Arteriosclerosis, Thrombosis, and Vascular Biology

BASIC SCIENCES

Endothelial HMGB1 Is a Critical Regulator of LDL

Transcytosis via an SREBP2–SR-BI Axis
Siavash Ghaffari, Erika Jang,* Farnoosh Naderinabi,* Rajiv Sanwal, Negar Khosraviani, Changsen Wang,
Benjamin E. Steinberg, Neil M. Goldenberg, Jiro Ikeda, Warren L. Lee

OBJECTIVE: LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor
class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in
the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease.
Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown.

APPROACH AND RESULTS: HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was
measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without
affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory
element-binding protein 2)—a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis
required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by
inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)—a cell surface receptor
for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of
HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization
of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the
aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis.
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CONCLUSIONS: Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI
expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.

GRAPHIC ABSTRACT: A graphic abstract is available for this article.

Key Words: atherosclerosis ◼ cell nucleus ◼ cytoplasm ◼ endothelial cells ◼ humans

A
therosclerosis is a disease remarkable for its
latency, beginning in the arteries of young adults
yet manifesting decades later. The accumulation of
LDL (low-density lipoprotein) cholesterol in the intimal
layer of arteries represents a first and pivotal step1 and
implies that circulating LDL has crossed the endothelial
barrier. Early on in the disease, the overlying endothelium
is healthy2 and precludes the passage of LDL between
cells; thus LDL passes through individual endothelial
cells by transcytosis.3
Until recently, almost nothing was known about
the mechanisms of LDL transcytosis in the systemic
See accompanying editorial on page 217
circulation. We and others have observed that the
scavenger receptor SR-BI (scavenger receptor class
B type 1)4 and the TGFβ family receptor ALK1 (activin
receptor-like kinase 1) mediate transcytosis,5 while the
high-affinity LDLR (LDL receptor) does not. The patho-
logical importance of SR-BI–mediated LDL transcyto-
sis to atherosclerosis has now also been established
using murine models of the disease.6 Nonetheless, it
remains largely unclear how the process is regulated.

Correspondence to: Warren L. Lee, MD, PhD, Keenan Centre for Biomedical Research, St. Michael’s Hospital, 30 Bond St, Toronto, Ontario, Canada. Email warren.
lee@unityhealth.to
*These authors contributed equally to this article.
The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.120.314557.
For Sources of Funding and Disclosures, see page 215.
© 2020 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb

