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Integrative Physiology
Loss of SR-BI Expression Leads to the Early Onset of
Occlusive Atherosclerotic Coronary Artery Disease,
Spontaneous Myocardial Infarctions, Severe Cardiac
Dysfunction, and Premature Death in
Apolipoprotein E–Deficient Mice
Anne Braun, Bernardo L. Trigatti, Mark J. Post, Kaori Sato, Michael Simons, Jay M. Edelberg,
Robert D. Rosenberg, Mark Schrenzel, Monty Krieger
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Abstract—Murine models of atherosclerosis, such as the apolipoprotein E (apoE) or the LDL receptor knockout mice,
usually do not exhibit many of the cardinal features of human coronary heart disease (CHD), eg, spontaneous myocardial
infarction, severe cardiac dysfunction, and premature death. Here we show that mice with homozygous null mutations
in the genes for both the high density lipoprotein receptor SR-BI and apoE (SR-BI/apoE double knockout [dKO] mice)
exhibit morphological and functional defects with similarities to those seen in human CHD. When fed a standard chow
diet, these hypercholesterolemic animals developed significant atherosclerotic lesions in the aortic sinus as early as 4
to 5 weeks after birth. We now show that they also exhibited extensive lipid-rich coronary artery occlusions and
spontaneously developed multiple myocardial infarctions and cardiac dysfunction (eg, enlarged hearts, reduced ejection
fraction and contractility, and ECG abnormalities). Their coronary arterial lesions, which were strikingly similar to
human atherosclerotic plaques, exhibited evidence of cholesterol clefts and extensive fibrin deposition, indicating
hemorrhage and clotting. All of the dKO mice died by 8 weeks of age (50% mortality at 6 weeks). Thus, SR-BI/apoE
dKO mice provide a new murine model for CHD and may help better define the role of lipoprotein metabolism and
atherosclerosis in the pathogenesis of myocardial infarction and cardiac dysfunction. Furthermore, these animals may
be useful for preclinical testing of potential genetic and/or pharmacological therapies for CHD. (Circ Res. 2002;90:270276.)
Key Words: SR-BI/apolipoprotein E knockout mice 䡲 atherosclerosis 䡲 myocardial infarction
䡲 coronary artery disease 䡲 lipoprotein metabolism
O
ne of the best understood risk factors for coronary artery
atherosclerosis, a leading cause of myocardial infarction
(MI) and death, is hypercholesterolemia. Unfortunately, few
hypercholesterolemia and atherosclerosis rodent models
spontaneously develop MIs and cardiac dysfunction. Fat-fed
LDL receptor (LDLR) knockout (KO)1 and chow-fed apolipoprotein E (apoE) KO mice,2– 4 standard models for hypercholesterolemia/atherosclerosis, do not usually exhibit MI or
reduced lifespans; although 24- to 40-week-old, fat-fed apoE
KO mice can develop coronary arterial plaques and, occasionally, small areas of myocardial fibrosis.4,5 ApoE/LDLR
double KO mice fed a high-fat/high-cholesterol diet for 7
months have occlusive coronary arterial atherosclerotic le-
sions and some associated perivascular myocardial scarring.6
Severe stress exacerbates the small ECG abnormalities seen
in these mice at rest and can induce endothelin-dependent
MIs,6 although such MIs were not reported to be lethal. Dahl
salt-sensitive, hypertensive rats expressing high levels of
human cholesteryl ester transfer protein7 and atherosclerosisprone male JCR:LA-corpulent rats,8 which have hyperlipidemia and atherosclerosis, develop coronary artery lesions
and MIs.
It would be useful to have additional, genetically manipulable, murine models of coronary heart disease (CHD) that
combine many of the cardinal features of human cardiovascular disease, including hypercholesterolemia, atherosclero-
Original received July 10, 2001; revision received December 19, 2001; accepted December 19, 2001.
From the Department of Biology (A.B., B.L.T., J.M.E., R.D.R., M.K.) and Division of Comparative Medicine (M. Schrenzel), Massachusetts Institute
of Technology, Cambridge, Mass; and Angiogenesis Research Center (M.J.P., K.S., M. Simons), Department of Medicine, Harvard Medical School and
Beth Israel Deaconess Medical Center, Boston, Mass. Present address for B.L.T. is the Department of Biochemistry, McMaster University, Hamilton,
Canada; for J.M.E., the Department of Medicine, Weill Medical College of Cornell University, New York, NY; for M.J.P. and M. Simons, the Department
of Medicine, Dartmouth Hitchcock Medical Center, Lebanon, NH; for K.S., the Division of Cardiovascular Research, St Elizabeth’s Medical Center,
Boston, Mass; and for M. Schrenzel, Zoological Society of San Diego, San Diego, Calif.
