Atherosclerosis, which underlies life-threatening cardiovascular disorders such as myocardial infarction and stroke1, is initiated by passage of low-density lipoprotein (LDL) cholesterol into the artery wall and its engulfment by macrophages, which leads to foam cell formation and lesion development2,3. It is unclear how circulating LDL enters the artery wall to instigate atherosclerosis. Here we show in mice that scavenger receptor class B type 1 (SR-B1) in endothelial cells mediates the delivery of LDL into arteries and its accumulation by artery wall macrophages, thereby promoting atherosclerosis. LDL particles are colocalized with SR-B1 in endothelial cell intracellular vesicles in vivo, and transcytosis of LDL across endothelial monolayers requires its direct binding to SR-B1 and an eight-amino-acid cytoplasmic domain of the receptor that recruits the guanine nucleotide exchange factor dedicator of cytokinesis 4 (DOCK4)4. DOCK4 promotes internalization of SR-B1 and transport of LDL by coupling the binding of LDL to SR-B1 with activation of RAC1. The expression of SR-B1 and DOCK4 is increased in atherosclerosis-prone regions of the mouse aorta before lesion formation, and in human atherosclerotic arteries when compared with normal arteries. These findings challenge the long-held concept that atherogenesis involves passive movement of LDL across a compromised endothelial barrier. Interventions that inhibit the endothelial delivery of LDL into artery walls may represent a new therapeutic category in the battle against cardiovascular disease.
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This work was supported by NIH grants R01HL131597 (P.W.S.), R01HL126795 (C.M.), R01HL084312 and R01HL129433 (E.A.F.), American Heart Association (AHA) Postdoctoral Fellowship Award 16POST30250019 (L.H.), AHA Innovative Research Grant 17IRG33410377 (W.-P.G.), AHA Grant-in-Aid 17GRNT33650076 (P.M.), Dan Adams Thinking Outside the Box Award from the Henrietta B and Frederick H. Bugher Foundation (W.-P.G.), the Rally Foundation (L.X.), and the Children’s Cancer Foundation (L.X.). We thank H. Cheng for providing albumin-Cre mice. TIRF assays were carried out in the UT Southwestern Live Cell Imaging Facility, and electron microscopy was performed in the UT Southwestern Electron Microscopy Core Facility with support from NIH Grant 1S10OD021685.
Nature thanks Carlos Fernandez-Hernando, Warren Lee and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, j, Schematics of the gene-targeting strategies to generate floxed alleles of SR-B1 (a) and Pdzk1 (j) to create SR-B1fl/fl and Pdzk1fl/fl mice. Exon 2 of SR-B1 and exons 2 and 3 of Pdzk1were floxed. The recombined alleles following introduction of Cre recombinase are also shown. b, k, Using primers depicted in a and j, PCR-based genotyping was performed on aortas with or without intact endothelial cells (ECs). Aortas were obtained from floxed mice and mice with SR-B1 or PDZK1 deleted selectively from the endothelium (SR-B1∆EC or PDZK1∆EC). In genotyping to evaluate SR-B1 excision (b), additional samples were obtained from SR-B1fl/fl mice expressing Cre recombinase globally (gCre). In genotyping to evaluate Pdzk1 excision (k), lung samples were also studied. c, l, qRT–PCR analysis of expression of SR-B1 (c) or Pdzk1 (l) in primary aortic endothelial cells (c, n = 7 and 3 for SR-B1fl/fl and SR-B1∆EC, respectively; l, n = 6 per group) and bone marrow-derived myeloid lineage cells (c, n = 5 and 4 for SR-B1fl/fl and SR-B1∆EC, respectively; l, n = 5 per group)). d–f, SR-B1 protein abundance (d, e, n = 4) and Alk1 transcript levels (f, n = 4) were evaluated in aortic endothelial cells from SR-B1fl/fl and SR-B1ΔEC mice; summary data for SR-B1 protein are in e. The uncropped versions of all immunoblots shown are provided in Supplementary Fig. 1. CNX, calnexin. g, m, Immunoblotting of SR-B1 (g) or PDZK1 (m) protein abundance in liver. h, n, Plasma cholesterol levels. For SR-B1 studies, n = 4, 3, 9 and 10 mice for bars from left to right; for PDZK1 studies, n = 4, 3, 6 and 6 mice. i, o, Representative lipoprotein profiles. Data are mean ± s.e.m.; in c, e, i, P values calculated by two-sided Student’s t-test; in h, n, P values calculated by ANOVA with Dunnett’s post-hoc test.