200   January 2021 Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557
 Ghaffari et al
Nonstandard Abbreviations and Acronyms
HCAEC human coronary artery endothelial cell
HMGB1 high mobility group box 1
LDL low-density lipoprotein
LDLR low-density lipoprotein receptor
ORO oil red O
PCR polymerase chain reaction
RAGE receptor for advanced glycation
endproducts
SR-BI scavenger receptor class B type 1
SREBP2 sterol regulatory element-binding protein 2
TBST Tris-buffered saline-0.1% Tween-20
TIRF total internal reflection fluorescence
The alarmin HMGB1 (high mobility group box
1)—the most abundant nonhistone structural protein
in the nucleus—is expressed abundantly in the endo-
thelial cells of normal arteries and is found in high
amounts extracellularly in atherosclerotic lesions.7 It is
ubiquitously expressed, including by endothelial cells
and leukocytes from which it is released by a poorly
characterized secretory pathway8,9; it is also released
passively by dying cells. Neutralization of HMGB1
by antibodies attenuates atherosclerosis in ApoE−/−
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mice,10 suggesting that a role for HMGB1 in advanced
atherosclerosis is likely. Whether HMGB1 is involved in
early disease—a period in which transcytosis of LDL is
critical—is unknown.
Another limitation of the existing literature is the
lack of distinction between intracellular and extracel-
lular HMGB1. While less studied, intracellular HMGB1
has nuclear structural functions and regulates gene
expression.11 This is potentially important since intra-
cellular HMGB1 has been reported to bind to and
enhance the function of SREBP (sterol regulatory
element-binding protein) 1 and 2, transcription fac-
tors involved in lipogenic and cholesterologenic gene
expression.12 Because of this, we hypothesized that in
early disease, intracellular HMGB1 in endothelial cells
might play an important role in regulating transcyto-
sis, over and above the contributions of extracellular
HMGB1, which may be derived from endothelial cells,
smooth muscle cells, or leukocytes.7
In this article, we report that nuclear HMGB1 enhances
SREBP2 stability and SR-BI expression, thereby promot-
ing LDL transcytosis by primary human coronary artery
endothelial cells (HCAECs). Mice deficient in endothelial
HMGB1 develop significantly fewer fatty streaks and are
protected from atherosclerosis.
Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557
HMGB1 Is a Critical Regulator of LDL Transcytosis
Highlights
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• Intracellular HMGB1 (high mobility group box 1)
regulates LDL (low-density lipoprotein) transcyto-
sis via SREBP2 (sterol regulatory element-binding
protein 2) and SR-BI (scavenger receptor class B
type 1).
• Nuclear HMGB1 stabilizes SREBP2 mRNA.
• Mice deficient in endothelial HMGB1 develop fewer
fatty streaks and are protected from atherosclerosis.
MATERIALS AND METHODS
Data for the study can be obtained upon reasonable request
to the corresponding author. Additional information on the
Methods can be found in the Major Resources Table in the
Data Supplement.
Cell Culture
Primary HCAECs were purchased from PromoCell (Heidelberg,
Germany) and maintained in EGM-2 (endothelial cell growth
medium-2) media with supplements (Lonza, Canada). Cells were
from 5 donors, 3 men (aged 27, 48, and 58 years) and 2 women
(aged 32 and 40 years). For all experiments, HCAECs were used
at passage 5 or 6. All cells were incubated under humidified air
containing 5% CO2 at 37 °C.
Isolation and Labeling of Human LDL
Blood was drawn from human volunteers after obtaining informed
consent and in accordance with a protocol approved by the
Institutional Research Ethics Board (19–314). Plasma was
obtained by centrifuging the blood at 2000g for 10 minutes at room
temperature. EDTA (EDT001; BioShop) and KBr (POB301.1;
BioShop) were added to the plasma at a final concentration of 1
mmol/L and 1.2 g/mL, respectively, and the mixture was mixed
on a shaker until dissolved. The plasma mixture was overlaid with
0.3 mmol/L EDTA in PBS and centrifuged at 65 000 rpm for
1.5 hours at 15 °C in vacuum. After centrifugation, the orange
LDL layer was kept. LDL was concentrated by filtration through
a 10k Molecular Weight cutoff filter (UFC901008; Millipore) and
centrifuging at 4000g for 1 hour at 4 °C. The concentrated LDL
was dialyzed in 0.3 mmol/L EDTA in PBS and filtered through a
0.45-μm pore filter. Concentration was measured using the BCA
assay (No. 23227; ThermoFisher). LDL was stored in the dark at
4 °C for up to 3 months. To generate DiI-LDL, human LDL (2 mg)
was mixed with 300 μg DiI (No. 145311; Abcam) and 4 mL of
lipoprotein-deficient serum. The mixture was incubated at 37 °C
for 15 to 16 hours. After incubation, 0.5 g of KBr was added and
mixed until dissolved. KBr solution (1.063 g/mL in 0.3 mmol/L
EDTA in PBS) was overlaid on top of the LDL mixture, and tubes
were centrifuged at 35 000 rpm for 24 hours at 4 °C in vacuum.
The pink DiI-LDL layer was collected and dialyzed against 0.3
mmol/L EDTA in PBS and filtered through a 0.45-μm pore filter;
the final concentration was measured using the BCA assay. To
generate Alexa Fluor 568–labeled LDL, human LDL (2 mg in 1
mL of 0.3 mmol/L EDTA/PBS) was mixed with 100 μL of 1 M
NaHCO3 and 100 μg of A568 Dye (Alexa Fluor 568 Succinimidyl
January 2021   201
 Ghaffari et al
Ester, No. A20003; ThermoFisher) in accordance with the manu-
facturer’s instructions; this was followed by mixing in the dark at
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room temperature for 1 hour. The mixture was dialyzed against
0.3 mmol/L EDTA/PBS.
Transfection
For siRNA transfections, a master mix was made containing
150 μL Opti-MEM (ThermoFisher, Canada), 4 μL Lipofectamine
RNAiMAX Reagent (ThermoFisher), and 32 nmol/L siRNA.
siRNAs were Hs_SREBF2 (NM_004599; Qiagen, Canada),
Hs_SCARB1 (NM_005505; Qiagen), and Hs_AGER
(NM_001206934; Qiagen) and nontargeting control siRNA
(1027310; Qiagen). A FlexiTube GeneSolution was used to
knock down HMGB1 (GS3146, 1027416; Qiagen) and con-
tains 4 different oligos: Hs_HMGB1_6 (S103650374), Hs_
HMGB1_4 (S102627842), Hs_HMGB1_3 (S102627835),
and Hs_HMGB1_2 (S102627828). HCAECs were transfected
at 70% confluency in complete serum EGM-2, and media was
changed 24 hours after transfection. All experiments on cells
transfected with siRNA were performed 36 to 72 hours after
transfection when the cells reached 100% confluency. Cell
lysates were collected after the experiment to determine the
degree of knockdown by immunoblot. In some experiments,
total RNA was isolated from similar cells that were seeded on
another plate and treated identically.
The green fluorescent protein-HMGB1 wild-type construct
was a generous gift from Dr Brian Ratliff. Transfection of plas-
mid DNA was performed on HCAECs that were 80% confluent
using Lipofectamine-3000 (ThermoFisher). A mix containing 1
μg/mL plasmid DNA was added to Opti-MEM and then added
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to HCAECs in serum-free EGM-2. After 2 hours, media was
changed to complete serum EGM-2. Experiments involving
plasmid-transfected cells were performed 24 hours after trans-
fection and at 100% confluency.
LDL and Albumin Transcytosis Assay
Total internal reflection fluorescence (TIRF) microscopy uses
an evanescent wave to illuminate just the proximal 100 nm or
so of the cell; thus, we can selectively image the basal mem-
brane of a live endothelial cell with minimal confounding from
the overlying cytoplasm and apical surface. This technique obvi-
ates confounding from paracellular gaps and does not require
a high transfection efficiency.4 Essentially, confluent endothelial
monolayers are exposed to a fluorophore-tagged ligand added
to the apical cell surface while the basal membrane of the cell
is imaged by TIRF. Cytoplasmic vesicles undergoing exocytosis
with the basal membrane are directly visualized and quantified.
TIRF microscopy was performed on a Leica DMi8 microscope
with 63×/1.47 (O) objectives; 405, 488, 561, and 637 nm
laser lines; 450/50, 525/50, 600/50, 610/75, and 700/75
emission filters; and run with Quorum acquisition software
(Quorum, Canada). Microscope settings were kept constant
between conditions.
Briefly, cells at 100% confluency were placed in a live
cell imaging chamber and treated with 20 μg/mL DiI-LDL in
cold HPMI (RMPI growth medium with HEPES) media for 10
minutes at 4 °C to allow apical membrane binding. Following
membrane binding, cells are washed twice with cold PBS+
to remove unbound ligand, and room temperature HPMI is
added. Cells are incubated on the live cell imaging stage at
202   January 2021 Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557
HMGB1 Is a Critical Regulator of LDL Transcytosis
37 °C for 2 minutes before initial image acquisition. Confluent
regions of the monolayer are selected by viewing the number
of nuclei in the DAPI (4′,6-diamidino-2-phenylindole) field of
view after staining with NucBlue Live ReadyProbes Reagent
(ThermoFisher), and TIRF microscopy of the basal membrane
is performed to visualize exocytosis. For each coverslip, 10 to
15 videos of 150 frames (100 ms exposure) are captured.
A single-particle tracking algorithm developed in MATLAB
(R2014b; Mathworks) was used to quantify the number of
transcytosis events by analyzing the videos.4,13
Glycyrrhizin—a direct inhibitor of extracellular HMGB114—
was purchased from Sigma Canada and was diluted in DMSO.
Cells were incubated with glycyrrhizin for 24 hours at 100
µmol/L in EGM-2 before measurement of transcytosis.
Similar to LDL, albumin transcytosis events were acquired
using TIRF microscopy as described previously.15 Cells were
treated with 10 μg/mL Alexa Fluor 488 (AF488)–conjugated
albumin (ThermoFisher Scientific).
Immunoblotting
Cell lysates were collected by lysing cells in SDS-containing
lysis buffer with 2 mmol/L sodium orthovanadate (Sigma,
Canada), 1 mmol/L PMSF (Sigma), 1X protease inhibitor cock-
tail (Roche, Canada), and 1X PhosSTOP (Sigma). Lysates were
resolved on 10% polyacrylamide gel and transferred onto a
nitrocellulose membrane and blocked for 1 hour in 5% BSA in
Tris-buffered saline-0.1% Tween-20 (TBST). Primary antibod-
ies were diluted in 0.5% BSA in TBST with the working concen-
tration of 1 µg/mL and incubated at 4 °C overnight on a rocker.
Primary antibodies were mouse anti–β-actin (sc-47778; Santa
Cruz, CA), rabbit anti–SR-BI (NB400-104; Novus Bio, Canada),
rabbit anti–α-actinin (No. 3134; Cell Signaling, Danvers, MA),
rabbit anti-HMGB1 (3935; Cell Signalling, Canada), mouse
anti-SREBP2 (NBP1-54446; Novus Bio), rabbit anti-CD36
(NB400-144; Novus Bio), and rabbit anti- caveolin1 (sc-894;
Santa Cruz, CA). After washing several times in TBST, mem-
branes were incubated with HRP-conjugated secondary anti-
bodies (1 μg/mL) for 1 hour at room temperature followed
by detection by enhanced chemiluminescence (Clarity ECL;
BioRad, Canada) and imaging on a ChemiDoc Imaging System
(BioRad). Protein abundance was quantified by densitometry
(ImageJ) after normalization to loading control.
Immunofluorescence
HCAECs were fixed/permeabilized in 100% ice-cold metha-
nol at −20 °C for 10 minutes followed by washing 3× with
PBS. Cells were blocked with 5% BSA in PBS for 30 min-
utes at room temperature followed by immunodetection
with primary antibodies diluted in PBS with the working
concentration of 10 µg/mL. Primary antibodies were rabbit
anti-HMGB1 (3935; Cell Signalling, Canada) and rabbit anti-
caveolin1 (sc-894; Santa Cruz, CA). After a 1-hour incuba-
tion at room temperature, cells were washed 3× in PBS and
treated with species-specific, chromophore-conjugated sec-
ondary antibody diluted 1:1000 (Jackson ImmunoResearch
Laboratories, West Grove, PA). Cells were then washed
and mounted on slides with DAPI-containing (1:5000)
Dako Fluorescence Mounting Medium (Agilent, Canada).
Negative controls (no primary antibody) were included in all
experiments. Slides were imaged by spinning disk confocal
 Ghaffari et al HMGB1 Is a Critical Regulator of LDL Transcytosis

microscopy performed on a Leica DMi8 microscope with set- Fluorescence Recovery After Photobleaching
tings kept constant between conditions.