Correspondence to Monty Krieger, Biology Department, Room 68-483, Massachusetts Institute of Technology, Cambridge, MA 02139. E-mail
[email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/hh0302.104462
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Braun et al
Murine Coronary Heart Disease
271
sis, occlusive fibrin-rich coronary artery lesions, ischemia,
MIs, and cardiac dysfunction. We now report that double
knockout (dKO) mice with homozygous null mutations in the
apoE and the HDL receptor scavenger receptor class B, type
I (SR-BI) genes, which exhibit hypercholesterolemia and
dramatically accelerated atherosclerosis,9 spontaneously develop extensive lipid- and fibrin-rich occlusive coronary
arterial lesions, multiple MIs, and cardiac dysfunction and die
prematurely at ⬇6 weeks of age. Thus, SR-BI/apoE dKO
mice represent a new model for the study of CHD.
Materials and Methods
Animals
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Mice (mixed C57BL/6x129 background) were generated at MIT and
were housed and fed a normal chow diet as previously described.9,10
All experiments were performed in accordance with proper guidelines for the care and use of laboratory animals and were subject to
prior approval by the Committee on Animal Care at MIT. Genotypes
were determined as described previously.9,10 Unless otherwise noted,
4- to 6-week old dKO mice, apoE KO littermates, and SR-BI KO and
wild type controls on the same background were studied. No
significant differences were observed between males and females.
Histology
Mice were euthanized and tissues prepared for cryosectioning as
previously described.9 Tissues for paraffin sections were immersionfixed in buffered 10% formalin (J.T. Baker). Sometimes heparin was
administered (450 U/20 g, IV) prior to euthanasia to prevent
coagulation.11 Tissue sections were stained with Masson’s trichrome
(Sigma),12 hematoxylin, and eosin (H&E) or Oil red O and hematoxylin.9 Immunohistochemistry was performed using anti-fibrin
(NYB-T2G1, 1 ␮g/mL, Accurate Chemical & Scientific Corp) or
anti-macrophage (F4/80, MCA 497, Serotec, diluted 1:10) antibodies
using M.O.M. immunodetection (AEC substrate) or Vectastain Elite
ABC (diaminobenzidine substrate) kits (Vector), respectively, with
hematoxylin counter-staining.
Figure 1. Survival of dKO mice and appearance of their hearts.
A, Percentage survival of control (dashed line) apoE KO (n⫽15),
SR-BI-het/apoE KO (n⫽19), and dKO (solid line, n⫽13) littermates, as a function of age. B and C, Posterior views of perfused hearts from apoE KO (6-weeks old, B) and dKO (5-weeks
old, C) mice. D, Lateral view of the right side of a heart from a
different 5-week old dKO mouse. Black and white arrows indicate left and right atria, respectively; arrowheads, myocardial
lesions. Original magnification: 7.5⫻.
Angiography
After median sternotomy, cannulation of the ascending aorta (PE50
polyethylene tubing: Becton Dickinson and Company), and opening
the right atrium for drainage, each heart was harvested, flushed with
PBS, and barium sulfate (E-Z-EM, Inc) was injected manually at a
maximum pressure of 80 mm Hg. Angiograms were obtained with a
Micro 50 (General Electric, 20 kV, 20-second exposure). Only the
left coronary arterial network could be routinely observed.
Gravimetry
Electrocardiography
Mice were euthanized, weighed, and perfused,9 and intact hearts or
the right ventricular (RV) free wall and the left ventricle
(LV)⫹septum (LV⫹S) were dissected and weighed.
For avertin-anesthetized mice, ECGs were recorded using 6 standard
limb leads with a Silogic EC-60 monitor (Silogic Design Limited,13).
For conscious mice, ECGs were recorded using AnonyMOUSE ECG
Screening Tools (Mouse Specifics, Inc).14
Magnetic Resonance Imaging (MRI)
Mice were anesthetized (chloral hydrate, 200 to 320 mg/kg IP;
Sigma) and placed in a 2T small bore magnet (Bruker Instruments) on a custom body coil containing ECG electrode patches.
Heart rates were adjusted to ⬇300 bpm with 1% to 2% isoflurane.
Scout, long-axis, and 6 to 7 1-mm thick short-axis images were
collected. Short-axis images spanning the entire heart were used
to measure LV tissue volume, LV end diastolic and end systolic
luminal volumes (LVEDV and LVESV), and ejection fractions
(EF⫽((LVEDV⫺LVESV)/LVEDV)⫻100%).
Hemodynamic Evaluation
Mice were heparinized (1 U/10 g IP), anesthetized with chloral
hydrate as above, intubated, and ventilated (Harvard Apparatus, Inc)
with room air (130 breaths/min; tidal volume: ⬇15 ␮L/g). Lidocaine
HCl (0.5%; Abbott) was administered locally. The right carotid
artery was exposed and a 1.4 Fr micromanometer catheter (Millar
Instruments) was advanced into the aorta and then the LV for
pressure measurements. LV pressures were measured before and
after cutting both vagal nerves. Data were recorded using a Windaq
DI 220 converter and analyzed using Windaq Pro software (Dataq
Instrument) with some manual intervention to correct for micromanometer drift and insure proper evaluation of LVEDP.
