A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis.
OBJECTIVE
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 transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved.


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 transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-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.


CONCLUSIONS
We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.
Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8 Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.
Susan M
SM
Armstrong
Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
Michael G
MG
Sugiyama
Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8.
Karen Y Y
KY
Fung
Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8.
Yizhuo
Y
Gao
Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8.
Changsen
C
Wang
Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8.
Andrew S
AS
Levy
Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8.
Paymon
P
Azizi
Toronto General Research Institute (TGRI), Toronto, Canada.
Mark
M
Roufaiel
Toronto General Research Institute (TGRI), Toronto, Canada.
Su-Ning
SN
Zhu
Hospital for Sick Children, Toronto, ON, Canada.
Dante
D
Neculai
Department of Microbiology and Immunology, The University of Western Ontario, London, ON, Canada.
Charles
C
Yin
Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8.
Steffen-Sebastian
SS
Bolz
Montreal Diabetes Research Centre, Montreal, QC, Canada.
Nabil G
NG
Seidah
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Toronto General Research Institute (TGRI), Toronto, Canada.
Myron I
MI
Cybulsky
Department of Microbiology and Immunology, The University of Western Ontario, London, ON, Canada.
Bryan
B
Heit
Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8 Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Interdepartmental Division of Critical Care Medicine and the Department of Medicine, University of Toronto, Toronto, ON, Canada LeeW@smh.ca.
Warren L
WL
Lee
D000818:Animals; D001011:Aorta / Q000378:metabolism; D002478:Cells, Cultured; D008078:Cholesterol, LDL / Q000378:metabolism*; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism; D051122:Scavenger Receptors, Class B / Q000502:physiology*; D012697:Serine Endopeptidases / Q000378:metabolism; D057900:Transcytosis
D008078:Cholesterol, LDL; D011973:Receptors, LDL; C495124:SCARB1 protein, human; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
Atherosclerosis; Endothelium; LDL; SR-BI; Transcytosis