For measurement of HMGB1 translocation, cells were pre-
treated with serum-free EGM-2 for 2 hours. Then unlabeled
LDL (40 µg/mL) was added to cells, and cells were incubated
at 37 °C with 5% CO2 for 15 minutes. A MATLAB script was
used to quantify nuclear/cytoplasmic intensity. Briefly, the
image of nuclei in the DAPI channel was thresholded to obtain
a binary image, and the whole field of view then divided into
nuclear and cytoplasmic area. Subsequently, the intensity of the
HMGB1 channel was quantified in each region, and nuclear/
cytoplasmic intensity was obtained.
RNA Isolation and Quantitative Polymerase
Chain Reaction
Total RNA was isolated from 100% confluent HCAECs seeded
on 6-well plates. For measurement of siRNA-mediated knock-
down efficiency by quantitative polymerase chain reaction
(PCR), RNA was isolated 48 hours after transfection. RNA iso-
lation was performed with the Qiagen RNeasy RNA Isolation
Kit according to manufacturer’s instructions. cDNA synthesis
was performed on 1 μg total RNA using Applied Biosystems
High Capacity cDNA Reverse Transcription Kit. Quantitative
PCR was performed on an Applied Biosystems QuantStudio
7 Flex Real-Time PCR system: (1) 95 °C for 3 minutes, (2) 95
°C for 15 seconds, (3) 58 °C for 30 seconds, (4) repeat from
2 an additional 39×, (5) melt curve from 65 °C to 95 °C at
0.5 °C increments. mRNA abundance is presented relative to
18S, which is a reference gene for our experiment. Primers for
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Analysis
HCAECs were seeded on 25-mm glass coverslips and trans-
fected with HMGB1-green fluorescent protein 2 days before
the experiment. On the day of experiment, unlabeled LDL (40
µg/mL) was added to cells that were then incubated at 37 °C
with 5% CO2 for 15 minutes. Fluorescence recovery after pho-
tobleaching analyses was performed with cells maintained in
EBM-2 growth medium at 37 °C with 5% CO2 supplied to the
microscope chamber. Images were captured on a ZEISS LSM
700 confocal microscope equipped with an environmental
chamber (37 °C, 5% CO2) using a 63× 1.4 NA objective. ZEN
lite software was used to measure the intensity of fluorescence
in the bleached region of interest and in the whole cell at each
time point. Two prebleach images were captured with excitation
at 488 nm before photobleaching. The whole nucleus was then
photobleached (100 ms, 100% laser power). Any remaining
fluorescence in the bleached area after the bleach was normal-
ized to zero. Fluorescence recovery was recorded at 1-second
intervals for the period of 24 seconds. Error bars in the recovery
curves were drawn showing SEM. The mobile fractions were
calculated from the end point of these curves.
mRNA Stability Assay
HCAECs were transfected with the HMGB1 siRNA or scram-
bled negative control siRNA. After 24 hours, cells were treated
with 5 μg/mL actinomycin D (Bishop, Canada) diluted in the
media to inhibit transcription. Total RNA was isolated just after
treatment with actinomycin D and once every 2 hours for 8

quantitative PCR are listed in Table 1, and the sequences were
provided by PrimerBank.
Table 1. Primers for Quantitative Polymerase Chain Reaction
hours. Subsequently, cDNA was synthesized, and real-time
quantitative PCR was performed. The data were fitted to a lin-
ear regression, and the regression was used to calculate the
half-life of the mRNA.

cDNA Forward primer (5′–3′) Reverse primer (5′–3′) Protein Half-Life Analysis
18S GATGGAAAATACAGCCAG- TTCTTCAGTCGCTCCAG- HCAECs were transfected with the HMGB1 siRNA or scram-
GTCCTA GTCTT bled negative control siRNA. After 48 hours, cells were treated
HMGB1 TATGGCAAAAGCGGA- CTTCGCAACATCAC- with 50 μg/mL cycloheximide (Bishop) to inhibit protein syn-
CAAGG CAATGGA thesis. Proteins were harvested at different times, and Western
ALK1 CGAGGGATGAA- GTCATGTCTGAGGCGAT- blot analysis was performed as described above. The data were
CAGTCCTGG GAAG fitted to a linear regression, and the regression was used to
SR-BI CTGTGGGTGAGATCAT- GCCAGAAGTCAACCTT- calculate the half-life of the protein.
GTGG GCTC
LDLR ACGGCGTCTCTTCCTAT- CCCTTGGTATCCGCAA-
GACA CAGA Mice
CD36 TCTTTCCTGCAGCCCAATG AGCCTCTGTTCCAACT-
Male mice with an endothelial cell–specific deletion of HMGB1
GATAGTGA (EC-HMGB1−/−; Fl/FlHMGB1 Tie2Cre+) were generated by
ICAM-1 ATGCCCAGACATCTGT- GGGGTCTCTATGCCCAA-
crossing C57BL/6J mice expressing Cre under the Tie2 pro-
GTCC CAA moter with C57BL/6J mice containing floxed HMGB1 (the
CAV-1 GCGACCCTAAACACCT- ATGCCGTCAAAACTGTGT-
latter a gift from Tadatsugu Taniguchi, University of Tokyo).
CAAC GTC HMGB1 floxed C57BL/6J mice not expressing Cre were
RAGE GTGTCCTTCCCAACGGCTC ATTGCCTGGCACCGGAAAA
used as controls. To generate HMGB1 knockout mice on an
LDLR−/− background, male EC-HMGB1−/− mice were crossed
SREBP2 AACGGTCATTCACCCAG- GGCTGAAGAATAGGAGTT-
GTC GCC
with female LDLR−/−, and the progeny were then crossed to
obtain EC-HMGB1−/−LDLR−/− mice. LDLR−/− mice with floxed
ALK1 indicates activin receptor-like kinase 1; CAV-1, caveolin-1; CD36, clus- HMGB1 not expressing Cre were used as controls. Genotyping
ter of differentiation 36; HMGB1, high mobility group box 1; ICAM-1, intercellular
adhesion molecule 1; LDLR, low-density lipoprotein receptor; RAGE, receptor for
was performed by ear notching and PCR using the KAPA
advanced glycation endproducts; SR-BI, scavenger receptor class B type 1; and Mouse Genotyping Kit (No. KK7301; Kapa Biosystems) and
SREBP2, sterol regulatory element-binding protein 2. primers for floxed HMGB1, LDLR, and Tie2Cre (Table 2).

Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557 January 2021   203
 Ghaffari et al
Table 2. Primers for Floxed HMGB1, LDLR, and Tie2Cre
Basic Sciences - AL
DNA Forward primer (5′–3′) Reverse primer (5′–3′)
HMGB1 TGT CAT GCC ACC CTG TGT GCT CCT CCC GGC
AGC AGT T AAG TT
CRE GCT CGA CCA GTT TAG TCG CGA TTA TCT TCT ATA
TTA CCC TCT TCA G
LDLR TAT GCA TCC CCA GTC CTA CCC AAC CAG CCC
TTT GG CTT AC (wild-type)
ATA GAT TCG CCC TTG TGT
CC (mutant)
CRE indicates Cre recombinase; HMGB1, high mobility group box 1; LDLR,
low-density lipoprotein receptor; and Tie2Cre, Cre recombinase expressed under
the Tie2 promoter.
Starting at 5 weeks old, EC-HMGB1−/− mice and their con-
trols were fed the Paigen high-fat, high-cholesterol diet con-
taining 15% cocoa butter, 1% corn oil, 1.25% cholesterol, and
0.5% cholic acid (Dyets 611695) for 18 weeks.16 Starting at
10 weeks old, EC-HMGB1−/−; LDLR−/− mice and their controls
were fed a high-fat, high-cholesterol diet (D12108C; Research
Diets) for 5 weeks. Mice were maintained in the St. Michael’s
Hospital Vivarium on a standard light-dark cycle and were pro-
vided access to food and water ad libitum. Experiments were
performed in accordance with the Animal Care Committee
guidelines at St. Michael’s Hospital and followed approved ani-
mal protocols (ACC899) and the American Heart Association
guidelines for experimental atherosclerosis studies.17 Although
endothelial cells from both male and female donors were
included in the study, the animal experiments were limited to
male mice due to cost. Follow-up work will include the study
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of female mice.
Blood Collection
Plasma lipid levels were measured at 3 time points: before
starting the high-fat diet, during the diet, and at the time of
euthanasia. Before blood collection, mice were fasted for 16
hours. Blood was collected via the saphenous vein and mixed
in tubes containing K2EDTA (BD Microtainer with K2EDTA, No.
365974). Plasma was obtained by centrifuging whole blood at
5000×g at 8 °C, for 10 minutes. Plasma was stored at −80 °C
before being sent to The Centre for Phenogenomics (Toronto,
Ontario) for analysis of total cholesterol, LDL, and triglycerides.
Oil Red O Staining
At experimental end point, mice on the high-fat diet were euth-
anized by exsanguination. A syringe was inserted into the left
ventricle, and the mouse was perfused with PBS and then 10%
buffered formalin. The aorta attached to the heart was fixed in
10% formalin for 1 hour. Using a dissecting microscope, the
adventitial tissue was removed, and the arch was separated
from the aortic root, the descending aorta, and the aortic arch
branches. Oil red O (ORO; No.O0625; Sigma-Aldrich) stock
solution was prepared by dissolving ORO (2.58 g) in 500 mL of
isopropanol. The working solution was prepared by diluting the
stock solution in double-distilled water (3:2) and then centrifug-
ing at 2000×g for 10 minutes. The arch was stained with ORO
working solution for 30 minutes, destained in 60% isopropanol
for 5 minutes twice, and then washed in PBS. The arch was cut
longitudinally along the greater curvature and pinned open en
204   January 2021 Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557
HMGB1 Is a Critical Regulator of LDL Transcytosis
face submerged in PBS. Photographs were taken with a Nikon
camera mounted to a dissecting microscope. Quantification
was performed on the images in ImageJ by setting the same
color threshold to all images and then quantifying the area of
positive pixels. Plaque area of knockout mice was normalized to
plaque area of cage mate control mice.
ORO Staining for Aortic Roots
After fixation, aortic roots were frozen in OCT on dry ice then
stored at −80 °C. Ten-micrometer cryosections were mounted
onto Superfrost Plus (12-550-15, Fisherbrand) microscope
glass slides at 70-μm intervals starting from the bottom of the
cusps until 8 sections were obtained per slide. Sections were
fixed in 10% formalin for 10 minutes and washed in ddH2O for
5 min. Sections were incubated in 60% isopropanol for 1 min-
ute, stained in ORO (No. O0625; Sigma-Aldrich) working solu-
tion for 12 minutes, destained in 60% isopropanol for 1 minute,
and washed in ddH2O for 30 seconds. Hematoxylin was used
as a counterstain for 1 minute, followed by washing in ddH2O
for 10 minutes. Coverslips were mounted on slides with aqua-
tex aqueous mounting media (Merck). Slides were imaged on
the Zeiss Axioscan.Z1 microscope with the 20× 0.8 NA objec-
tive lens with the same settings for all samples. Images were
quantified using Halo analysis software (Indica Labs) with set-
tings kept constant between groups.
Evans Blue Dye Assay
At experimental end point, EC-HMGB1−/− mice and controls
on the high-fat diet were injected via retro-orbital vein with
1% Evans blue dye (206334; Millipore Sigma) dissolved in
sterile PBS and then euthanized after 30 minutes by exsan-
guination. The mice were perfused and aortic arches were
cleaned as described above. Evans blue dye was extracted
from aortic arches by incubating in formamide at 56 °C for 4
days before measuring absorbance at 620 nm. Absorbance
measurements from EC-HMGB1−/− mice were normalized to
that of cage mate control mice.
Analysis of LDL Transcytosis In Vivo
To measure LDL transcytosis in vivo, we adapted a published
method.5 Briefly, EC-HMGB1−/− mice and EC-HMGB1−/−;
LDLR−/− mice or littermate controls on a standard laboratory
diet (Teklad 2918) were anesthetized and injected via the
retro-orbital vein with 200 μg of Alexa Fluor 568-LDL; Alexa-
conjugated LDL was used as it is fixable and the fluorophore
is conjugated covalently to LDL. After 30 minutes, mice were
euthanized by exsanguination; the short time point was chosen
to focus on endothelial permeability to LDL and to minimize
confounding from leukocytes. A syringe was inserted into the
left ventricle, and the mouse was perfused with PBS and then
10% buffered formalin. The aorta attached to the heart was
fixed in 10% formalin for 1 hour. Using a dissecting micro-
scope, the adventitial tissue was removed, and the arch was
separated from the aortic root, the descending aorta, and the
aortic arch branches. Aortas were permeabilized for 5 minutes
at room temperature in 0.5% Triton X-100 (TRX506.500;
BioShop) and 10% DMSO (DMS666.100; BioShop) in PBS.
They were washed in PBS 3× for 5 minutes. Aortas were incu-
bated with 1:100 (10 μg/mL) rabbit anti-mouse CD31 primary
 Ghaffari et al
antibody (NB100-2284; Novus) in 5% BSA in PBS overnight
at 4 °C. After washing, they were incubated with 1:200 (7.5 μg/
mL) anti-rabbit Alexa Fluor 488 secondary antibody (Jackson
ImmunoResearch) in 5% BSA in PBS for 1 hour at room tem-
perature. After washing, aortas were incubated in DAPI (1:400
in PBS; No. 62248; Thermo Scientific) for 20 minutes at room
temperature. Aortas were washed in PBS 5× for 3 minutes
and then mounted on slides with Dako fluorescence mounting
medium (S3023; Agilent). Slides were imaged with a ZEISS
LSM700 confocal microscope using a 20× 0.8 NA objective
lens and acquired with the ZEN Black software; settings were
kept the same between groups. Using ImageJ, background
fluorescence was subtracted, and the integrated density of the
Alexa Fluor 568-LDL signal was quantified and normalized to
tissue area and then to control mice.
Flow Cytometry and Histology
Mice on a standard laboratory diet (Teklad 2918) ranging from
5 to 10 weeks of age were euthanized via exsanguination by
cardiac puncture. Blood was collected in tubes containing a final
concentration of 10% v/v of 0.5M EDTA, pH 8. Blood was incu-
bated with 1× red blood cell lysis buffer (1:10 blood to RBC lysis
buffer; 10X RBC lysis buffer: 0.155 M NH4Cl; 0.01 M KHCO3;
0.1 mmol/L EDTA; dissolved in ddH2O and filter sterilized) on
a shaker at room temperature for 5 minutes. Blood was cen-
trifuged at 300g for 5 minutes at 4 °C. Supernatant was dis-
carded. Cells were washed in 2% FBS in PBS and spun at 500g
for 5 minutes at 4 °C. Cells were counted on a hemocytometer
and resuspended at a concentration of 1 to 2×106 cells/mL.
Cells were stained with 1:100 (5 μg/mL) Alexa Fluor 488 anti-
Downloaded from http://ahajournals.org by on November 17, 2022
CD115 (No. 135511; BioLegend), 1:100 (5 μg/mL) Alexa Fluor
647 anti-Ly6G (No. 127609; BioLegend), and 1:1000 Fixable
Viability stain 780 (No. 565388; BD Bioscience) in 2% FBS in
PBS for 30 minutes on ice. After washing, cells were fixed with
2% PFA (No. 15710; Electron Microscopy Sciences) in PBS for
15 minutes on ice. After washing, cells were permeabilized with
0.1% Triton X-100 in PBS for 15 minutes on ice. After washing,
cells were stained with 1:25 (36 μg/mL) Alexa Fluor 405 anti-
HMGB1 antibody (NB100-2322AF405; Novus) in 2% FBS in
PBS for 15 minutes on ice. Cells were washed and then filtered
through a 0.70-μm filter. Single-stain controls were prepared for
each antibody with the UltraComp eBeads Plus Compensation
beads (01-3333-42; Invitrogen), and the single stain for the
viability dye was prepared with cells that were half dead and
half live. Fluorescence-minus-one controls were prepared for
each group. Samples were analyzed by the Sick Kids/University
Health Network Flow Cytometry Facility in Toronto, Canada. Cells
were gated for single cells in the forward scatter and then side
scatter channels, then gated for live cells using the gating from a
live/dead control sample. Positive CD115 or Ly6G was graphed
against forward scatter. The positive populations were then
graphed as HMGB1 versus forward scatter to obtain double-
positive CD115+/HMGB1+ or Ly6G+/HMGB1+ populations.
For lung histology, mice were euthanized via cardiac punc-
ture. Lungs were perfused with 600 μL of 10% formalin, dis-
sected and fixed in 10% formalin for 2 days, followed by tissue
processing and embedding in paraffin wax. The tissue was cut
in serial 5-μm-thick sections and mounted on glass slide. The
sections were deparaffinized and then boiled for 20 minutes
in sodium citrate buffer (10 mmol/L sodium citrate, 0.05%
Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557
HMGB1 Is a Critical Regulator of LDL Transcytosis
Tween-20, pH 6.0) for antigen retrieval. Slides were washed
in TBST twice for 5 minutes, followed by permeabilization with
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PBS-1% Triton X-100 for 30 minutes at room temperature.
Samples were circled using a hydrophobic pen and blocked
using 10% goat serum with 1% BSA in 1X TBS for 1 hour.
Slides were washed 3× for 2 minutes, followed by blocking with
AffiniPure Fab Fragment Goat Anti-Mouse IgG (115-007-003;
Jackson ImmunoResearch) for 1 hour at room temperature.
Slides were washed once with TBST and incubated overnight
at 4° with primary antibody: 1:50 (4 μg/mL) rabbit anti-mouse
Tie-2 (H-176, sc-9026; Santa Cruz Biotechnology), 1:250 (4
μg/mL) rabbit anti-mouse AQP5 (ab78486; Abcam), or 1:200
(5 μg/mL) mouse anti-HMGB1 (ab190377; Abcam). Slides
were washed twice with TBST for 5 minutes and incubated
with conjugated secondary antibody Cy3-conjugated goat anti-
mouse IgG (115-165-003; Jackson ImmunoResearch) and
Alexa Fluor-647–conjugated goat anti-rabbit IgG (111-605-
003; Jackson ImmunoResearch) for 1 hour at room tempera-
ture. Slides were counterstained with DAPI.
For heart sections, hearts were flushed with PBS and flash
frozen using isopentane for 20 seconds before being stored
at −80 °C for further processing. Serial 5-μm cryosections
were mounted onto Superfrost microscope glass slides and
were allowed to dry for 10 minutes. Sections were fixed and
permeabilized with methanol at −20 °C for 20 minutes and
then washed with PBS +0.1% Tween-20 3× for 5 minutes.
After blocking and washing, slides were incubated with primary
antibody overnight at 4 °C 1:100 (10 μg/mL) mouse anti-
HMGB1 (ab190377; Abcam). Excess primary antibody was
washed off and secondary antibody applied as with lung sec-
tions. After washing, the next primary antibodies were applied
for 1 hour at room temperature: 4 μg/mL rabbit anti-mouse
Tie-2 (H-176; Santa Cruz Biotechnology) or 1:100 (4 μg/mL)
rabbit anti-mouse cardiac troponin I (ab47003; Abcam). These
were labeled with appropriate secondary antibody for 1 hour.
Finally, the slides were coated with 20 μL per section of DAPI-
containing (1:5000) Dako fluorescence mounting medium
(S3023; Agilent), and coverslips were added. For both lung and
heart sections, a no-primary-antibody control was performed
(ie, incubation with secondary antibody only).
Statistics
Unless indicated otherwise, each experiment was performed at
least 4× independently. Statistical tests were performed using
GraphPad Prism software, version 6.0 (La Jolla, CA); data were
tested for normality and equal variance to confirm appropri-
ateness of parametric tests. For experiments with 2 indepen-
dent groups, P values were determined by paired or unpaired
Student t tests where appropriate. P<0.05 was deemed statis-
tically significant. For experiments with >2 independent groups,
P values were determined by 1-way ANOVA with significance
set at P<0.05; individual comparisons between groups were
determined by Tukey post hoc test. For the time point experi-
ments, P values were corrected for multiple comparisons using
the Holm-Sidak method.
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Figure 1. HMGB1 is required for LDL transcytosis by human coronary artery endothelial cells.
A, HMGB1 (high mobility group box 1) was knocked down by siRNA in human coronary artery endothelial cells (HCAECs) achieving a
reduction of 80% to 90% in protein levels. Representative blots (left); quantification (right). n=4 independent experiments; ***P<0.001 by
t test. Error bars represent SEM. B, Transcytosis of DiI-tagged LDL (low-density lipoprotein) across confluent HCAEC from male donors is
measured in real time using total internal reflection fluorescence microscopy. Knockdown of HMGB1 led to a decrease in LDL transcytosis;
each point represents 1 cell. n=4 experiments; ***P<0.001 by t test; error bars represent SD. C, Knockdown of HMGB1 led to a decrease in
LDL transcytosis across confluent cells from female donors; each point represents 1 cell. n=4 experiments; ***P<0.001 by t test; error bars
represent SD. D, Knockdown of HMGB1 had no effect on albumin transcytosis. Transcytosis of Alexa 488–albumin across confluent cells from
a male donor was measured; each point represents 1 cell. n=4 experiments; error bars represent SD. E, Knockdown of HMGB1 in human
coronary artery endothelial cells had no effect on transendothelial electrical resistance (ER); PAF (platelet-activating factor) is a positive control.
n=4; ***P<0.001 by 1-way ANOVA; ns indicates nonsignificant by Tukey multiple comparison test. SCRM indicates non-targeting control RNA.