Statistical Analysis
A value of Pⱕ0.05 was considered significant (2-tailed, unpaired
Student’s t test or ANOVA test, StatView).
Results
SR-BI/apoE double KO mice (dKO mice) fed a standard low
fat/low cholesterol diet develop extensive aortic sinus atherosclerosis by 5 weeks of age and die prematurely.9 All of the
dKO mice died between 5 to 8 weeks of age (50% mortality
at 6 weeks; Figure 1A, solid line). No control mice died
during this period (Figure 1A, dashed line). Before death, the
dKO mice exhibited a 1- to 2-day period of progressively
reduced activity and altered appearance (ruffled fur, abnormal
gate, and occasionally labored breathing). We therefore
explored the possibility that the dKO mice had cardiac
abnormalities.
Extensive Myocardial Fibrosis
dKO hearts (Figures 1C and 1D) were enlarged relative to
controls (eg, apoE KO, Figure 1B; see below) and exhibited
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Circulation Research
February 22, 2002
Figure 3. Gravimetric (A) and MRI (B) analyses. A, Mean values
of intact heart, left ventricular wall⫹septum (LV⫹S), and right
ventricular (RV) wall weights normalized to body weights.
**P⬍0.0001 by ANOVA. B, Mean values of LV⫹S tissue volumes
and LV end diastolic luminal volumes (LVEDV) determined by
MRI and normalized to body weights. *P⫽0.003 and **P⬍0.0001
by Student’s t test and ANOVA. Error bars represent 1 standard
deviation.
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Figure 2. Histological analysis of dKO hearts. A, Longitudinal
section of a dKO heart stained with trichrome (healthy myocardium, red; fibrotic tissue, blue). Arrow and B/C box indicate
fibrotic lesions in the left ventricular outflow tract area; arrowheads, right ventricular wall lesions. *Left ventricular lumen, filled
with blood (not perfused). Scale bar: 1 mm. B and C, Higher
magnification views of the lesion shown in B/C box of panel A
stained with trichrome (B) and H&E (C). Arrow indicates fibrotic
tissue (B) and abnormal myocardial cell (C); Arrowheads, dilated
cells. D, Higher magnification view of a right ventricular wall
lesion (trichrome). E, Trichrome-stained apical section of a heart
from a different dKO mouse exhibiting myocardial scarring. F
through H, Serial cross-sections through the left ventricular outflow tract area stained with either trichrome (F), F4/80 antibody
(macrophages, G), or Oil red O (lipid, H). A mitral valve leaflet
(top left corner in G and H) and the corresponding attachment
site can be seen (bottom left corner in F through H). Arrows
indicate areas of intense fibrosis (F), macrophage staining (G),
and globular, apparently intracellular, lipid staining (H). Solid
black arrowheads indicate areas virtually devoid of cells (F and
G) containing small punctate, apparently extracellular lipid staining (H); white-filled arrowheads, less fibrotic, less damaged
areas (F, G) that exhibit more diffuse, less intense lipid staining
(H). I through K, Serial cross-sections through a LV papillary
muscle (same mouse as in F through H), stained with either
trichrome (I), F4/80 antibody (J), or Oil red O (K). Blue fibrotic
tissue (I), brown macrophage staining (J), and red lipid staining
(K) are seen in the central area of the papillary muscle. Scale
bars (B through K): 50 ␮m.
pale, discolored patches (arrowheads, Figures 1C and 1D) not
seen in any controls (eg, Figure 1B), suggesting extensive MI
and scarring. These lesions were always present in the
atrioventricular (AV) groove of the left ventricle (LV, Figure
1C) and frequently present at various locations on the right
ventricular (RV) wall (Figure 1D), the LV wall, and/or the
apex (not shown). Figure 2A shows a representative longitudinal section of a dKO heart (unperfused) stained with
trichrome (healthy myocardium red, fibrotic tissue blue).
Regions surrounding the mitral valves (not the valves themselves) and the LV outflow tract were invariably fibrotic
(Figure 2A, B/C box and arrow). Higher magnification views
(Figures 2B and 2C) show lesions contained fibrotic connective tissue (Figure 2B, arrow), few remaining myocytes
(Figure 2C, arrow), and numerous large, dilated, apparently
mononuclear inflammatory cells (Figures 2B and 2C, arrow-
heads), some of which were macrophages (brown, punctate
F4/80 antibody staining, Figure 2G). Lesions in the RV free
wall and more apical regions (Figure 2A, arrowheads, and
Figures 2D and E) appeared more well-organized and contained fewer dilated cells than those in the outflow tract area
and were characterized by extensive fibrosis, inflammation
(Figure 2D), and in some cases, diffuse necrosis and myocardial scarring typical of healed infarcts (Figure 2E). Numerous macrophages were detected in these lesions and
lesions in the papillary muscle (Figures 2G and 2J). Thus,
macroscopic and microscopic observations revealed multiple
MIs in the dKO mice.
