDL, while degradation of the LDL receptorby PCSK9had no effect. Instead, LDL colocalized partially with the scavenger receptorSR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI -deficient mice were perfused ex vivo with LDL and accumulated significantly less sub-endothelial LDL compared with wild-type littermates. Conclusion We developed an assay to quantify LDL transcytosisacross endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosismay identify novel targets for the prevention or therapy of atherosclerosis.