206   January 2021 Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557
 Ghaffari et al
RESULTS
HMGB1 Is Required for LDL Transcytosis by
HCAECs
Primary HCAECs were depleted of HMGB1 by siRNA,
resulting in ≈80% to 90% reduction in protein levels (Fig-
ure 1A). Loss of HMGB1 caused a significant inhibition
of LDL transcytosis by cells from both male and female
donors (Figure 1B and 1C). The effect was not due to
a reduction in cell viability or generalized impairment in
vesicular traffic as albumin transcytosis was unaffected
(Figure 1D). Finally, depletion of HMGB1 had no effect
on endothelial barrier function in vitro as measured by
transendothelial electrical resistance (Figure 1E).
Deficiency of HMGB1 Leads to Downregulation
of SR-BI But Not ALK1
The inhibition of LDL but not albumin transcytosis by
depletion of HMGB1 made us consider whether the
mechanism involved a receptor. Knockdown of HMGB1
resulted in a significant reduction in SR-BI expression
at both the protein (Figure 2A) and the mRNA levels
(Figure 2B). However, levels of ALK1, caveolin-1, and
CD36 were unaffected (Figure 2B and 2C), and the sub-
cellular distribution of caveolin-1 was also unchanged
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(Figure I in the Data Supplement). Interestingly, mRNA
for SREBP2 and LDLR were significantly reduced in
HMGB1-depleted cells.
These data suggested that the effect of HMGB1 deple-
tion on LDL transcytosis might be through regulation of
SR-BI. To test this notion, we depleted both HMGB1 and
SR-BI in coronary artery endothelial cells. While knock-
down of either SR-BI or HMGB1 significantly inhibited
LDL transcytosis, combinatorial depletion of both proteins
did not achieve any further reduction, consistent with a
shared pathway (Figure 2D). To explore the contribution of
HMGB1 to LDL transcytosis by an alternative approach,
we overexpressed HMGB1 in coronary endothelial cells
by transfection. Transfected cells displayed a significant
increase in LDL transcytosis (Figure 2D). To determine
whether this effect was mediated by intracellular versus
extracellular (ie, secreted) HMGB1, we treated trans-
fected cells with glycyrrhizin14—an inhibitor of HMGB1
and measured LDL transcytosis. We also treated trans-
fected cells with siRNA to RAGE (receptor for advanced
glycation endproducts)—the main cell surface receptor for
HMGB1.18 Neither of these interventions attenuated the
effect of HMGB1 overexpression on LDL transcytosis
(Figure 2E). Because of this, we decided to focus on iden-
tifying a potential intracellular pathway by which endothe-
lial HMGB1 might regulate LDL transcytosis.
Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557
HMGB1 Is a Critical Regulator of LDL Transcytosis
HMGB1 Regulates LDL Transcytosis via
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SREBP2
Given the role of HMGB1 as a nuclear structural protein,
we wondered whether the connection between intracel-
lular HMGB1 and SR-BI expression reflected transcrip-
tional regulation of the receptor. Knockdown of HMGB1
caused a significant reduction in protein levels of both
mature and immature (precursor) SREBP2 protein (Fig-
ure 3A), as well as mRNA (Figure 2B). SREBP2 is a
critical transcription factor that regulates expression of
cholesterologenic genes, including LDLR and SR-BI.19
While depletion of either HMGB1 or of SREBP2 signifi-
cantly attenuated LDL transcytosis, combinatorial knock-
down had no additional effect (Figure 3B and 3C). In
addition, the effect of HMGB1 overexpression to induce
LDL transcytosis required SREBP2 as it was prevented
by knockdown of the transcription factor (Figure 3D).
HMGB1 Translocates to the Nucleus in an LDL-
and SR-BI–Dependent Manner and Regulates
SREBP2 Half-Life
We examined the cellular localization of HMGB1 by immu-
nofluorescence before and after coronary artery endothe-
lial cells were incubated with LDL. As expected, at baseline,
most HMGB1 was localized to the nucleus. However, upon
addition of LDL, we observed a significant increase in the
ratio of nuclear-to-cytoplasmic HMGB1 (Figure 4A). This
effect was partially inhibited by knockdown of SR-BI (Fig-
ure 4A) but not of LDLR (not shown). To verify this finding
by a complementary approach, we transfected endothe-
lial cells with green fluorescent protein–tagged HMGB1
and performed fluorescence recovery after photobleach-
ing experiments of nuclear HMGB1 in the presence or
absence of LDL in the media. The addition of LDL sig-
nificantly increased the recovery of nuclear HMGB1 after
photobleaching (Figure 4B). Theorizing that HMGB1 lev-
els in endothelial cells might be regulated by sterols, we
loaded endothelial cells with LDL and measured HMGB1
protein levels 24 hours later. Incubation with LDL caused
a significant increase in total HMGB1 expression (Fig-
ure 4C). We then speculated that one of the functions of
nuclear HMGB1 might be to stabilize SREBP2, prolonging
its half-life and, therefore, its activity upstream of SR-BI.
We measured the half-life of SREBP2 mRNA and protein
in control cells and in cells depleted of HMGB1, using acti-
nomycin to inhibit transcription and cycloheximide to inhibit
protein synthesis. In cells depleted of HMGB1, the half-
life of SREBP2 mRNA and protein was reduced by ≈50%
(Figure 4D and 4E).
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Figure 2. Deficiency of HMGB1 leads to down-regulation of SR-BI but not ALK1.

