Inhibition of Low density Lipoprotein Internalization and Transcytosis by HDL; an alternative role for “good” cholesterol

Atherosclerosis results from the build‐up of low‐density lipoprotein (LDL) cholesterol and immune cells in the arteries leading to their occlusion. High‐density lipoprotein (HDL) is believed to reverse this process by removing the arterial cholesterol from the body. Both LDL and HDL reach beneath the artery by crossing the endothelium through Scavenger receptor B1 (SR‐B1) mediated transcytosis. ApoAI, the protein exclusively found on HDL, mediates the interaction between HDL and SR‐B1. One natural ApoAI variant called Milano (ApoAI‐Mil) is of interest because injections of lipidated ApoAI‐Mil led to enhanced plaque regression. We hypothesize that the natural ability of ApoAI‐Mil to dimerize leads to better interaction with SR‐B1. This could result in better inhibition of LDL transcytosis since both HDL and LDL bind to SR‐B1 for transcytosis and this could also improve HDL transcytosis so that more HDL will reach the target tissue to extract cholesterol. We took an in vitro microscopy approach to quantify internalization of fluorescently labeled LDL in coronary endothelial cells. The ApoAI variants were also lipidated to form fluorescent HDL‐like particles (DiI‐DMPC‐WT or DiI‐DMPC‐Mil) to measure its association with SR‐B1 overexpressing cells. We observed that recombinant ApoAI‐WT can inhibit LDL internalization in coronary endothelial cells. There was also about an 8x‐fold increase of DiI‐DMPC‐WT fluorescence signal in HeLas overexpressing SR‐B1 compared to GFP alone. Furthermore, there was an even higher fold increase (~11x) of DiI‐DMPC‐Mil fluorescence signal compared to GFP alone. Similarly, excess recombinant ApoAI‐Mil led to greater inhibition of LDL internalization compared to ApoAI‐WT. There was no difference in the amount of DiI‐DMPC‐WT or ‐Mil associated with HeLa cells overexpressing Alk1, a LDL‐specific transcytosis receptor. Together this suggests that compared to ApoAI‐WT, ApoAI‐Mil more readily associates with only SR‐B1 which may lead to better inhibition of LDL internalization and to more HDL available to remove arterial cholesterol. Support or Funding Information Early Research Award & CIHR