Miriam Lee-Rueckert1, Francisco Blanco-Vaca2,3,4, Joan Carles Escolà-Gil2,3,4, Lídia Cedó2,4, Jari Metso5, David Santos2,4, Annabel García-León2, Núria Plana4,6, Sonia Sabate-Soler2, Noemí Rotllan2, Andrea Rivas-Urbina2,3, Karen A Méndez-Lara2,3, Mireia Tondo2, Josefa Girona6, Josep Julve2,3,4, Victor Pallarès2, Aleyda Benitez-Amaro7, Vicenta Llorente-Cortes7,8, Antonio Pérez2,3,4, Diego Gómez-Coronado9,10, Anna-Kaisa Ruotsalainen11, Anna-Liisa Levonen11, José Luis Sanchez-Quesada2,3,4, Luís Masana4,6, Petri T Kovanen1, Matti Jauhiainen5. 1. and Wihuri Research Institute, Helsinki, Finland (P.T.K., M.L.-R.). 2. From the Institut d'Investigacions Biomèdiques Sant Pau, Barcelona, Spain (L.C., D.S., A.G.-L., S.S.-S., N.R., A.R.-U., K.A.M.-L., M.T., J.J., V.P., A.P., J.L.S.-Q., F.B.-V., J.C.E.-G.). 3. Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Spain (A.R.-U., K.A.M.-L., J.J., A.P., J.L.S.-Q., F.B.-V., J.C.E.-G.). 4. CIBER de Diabetes y Enfermedades Metabólicas Asociadas, CIBERDEM, Madrid, Spain (L.C., D.S., N.P., J.J., A.P., J.L.S.-Q., L.M., F.B.-V., J.C.E.-G.). 5. Minerva Foundation Institute for Medical Research and National Institute for Health and Welfare, Genomics and Biomarkers Unit, Biomedicum, Helsinki, Finland (J.M., M.J.). 6. Vascular Medicine and Metabolism Unit, Research Unit on Lipids and Atherosclerosis, Sant Joan University Hospital, Rovira i Virgili University, IISPV, Reus, Spain (N.P., J.G., L.M.). 7. CIBER en Bioingeniería, Biomateriales y Nanomedicina, Institut de Recerca Josep Carreras, Barcelona, Spain (V.P.); Biomedical Research Institute Sant Pau (IIB Sant Pau), Institute of Biomedical Research of Barcelona-Spanish National Research Council (A.B.-A., V.L.-C.). 8. Centro de Investigación Biomédica en Red Enfermedades Cardiovasculares, Instituto de Salud Carlos III, Madrid, Spain (V.L.-C.). 9. Servicio de Bioquímica-Investigación, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain (D.G.-C.). 10. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain (D.G.-C.). 11. University of Eastern Finland, A.I. Virtanen Institute for Molecular Sciences, Kuopio (A.-K.R., A.-L.L.).
Abstract
RATIONALE: The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown. OBJECTIVE: We investigated LDL contributions to the m-RCT pathway in hypercholesterolemic mice. METHODS AND RESULTS: Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (human APOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B-containing lipoproteins. CONCLUSIONS: Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces. Graphical Abstract: A graphical abstract is available for this article.
RATIONALE: The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown. OBJECTIVE: We investigated LDL contributions to the m-RCT pathway in hypercholesterolemicmice. METHODS AND RESULTS: Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (humanAPOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B-containing lipoproteins. CONCLUSIONS: Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces. Graphical Abstract: A graphical abstract is available for this article.
Authors: Andrea Rivas-Urbina; Noemi Rotllan; David Santos; Josep Julve; Jose Luis Sanchez-Quesada; Joan Carles Escolà-Gil Journal: Methods Mol Biol Date: 2022