A, Knockdown of HMGB1 (high mobility group box 1) in human coronary artery endothelial cells by siRNA led to a decrease in SR-BI
(scavenger receptor class B type 1) protein levels. n=4; *P<0.05 by t test; error bars represent SEM. B, mRNA level by quantitative
polymerase chain reaction in HMGB1-depleted cells. n=4; *P<0.05 for LDLR (LDL [low-density lipoprotein] receptor), **P<0.01 for SR-BI and
SREBP2 (sterol regulatory element-binding protein 2); error bars represent SEM. C, Depletion of HMGB1 by siRNA had no effect on levels
of CAV-1 (caveolin-1) or CD36. Blot is representative of 4 experiments, and quantification is normalized to cells receiving nontargeting control
RNA (SCRM). D, Left, Knockdown of either HMGB1 or SR-BI significantly inhibited LDL transcytosis, but depletion of both did not achieve
any further reduction; each point represents 1 cell. n=4 experiments; ***P<0.001 by 1-way ANOVA. Right, Cells were transfected (Continued )

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 Ghaffari et al
Mice Deficient in Endothelial HMGB1 Develop
Fewer Fatty Streaks on a High-Fat Diet
To validate our data from cell culture, we generated EC-
HMGB1−/− mice using a Tie2Cre promoter.20 By histology
of lung and heart sections, we observed complete loss of
endothelial HMGB1 but retention of the protein in the adja-
cent epithelium and myocardium. Expression of HMGB1
in circulating monocytes and neutrophils (as well as the
absolute number of these leukocytes) was not significantly
reduced in the knockout animals (Figure II in the Data
Supplement) despite literature describing expression of
Tie2 by hematopoietic cells.21,22 To determine whether loss
of endothelial HMGB1 reduced LDL transcytosis in vivo,
we injected EC-HMGB1−/− and littermate control animals
with fluorophore-tagged (Alexa-568) LDL and measured
its accumulation 30 minutes later in the inner curvature
of the aortic arch.5 We observed a significant reduction in
LDL accumulation in the knockout animals (Figure 5A); the
reduction over such a short period of time is consistent
with impairment of endothelial transcytosis of LDL.
We next assessed the effect of HMGB1 deletion on
the earliest stage of atherosclerosis—the formation of
fatty streaks. Mice deficient in endothelial HMGB1 and
wild-type littermates were fed a high-fat diet for 18 weeks
followed by staining of the inner curvature of the aorta
with ORO. As expected, not all mice (≈50%) developed
fatty streaks, and these were modest in size.16 However,
EC-HMGB1−/− mice displayed significantly smaller fatty
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streaks in the aortic arch compared with controls (Fig-
ure 5B), whereas there was no difference in the aortic
root (Figure 5C). This reduction in neutral lipid accumu-
lation in the aortic arch and the decrease in fluorescent
LDL uptake in the aorta (ie, Figure 5A) were not due to
enhanced endothelial barrier function in EC-HMGB1−/−
animals as permeability to albumin (a smaller molecule
than LDL15) was not significantly altered (Figure 5D).
The effect was not attributable to lower cholesterol levels
as deficiency of endothelial HMGB1 did not significantly
affect total cholesterol or LDL cholesterol at baseline or
on the high-fat diet. Triglyceride levels were also similar
between groups (Figure III in the Data Supplement).
Mice Lacking Endothelial HMGB1 in the
Setting of LDLR Deficiency Are Protected From
Atherosclerosis
Although informative about the earliest stages of dis-
ease, a drawback of using wild-type mice is that they only
develop lesions on a high-fat diet that contains cholic
Figure 2 Continued. with GFP alone or GFP-HMGB1. Overexpression of HMGB1 significantly increased LDL transcytosis; each point
represents 1 cell. n=4 experiments; ***P<0.001 by t test; error bars represent SD. E, Neither glycyrrhizin (GCZ) nor RAGE (receptor for
advanced glycation endproducts) siRNA blocked the effect of HMGB1 overexpression on LDL transcytosis. Cells were transfected with GFP
alone or GFP-HMGB1 before treatment with GCZ; each point represents 1 cell. n=4 experiments; ***P<0.001 by 1-way ANOVA; error bars
represent SD; ns indicates nonsignificant by Tukey multiple comparison test. GFP indicates green fluorescent protein.
Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557
HMGB1 Is a Critical Regulator of LDL Transcytosis
acid. As cholate can be hepatotoxic,23 this may confound
interpretation of the results. To study the contribution of
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endothelial HMGB1 to more advanced atherosclerotic dis-
ease and without the confounder of dietary cholic acid, we
generated EC-HMGB1−/− mice on a LDLR−/− background.
As before, we injected these double knockout mice and
LDLR−/− littermate controls with Alexa-568–tagged LDL
and measured its accumulation in the inner curvature of
the aortic arch 30 minutes later. Far less Alexa-685–LDL
entered the aorta of EC-HMGB1−/−:LDLR−/− mice than
LDLR−/− controls (Figure 6A). Double knockout mice and
LDLR−/− littermate controls were then fed a high-fat diet
for 5 weeks. EC-HMGB1−/−:LDLR−/− mice developed sig-
nificantly less disease than controls (Figure 6B and 6C)
despite similar or even slightly higher circulating LDL and
triglyceride levels (Figure IV in the Data Supplement).
DISCUSSION
Although the existence of endothelial transcytosis has
been postulated for decades24 until recently, its mecha-
nisms and pathological importance have remained con-
troversial. Technical limitations have hindered its study:
morphological experiments using electron microscopy
provided elegant images but were not feasible for mech-
anistic studies. Experiments using animals were limited
by gaps in our understanding of the regulation of trans-
cytosis, as targets for gene knockout were unclear.
Recent advances in live cell imaging using TIRF
microscopy4 and sensitive studies using radionuclear iso-
topes25 have now elucidated some of the earliest stages
of transcytosis. Two receptors have been identified (SR-
BI and ALK15), and there is a proven pathological role for
endothelial SR-BI–mediated LDL transcytosis in athero-
genesis.6 Determining the regulation of SR-BI–mediated
transcytosis is now a priority.
Our data demonstrate that endothelial HMGB1 plays
an unsuspected role upstream of SR-BI. While HMGB1
is best studied as an alarmin—a protein with cytokine-
like properties that is secreted or released by dying
cells26,27—it is also an abundant and ubiquitous nuclear
protein. Here, we show that HMGB1 translocates to
the nucleus in an LDL- and SR-BI–dependent manner
where it regulates the half-life of SREPB2. This in turn
regulates SR-BI expression and LDL transcytosis. The
effect of HMGB1 depletion on LDL transcytosis was not
due to nonspecific impairment of cell health or vesicu-
lar traffic as albumin transcytosis was unaffected. Fur-
thermore, while it is well established that caveolin-1 is a
critical regulator of LDL (and albumin) transcytosis,15,28,29
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Figure 3. HMGB1 regulates LDL transcytosis via SREBP2.