Extended Data Fig. 2 Atherosclerosis is promoted by endothelial SR-B1 but not by endothelial PDZK1, and neither endothelial SR-B1 nor PDZK1 affects circulating lipids.
Findings are shown for SR-B1 in mixed background females (a–g) and C57BL/6 males (h–n), and for PDZK1 in C57BL6 males (o–u). a, h, o, Representative in situ aortic arch images of atherosclerotic plaque (yellow arrows); b, i, p, representative images for lipid-stained lesions in en face aortas; d, k, r, representative images for lipid and haematoxylin-stained aortic root sections (lesions outlined by yellow dashed line, magnification 40×). c, j, q, Quantification of lesion areas in en face aortas (per cent total surface area); c, n = 13 and 14 for SR-B1fl/fl and SR-B1∆EC, respectively; j, n = 10 and 9, respectively; q, n = 14 per group. e, l, s, Quantification of lesion areas in in aortic root sections; e, n = 9 per group; l, n = 10 and 9 for SR-B1fl/fl and SR-B1∆EC, respectively; s, n = 10 and 11, respectively. f, m, t, Plasma total cholesterol (TC), triglyceride, and HDL cholesterol (HDL-c). f, n = 12 per group for TC and TG, 10 per group for HDL-c; m, n = 16 and 9 for SR-B1fl/fl and SR-B1∆EC, respectively, for TC and TG, and n = 10 and 9, respectively, for HDL-c; t, n = 14 per group for TC and TG, and 10 per group for HDL-c. Findings for SR-B1fl/fl, SR-B1ΔEC and PDZK1ΔEC are shown in blue, red and orange, respectively. g, n, u, Representative lipoprotein profiles. Data are mean ± s.e.m; in c, e, j, l, P values calculated by two-sided Student’s t-test.
Extended Data Fig. 3 Endothelial SR-B1 promotes atherosclerosis in LDLR null mice by driving LDL entry into the artery wall.
a, Representative in situ aortic arch images of atherosclerotic plaque (yellow arrows) in male Ldlr−/−SR-B1fl/fl and Ldlr−/−SR-B1∆EC mice. b, Representative lipid-stained en face images of aortas. c, Lesion areas in en face aortas (per cent of total surface area); n = 10 and 8 for Ldlr−/−SR-B1fl/fl and Ldlr−/−SR-B1∆EC, respectively. d, Representative lipid and haematoxylin-stained aortic root sections (lesions outlined by yellow dashed line, magnification 40×). e, Lesion areas in aortic root sections; n = 10 and 8, respectively. f–h, Plasma total cholesterol (f), triglyceride (g), and HDL cholesterol (h); n = 10 and 8, respectively. i, Representative lipoprotein profiles. j, k, DiI–nLDL uptake in the aorta. Human apolipoprotein B abundance (j) or DiI fluorescence intensity (k) was evaluated in aorta homogenates 4 h after intravenous DiI–nLDL injection. j, Left, representative immunoblot with three samples per group; j, k, n = 5 and 4 for Ldlr−/−SR-B1fl/fl and Ldlr−/−SR-B1∆EC, respectively. l, m, Uptake of DiI-labelled mouse LDL (l) or mouse VLDL/IDL (m) in aorta from Ldlr−/−SR-B1fl/fl and Ldlr−/−SR-B1∆EC mice (n = 4 and 5, respectively). Data are mean ± s.e.m., P values calculated by two-sided Student’s t-test.
Extended Data Fig. 4 Endothelial SR-B1 and hepatocyte SR-B1 have opposing effects on atherosclerosis.
Using AAV8-PCSK9, hypercholesterolaemia was induced in male SR-B1fl/fl mice, and in SR-B1∆EC or SR-B1∆HEP mice, which lack SR-B1 selectively in endothelial cells or hepatocytes, respectively. a–l, Findings in SR-B1fl/fl versus SR-B1∆EC mice; m–v, findings in SR-B1fl/fl versus SR-B1∆HEP mice. a, Representative in situ aortic arch images of atherosclerotic plaque (yellow arrows) in SR-B1fl/fl and SR-B1∆EC mice. b, Representative lipid-stained en face images of aortas. c, Lesion areas in en face aortas (per cent of total surface area); n = 12 and 10 for SR-B1fl/fl and SR-B1∆EC mice, respectively. d, Representative lipid and haematoxylin-stained aortic root sections (lesions outlined by yellow dashed line, magnification 40×). e, Lesion areas in aortic root sections; n = 10 and 9, respectively. f–h, Plasma total cholesterol (f), triglyceride (g), and HDL cholesterol (h), n = 12 and 11, respectively. i, Representative lipoprotein profiles. j, k, Uptake of DiI–nLDL in the aorta. Human apolipoprotein B abundance (j) or DiI fluorescence intensity (k) was evaluated in aorta homogenates 4 h after intravenous injection (n = 6 and 5, respectively). j, Left, representative immunoblot with three samples per group. l, LDLR abundance in livers of control, SR-B1fl/fl and SR-B1∆EC mice administered AAV-PCSK9. Immunoblot depicts protein abundance for two samples per group. m, Survival curves; n = 5 and 4 for SR-B1fl/fl and SR-B1∆HEP mice, respectively. n, Representative lipid-stained en face images of aortas. o, Lesion areas in en face aortas. Aortas and plasma were available from only two SR-B1∆HEP mice. p, Longitudinal sections of SR-B1fl/fl and SR-B1∆HEP hearts stained with haematoxylin and eosin (H&E) or trichrome (healthy myocardium, red/brown; fibrotic tissue, blue; yellow asterisks and arrows indicate areas of severe fibrosis). Images shown are representative of those obtained in all three hearts per group that underwent histological analysis. q, Coronary arteries of SR-B1fl/fl and SR-B1∆HEP mice stained with H&E or trichrome, and coronary artery of SR-B1∆HEP mouse stained with anti-CD68 to detect macrophages. r–t, Plasma total cholesterol, triglyceride, and HDL cholesterol. u, Representative lipoprotein profiles. v, Immunoblotting of SR-B1 abundance in liver, showing findings for two samples per group. Data are mean ± s.e.m; P values calculated by two-sided Student’s t-test.
a, qRT–PCR was used to compare Cd68 transcript levels in aortas from Apoe−/−SR-B1fl/fl and Apoe−/−SR-B1∆EC male mice, n = 8 per group. b–i, mRNA abundance was also evaluated for the following genes, using Hprt1 as a housekeeping gene and normalizing expression to Cd68 levels: E-selectin (Sele; b), P-selectin (Selp; c), Vcam1 (d), Icam1 (e), Tgfb1 (f), Tnf (g), Il6 (h), and Il10 (i), with n = 8 per group. j, Representative still images of leukocyte–endothelial cell adhesion evaluated by intravital microscopy in the mesenteric microcirculation of Apoe−/−SR-B1fl/fl and Apoe−/−SR-B1∆EC male mice administered vehicle (n = 10 and 11, respectively) or TNF (n = 5 and 6, respectively). k, Summary data for leukocyte velocity in four study groups in j. l, Gating strategy for evaluation of CD45+F4/80+ cell number and uptake of DiI–LDL in the aorta. Following digestion of the aorta, all cells were first gated in FSC/SSC according to cell size and granularity. The resulting population was gated according to cell viability using DAPI. DAPI-negative live cells were gated for positivity for CD45, and CD45+ cells were then gated for positivity for F4/80 and the DiI label. Data are mean ± s.e.m.; in k, P values calculated by ANOVA with Dunnett’s post-hoc test.