A, HMGB1 (high mobility group box 1) knockdown in human coronary artery endothelial cells by siRNA led to a decrease in both mature (m)
and precursor (p) forms of SREBP2 (sterol regulatory element-binding protein 2) by Western blot. Representative blot (left); quantification
(right). n=4; *P<0.05 by t test; error bars represent SEM. B, Knockdown of either HMGB1 or SREBP2 significantly inhibited LDL (low-density
lipoprotein) transcytosis, but depletion of both did not achieve any further reduction; each point represents 1 cell. n=4 experiments; ***P<0.001
by 1-way ANOVA; error bars represent SD. C, SREBP2 was knocked down by siRNA achieving a reduction in protein levels of around 80%
(quantification is of precursor form). n=4; *P<0.05 by t test; error bars represent SEM. D, Depletion of SREBP2 prevented induction of LDL
transcytosis caused by HMGB1 overexpression. Each point represents 1 cell. n=4 experiments; ***P<0.001 by 1-way ANOVA; error bars
represent SD; ns indicates nonsignificant by Tukey multiple comparison test.

cells depleted of HMGB1 displayed no change in the these proteins functioning in the same pathway. Over-
total levels or the subcellular distribution of caveolin-1. expression of HMGB1 stimulated LDL transcytosis in
Instead, combinatorial siRNA knockdown of HMGB1 an SREBP2-dependent manner and was unaffected by
and SR-BI, as well as SR-BI and SREBP2, supports inhibition of extracellular HMGB1; conversely incubation

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Figure 4. HMGB1 translocates to the nucleus in an LDL and SR-BI-dependent manner and regulates SREBP2 half-life.
A, Human coronary artery endothelial cells (HCAECs) were immunostained for HMGB1 (high mobility group box 1; red) before and after incubation
with LDL (low-density lipoprotein; 40 μg/mL) for 15 min. LDL significantly increased the nuclear-to-cytoplasmic ratio, and SR-BI (scavenger receptor
class B type 1) depletion by siRNA partially inhibited this effect. Representative images (left); quantification (right). n=4; ***P<0.001 by 1-way
ANOVA, ***P<0.001 and *P<0.05 by Tukey multiple comparison test; error bars represent SEM. Scale bars=30 µm. B, FRAP of nuclear HMGB1
in HCAECs expressing GFP-HMGB1 in the presence or absence of 40 µg/mL unlabeled LDL; ***P<0.001 by Holm-Sidak multiple comparisons
test for each time point. Error bars represent SD. C, Incubation of serum-starved endothelial cells with LDL (40 μg/mL, 24 h) induced HMGB1
expression. Western blot is representative of 4 independent experiments; **P<0.01 by paired t test with data normalized to control. D, The stability
of SREBP2 (sterol regulatory element-binding protein 2) mRNA was assessed in HCAECs. The half-life of SREBP2 mRNA in control cells and cells
depleted of HMGB1 by siRNA was measured in the presence of actinomycin. E, The half-life of SREBP2 protein was assessed in cells depleted
of HMGB1 by siRNA; cycloheximide (CHX) was used to inhibit protein synthesis for the indicated number of hours (h). *P<0.05 and **P<0.01 by
Holm-Sidak multiple comparisons test for each time point. n=4 experiments; error bars represent SEM. M indicates mature; and p, precursor.

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 Ghaffari et al HMGB1 Is a Critical Regulator of LDL Transcytosis
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Figure 5. Mice deficient in endothelial HMGB1 develop fewer fatty streaks on a high-fat diet.
A, Mice deficient in endothelial HMGB1 (high mobility group box 1) and wild-type littermates were injected retro-orbitally with 200 μg of AlexaFluor
(AF) 568–conjugated LDL (low-density lipoprotein) and euthanized 30 min later. The accumulation of LDL was measured and normalized to the
imaged area and to the paired control animal. Representative images of the inner curvature of the aorta en face (left); quantification with each
point representing 1 animal (right); *P<0.05 by paired, nonparametric t test. B, Mice deficient in endothelial HMGB1 and wild-type littermates
were fed a high-fat diet for 18 wk followed by staining of the inner curvature of the aorta with oil red O (ORO). Of 12 wild-type mice, 6 developed
visible plaque compared with 5 (of 11) knockout mice. Of those displaying plaque, endothelial cell (EC)–HMGB1−/− mice showed significantly
smaller fatty streaks than littermate controls (scatterplot). Representative images (left); quantification (right); each point represents 1 animal;
**P<0.01 by t test. C, Lipid accumulation at the aortic root was also measured (representative image at left) and quantified (right); data are from
7 knockout animals and 9 controls. D, EC-HMGB1−/− mice and controls were injected via retro-orbital vein with 1% Evans blue dye and then
euthanized after 30 min. Permeability to Evans blue dye was not significantly altered. n=4 to 5. ns indicates nonsignificant.