a, Three-dimensional depiction of localization of DiI–oxLDL determined by confocal fluorescence microscopy of the luminal surface of the ascending aorta of Apoe−/−SR-B1fl/fl and Apoe−/−SR-B1∆EC mice. Red, DiI; blue, Hoechst staining of nuclei. b, Representative cumulative images of the x–y plane parallel to the luminal surface. c, Summation of DiI–oxLDL signal in the superficial ascending aorta. Two areas encompassing at least 100 cells each were counted per mouse in three mice per group for a total of n = 6 areas per genotype group. d, e, Uptake of DiI–oxLDL in the aorta. Human apolipoprotein B abundance (d) and DiI fluorescence intensity (e) were evaluated in aorta homogenates 4 h after intravenous DiI–oxLDL injection; n = 8 mice per group. f, g, Using the same approaches as in d, e, uptake of DiI–oxLDL in the aorta was evaluated in Apoe−/− mice treated with control or SR-B1-blocking antibodies given intraperitoneally before intravenous injection of DiI–oxLDL (n = 6 and 7 for control and anti-SR-B1 antibodies, respectively). h, Quantification of CD45+F4/80+ macrophages in the aorta (n = 6 aortas per group). Results are expressed relative to abundance in Apoe−/−SR-B1fl/fl control mice. i, Distribution of DiI–oxLDL in CD45+F4/80+ macrophages in the aorta; n = 6 mice per group. j, k, Uptake of DiI–nLDL in the aorta. Human apolipoprotein B abundance (j) or DiI fluorescence intensity (k) was evaluated in aorta homogenates 4 h after intravenous DiI–nLDL injection; n = 7 and 8 for Apoe−/−SR-B1fl/fl and Apoe−/−SR-B1∆EC mice, respectively. l, m, Using the same approaches as in j, k, uptake of DiI–nLDL in the aorta was evaluated in Apoe−/− mice treated with control or SR-B1 blocking antibodies given intraperitoneally before intravenous injection of DiI–nLDL (n = 5 mice per group). Left panels in d, f, j, l show representative immunoblots with three samples per group; data are mean ± s.e.m.; P values calculated by two-sided Student’s t-test. See also Supplementary Videos 1, 2.
Extended Data Fig. 7 Low-power electron micrograph images of gold-labelled LDL and immunogold-labelled SR-B1 in aortic endothelial cells in vivo.
Following intravascular administration in wild-type mice, gold-labelled LDL particles and SR-B1 were localized in aortic endothelial cells by electron microscopy. The panels show images for different endothelial cells, each bordered by the lumen and elastic lamina. Shown are the locations of the high-power fields provided in Fig. 1n, with gold-labelled LDL (large particles) highlighted by yellow arrows and immunogold-labelled SR-B1 (small particles) highlighted by red arrows.
Extended Data Fig. 8 SR-B1 governs LDL transcytosis in endothelial cells independent of effects on caveolae function.
a, Uptake of DiI–nLDL and DiI–oxLDL in endothelial cells after RNAi knockdown of SR-B1 or Pdzk1. Left, red, DiI–oxLDL; blue, DAPI-stained nuclei. n = 6 per group. b, c, Uptake (b) and transcytosis (c) of DiI–nLDL and DiI–oxLDL in cells treated with control IgG, SR-B1 blocking antibody or BLT1. n = 6 per group. d, Transcytosis of DiI–nLDL and DiI–oxLDL in endothelial cells after RNAi knockdown of SR-B1. n = 3 per group. e, f, Activation of NOS activation by VEGF (100 ng ml–1) or HDL (20 μg ml–1) with or without RNAi knockdown of SR-B1 (e, n = 10 per group) or disruption of caveolae by methyl-β-cyclodextrin (CD) treatment (f, 10 mM for 60 min, n = 8 per group). g, Abundance of target protein following RNAi knockdown of SR-B1, Pdzk1, Ldlr or Cd36 in HAECs. Findings for three samples per condition are shown. In all studies, expression of SR-B1, LDLR and CD36 was evaluated. h, i, Uptake of DiI–nLDL and DiI–oxLDL in cells depleted of LDLR by RNAi (h, n = 12 for nLDL and 13 for oxLDL) or treated with control versus LDLR blocking antibody (i, n = 6 per group). j, Transcytosis of DiI–nLDL and DiI–oxLDL in cells treated with control versus LDLR blocking antibody. n = 6 for nLDL and 3 for oxLDL. k, l, Uptake of DiI–nLDL and DiI–oxLDL in cells depleted of CD36 by RNAi (k, n = 6 per group) or treated with control versus CD36 blocking antibody (l, n = 9 and 12 for nLDL, respectively, and n = 6 for oxLDL). m, Transcytosis of DiI–nLDL and DiI–oxLDL in cells treated with control versus CD36 blocking antibody. n = 6 per group. Data are mean ± s.e.m.; P values calculated by ANOVA with Dunnett’s post-hoc test (a–c) and two-sided Student’s t-test (d–f, h, i, k, l).
a, Abundance of SR-B1, LDLR, and CD36 protein following RNAi knockdown of SR-B1, or following reconstitution of wild-type SR-B1 expression in cells depleted of the endogenous receptor. Expression of caveolin-1 (Cav1) was also evaluated. Findings for two samples per condition are shown. b, Alk1 transcript levels in cells manipulated as in a. n = 3, 4 and 4, respectively. c, Abundance of SR-B1, LDLR, and CD36 protein following RNAi knockdown of Alk1, SR-B1, or both. Findings for two samples per condition are shown. d, Alk1 transcript levels in cells manipulated as in c. n = 4 per group. e, Phosphorylation of SMAD1/5 in response to BMP9 (10 ng ml–1 for 0–120 min) after RNAi knockdown of SR-B1 or Alk1. The abundance of Ser463/465 phosphorylation of SMAD1/5 (p-SMAD1/5) and total SMAD1 (t-SMAD1) were evaluated by immunoblotting. f, Transcytosis of DiI–nLDL following RNAi knockdown of Alk1, SR-B1, or both. n = 9 per group. g, h, Uptake of nLDL was evaluated using 125I-nLDL in the absence or presence of a 50-fold excess of unlabelled nLDL (g), and following RNAi knockdown of SR-B1 or Dock4, or reconstitution of wild-type SR-B1 expression in cells depleted of the endogenous receptor (h). n = 8 per group. i, j, Transcytosis of nLDL was evaluated using 125I-nLDL in the absence or presence of a 50-fold excess of unlabelled nLDL (i), and following manipulation of SR-B1 or DOCK4 expression as in h (j). n = 3 per group. k, l, Transcytosis of nLDL was evaluated using TIRF microscopy in cells treated with Dyngo4A (k, 30 μM) or following RNAi knockdown of SR-B1 or Dock4 (l). n = 6 per group. m, Activation of RAC1 in response to oxLDL in cells expressing GFP control versus dominant-negative RAC1, and in untreated cells versus cells incubated with the RAC1 inhibitor NSC23766. Data are mean ± s.e.m.; P values calculated by ANOVA with Dunnett’s post-hoc test (d, f, h, j, l) or two-sided Student’s t-test (g, i, k).