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Figure 6. Mice lacking endothelial HMGB1 in the setting of LDLR deficiency are protected from atherosclerosis.
A, Endothelial cell (EC)–HMGB1−/−:LDLR−/− and LDLR−/− littermate controls were injected retro-orbitally with 200 μg of AlexaFluor (AF)
568–conjugated LDL (low-density lipoprotein) and euthanized 30 min later. The accumulation of LDL was measured and normalized to the
imaged area and to the paired control animal. Representative images of the inner curvature of the aorta en face (left); quantification with each
point representing 1 animal (right); *P<0.05 by paired, nonparametric t test. B, Double knockout mice (EC-HMGB1−/−:LDLR−/−) and LDLR−/−
littermate controls were fed a high-fat diet for 5 wk followed by staining of the inner curvature of the aorta with oil red O (ORO). EC-HMGB1−
/−
:LDLR−/− mice showed significantly smaller fatty streaks than controls. Representative images (left), quantification (right). n=15 to 17 (each
point represents 1 mouse); *P<0.05 by t test. C, Lipid accumulation at the aortic root was also measured (representative image at left) and
quantified (right); data are from 15 knockout animals and 18 controls; P<0.05 by unpaired t test. HMGB1 indicates high mobility group box 1;
and LDLR, low-density lipoprotein receptor.

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 Ghaffari et al
with LDL induced HMGB1 expression. Finally, mice
deficient in endothelial HMGB1 developed smaller fatty
Basic Sciences - AL
streaks and (when on a LDLR−/− background) signifi-
cantly less atherosclerosis.
While HMGB1 has been implicated previously in ath-
erosclerosis, prior literature has focused on its function
extracellularly where major sources include leukocytes7
and smooth muscle cells.30 Our data indicate an important
role for endothelial HMGB1 that occurs far earlier in the
disease and is independent of its paracrine functions.
Under low sterol conditions, SREPB2 is translocated to
the nucleus; otherwise, it resides in its immature form in the
endoplasmic reticulum.31,32 The enhanced recruitment of
HMGB1 to the nucleus upon addition of LDL may, there-
fore, seem paradoxical. Our data suggest that a function
of HMGB1 is to ensure the stability of transcription fac-
tors, including SREBP2. We speculate that this would per-
mit continuing expression of SR-BI and other downstream
genes in response to elevated circulating cholesterol. At
this point, it is unknown whether this system is specific to
LDL transcytosis or whether it might also have the effect
of enhancing HDL transcytosis (and potentially reverse
cholesterol transport33). While it is known that HMGB1
can enhance the function of other transcription factors
(eg, p5334; glucocorticoid receptor35), in our experiments,
the effect of HMGB1 overexpression and HMGB1 knock-
down on LDL transcytosis required SR-BI and SREBP2.
This supports the notion of a specific axis linking HMGB1–
SREBP2–SR-BI that regulates LDL transcytosis (Sche-
Downloaded from http://ahajournals.org by on November 17, 2022
matic). The mechanism by which nuclear HMGB1 regulates
the stability of SREBP2 mRNA remains unknown as does
its specificity; the effect may be via RNA binding or through
the recruitment of other proteins.36
The translocation of HMGB1 from the nucleus to
the cytoplasm requires posttranslational modifications
such as hyperacetylation and phosphorylation.11 Further
research will be needed to determine the mechanisms by
which HMGB1 accumulates in the endothelial nucleus
in response to LDL, for instance, whether they are sim-
ply reciprocal to what has been reported (deacetylation,
dephosphorylation) or whether specific and additional
modifications are required. Our data also raise the ques-
tion of how the endothelial cells sense the addition of LDL;
while depletion of SR-BI significantly attenuated HMGB1
translocation, the effect was only partial. This suggests
the potential involvement of another receptor or sensor.
Our preliminary experiments suggest that LDLR is not
required, and we are currently examining other candidates.
Our experiments in mice deficient in endothelial
HMGB1 support a role for the protein in regulating early
disease: we observed a significant reduction in accu-
mulation of LDL in the aortic arch of knockout animals
within 30 minutes of injection into the circulation. This
supports the notion that LDL transcytosis was reduced
in vivo, consistent with our in vitro data. On a high-fat
diet, circulating cholesterol levels in the wild-type and
214   January 2021 Arterioscler Thromb Vasc Biol. 2021;41:200–216. DOI: 10.1161/ATVBAHA.120.314557
HMGB1 Is a Critical Regulator of LDL Transcytosis
knockout animals were similar; yet knockout mice had a
significant reduction in fatty streaks. Despite the incom-
plete specificity of the Tie2Cre promoter for endothe-
lial cells, we found no significant reduction in HMGB1
expression in monocytes or neutrophils. Furthermore,
the attenuation in LDL accumulation in the aortic arch of
knockout animals within 30 minutes of injection is most
consistent with an impairment of endothelial LDL trans-
cytosis rather than a leukocyte-driven phenotype.
We obtained similar results in a double knockout
mouse model in which endothelial HMGB1 was deleted
in a LDLR−/− background. Unlike the ApoE−/− mouse
model, LDLR-deficient animals develop elevated LDL
levels appropriate for studying LDL transcytosis. How-
ever, the contribution of HMGB1 to LDL transcytosis by
the endothelium is harder to interpret in advanced athero-
sclerotic disease given the protein’s established role in
endothelial activation and leukocyte recruitment, as well
as the presence of extracellular HMGB1. This makes it
impossible to attribute the reduction in atherosclerosis to
impaired transcytosis alone. As with the single knockout
animals, double knockout mice displayed a significant
reduction in the accumulation of LDL in the aorta within
30 minutes of injection. As these mice and their controls
both lack LDLR, this observation supports the notion that
SR-BI–mediated LDL transcytosis is reduced by deletion
of HMGB1 in the endothelium.
In contrast to its proinflammatory characteristics once
secreted or released, HMGB1 that is intracellular has been
reported to be anti-inflammatory by promoting autophagy
and preventing apoptosis.37,38 These studies have focused
on the role of the protein when in the cytosol rather than
on its nuclear functions. Given that the vast majority of
HMGB1 in cells is localized to the nucleus, the reduction
in LDL transcytosis caused by siRNA to HMGB1 likely
reflects the loss of nuclear rather than cytoplasmic pro-
tein. As autophagy suppresses LDL transcytosis,39,40 loss
of cytoplasmic HMGB1 would be expected to enhance
rather than inhibit LDL transcytosis. While our data impli-
cate nuclear HMGB1 as a positive regulator of transcyto-
sis, our work leaves open the possibility that extracellular
and intracellular HMGB1 may work additively or synergis-
tically to accelerate atherogenesis.
In closing, our findings add to the growing body of litera-
ture showing that LDL transcytosis is a regulated (and thus
potentially therapeutically tractable) process.41 For instance,
we recently reported that estrogen significantly attenuates
LDL transcytosis by endothelial cells from male donors13;
interestingly, the mechanism of this effect was also through
SR-BI. Further understanding of the regulation of SR-BI–
mediated LDL transcytosis is likely to provide insights into
the therapy and prevention of atherosclerosis.
ARTICLE INFORMATION
Received April 23, 2020; accepted October 5, 2020.
 Ghaffari et al
Affiliations
Keenan Centre for Biomedical Research, St. Michael’s Hospital, Toronto, Canada
(S.G., F.N.N., R.S., N.K., C.W., W.L.L.). Department of Laboratory Medicine and Patho-
biology (E.J., R.S., W.L.L.) and Department of Biochemistry (W.L.L.), University of
Toronto, Canada. Hospital for Sick Children, Toronto, Canada (B.E.S., N.M.G.). To-
ronto General Hospital Research Institute, University Health Network, Canada (J.I.).
Acknowledgments
We thank Dr Monika Lodyga from the Research Core Facility of the Keenan
Research Centre for assistance with flow cytometry.
Sources of Funding
This work was funded by grants from the Heart and Stroke Foundation of Canada
(G-16-00013521) and the Canadian Institutes of Health Research (CIHR; PJT
168947), both to W.L. Lee. S. Ghaffari was supported by a postdoctoral schol-
arship from the Research Training Centre of the Keenan Research Centre for
Biomedical Science; E. Jang was supported by a Canada Graduate Scholarship–
Master’s Award from the CIHR. W.L. Lee is supported by a Canada Research
Chair in Mechanisms of Endothelial Permeability.
Disclosures
None.
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