Extended Data Fig. 10 Lentiviral reconstitution of wild-type and mutant SR-B1 expression in human endothelial cells.
a–c, Studies of reconstituted wild-type SR-B1, extracellular point mutants of SR-B1, or SR-B1(Q445A). The extracellular point mutants were: SR-B1(M159E), SR-B1(T165E), SR-B1(F171A), SR-B1(T175A), and SR-B1(E178A). Whole cell lysate abundance (a), cell surface abundance (b; except for Q445A, which was previously evaluated), and nLDL and oxLDL binding (c) were evaluated. d–f, Studies of reconstituted wild-type SR-B1 or C-terminal cytoplasmic tail deletion mutants of SR-B1. The mutants were: SR-B1(∆C15) (∆495–509), SR-B1(∆C23) (∆487–509) and SR-B1(∆C30) (∆480–509). Whole cell lysate abundance (d), cell surface abundance (e), and nLDL and oxLDL binding (f) were evaluated. g, Top, sequence alignment of amino acids in the C-terminal cytoplasmic tail of SR-B1 homologues (residues 487–494) from human (Homo sapiens, Hs, Q8WTVO, Swiss-Prot), mouse (Mus musculus, Mm, Q61009, Swiss-Prot), rat (Rattus norvegicus, Rr, P97943, Swiss-Prot), bovine (Bos taurus, Bt, O18824, Swiss-Prot), pig (Sus scrofa, Ss, Q8SQC1, Swiss-Prot), and Chinese hamster (Cricetulus griseus, Cg, Q60417, Swiss-Prot). Fully conserved residues are shown in bold. Bottom, comparison of human SR-B1 residues 487–494 and entire human CD36 C-terminal cytoplasmic tail. Residues of SR-B1 not shared with CD36 are shown in bold. h–j, Studies of reconstituted wild-type or C-terminal cytoplasmic tail substitution mutants of SR-B1. The mutants were: SR-B1(IQAY), SR-B1(SESL), SR-B1(Y490A), SR-B1(Q488A), SR-B1(S491A), SR-B1(E492A), SR-B1(S493A), and SR-B1(L494A). Whole cell lysate abundance (h), cell surface abundance (i), and nLDL and oxLDL binding (j) were evaluated. k–m, Binding (k), uptake (l) and transcytosis (m) of nLDL were evaluated with the various mutants shown at an nLDL concentration of 100 μg ml–1. n, Whole cell lysate abundance of CD36 and LDLR following reconstitution with the SR-B1 mutants tested in k–m. Data are mean ± s.e.m. For cell surface abundance by flow cytometry, n = 3 or 4. For LDL binding, n = 4 or 8. For LDL transcytosis, n = 3. In c, k–m, P values for comparison with wild-type calculated by two-sided Student’s t-test.
This file contains uncropped scans of source data immunoblots.
Three-dimensional imaging of LDL uptake in the ascending aorta in mice with versus without SR-B1 in endothelium, related to Figure 2. Male Apoe−/−SR-B1fl/fl mice previously placed on an atherogenic diet for one week received an IV injection of DiI–oxLDL, and 4 hours later the aortas were perfused, isolated and immediately subjected to Hoechst staining and confocal fluorescence microscopy on a randomly selected region on the luminal surface of the ascending aorta to a depth of 50um. DiI is shown in red and Hoechst staining of nuclei is shown in blue. At the start of the representative videos shown, the lumen is on the left side.
Three-dimensional imaging of LDL uptake in the ascending aorta in mice with versus without SR-B1 in endothelium, related to Figure 2. Male Apoe−/−SR-B1∆EC mice previously placed on an atherogenic diet for one week received an IV injection of DiI–oxLDL, and 4 hours later the aortas were perfused, isolated and immediately subjected to Hoechst staining and confocal fluorescence microscopy on a randomly selected region on the luminal surface of the ascending aorta to a depth of 50 μm. DiI is shown in red and Hoechst staining of nuclei is shown in blue. At the start of the representative videos shown, the lumen is on the left side.
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Huang, L., Chambliss, K.L., Gao, X. et al. SR-B1 drives endothelial cell LDL transcytosis via DOCK4 to promote atherosclerosis. Nature 569, 565–569 (2019). https://doi.org/10.1038/s41586-019-1140-4
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