In hypercholesterolemic animals, macrophages can accumulate extensive cytosolic lipid deposits (foam cells).4 Neutral lipid staining (oil red O, Figures 2H and 2K) of dKO
hearts was particularly intense in macrophage-rich, fibrotic
regions (Figures 2F through 2K) and appeared both in a
concentrated, intense, globular pattern reminiscent of intracellular lipid (Figure 2H, arrow) and in a punctate pattern
reminiscent of extracellular lipid (Figure 2H, black arrowheads).15 More diffusely distributed lipid was detected in
non-fibrotic tissue throughout the heart between myocardial
fibers (eg, Figure 2H, white-filled arrowheads). The codistribution of lipid and macrophages suggested the presence of
macrophage foam cells. Future studies will determine if
macrophage infiltration into fibrotic lesions is a consequence
of and/or contributes to lesion development and if these
lesions are similar to those in human inflammatory
cardiomyopathies.
Heart Function
Intact hearts and LVs⫹septa (LV⫹S) and RVs from dKO
mice were larger than those from age-matched controls
(Figures 1B through 1D and Figure 3A, 1.6- to 1.8-fold
greater mean heart-to-body weight ratios). Furthermore, dKO
mice had a significantly lower body weight (15.3⫾2.0 g) than
control animals (wild type: 20.7⫾4.3 g; SR-BI KO: 21.3⫾3.8
g; apoE KO: 18.8⫾2.3 g. P⫽0.002). This was confirmed by
MRI analysis of LV⫹S tissue volume (Figure 3B). Increased
tissue volume reflects a thicker LV⫹S wall (assuming no
change in ventricular length). In contrast, the body weight–
Braun et al
Murine Coronary Heart Disease
273
Hemodynamic Analyses of Heart Function in Control and dKO Mice
Genotype
Aortic Diastolic
Pressure,
mm Hg
Aortic Systolic
Pressure,
mm Hg
LVEDP,
mm Hg
LVSP,
mm Hg
⫹dP/dt,
mm Hg/s
⫺dP/dt,
mm Hg/s
Heart Rate, bpm
Wild type
61⫾17 (6)
86⫾17 (6)
5.7⫾0.9 (6)
88⫾17 (6)
3800⫾900 (6)
⫺3400⫾800 (6)
509⫾71 (6)
SR-BI KO
55⫾22 (5)
79⫾20 (5)
7⫾4.4 (3)
73⫾11 (3)
3200⫾900 (3)
⫺2900⫾600 (3)
565⫾122 (5)
apoE KO
52⫾12 (5)
77⫾9 (5)
6.1⫾1.7 (5)
82⫾7 (5)
3500⫾500 (5)
⫺3300⫾500 (5)
524⫾77 (5)
dKO
39⫾6 (6)
54⫾5 (6)†
9.6⫾2.2 (6)
40⫾19 (6)†
1100⫾500 (6)‡
⫺1100⫾400 (6)‡
390⫾39 (6)
0.1
0.005
0.49
0.0002
⬍0.0001
⬍0.0001
0.01
P (ANOVA)*
Data shown here were obtained before cutting the vagal nerves (see Materials and Methods). LVEDP indicates LV end diastolic pressure; LVSP,
LV systolic pressure. Values represent mean⫾1 SD. Numbers of animals per group are indicated in parentheses.
*ANOVA test of probability that all samples belong to the same group. †,‡Pairwise comparisons with each of the controls by unpaired Student’s
t test; †P⬍0.03 and ‡P⬍0.003.
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corrected LVEDVs were only slightly higher for dKO hearts
(Figure 3B), suggesting only minor dilation. Thus, the increased size of dKO hearts was due primarily to increased
ventricular tissue mass, possibly resulting from thickening of
the wall near the outflow tract and compensatory thickening
of the ventricular wall in response to reduced contractility
(see following paragraph).
Hemodynamic analysis revealed that aortic systolic blood
pressure and heart rate (HR) were significantly lower in dKO
than in control mice (Table). dKO mice also had substantially
lower LV systolic pressure (LVSP) and contractility (⫹dP/
dt), indicating LV systolic dysfunction. A similar (3-fold)
reduction in ⫺dP/dt indicated impaired LV relaxation. The
somewhat lower HR of dKO mice relative to controls (not
observed in nonanesthetized mice) was not due to extracardiac neuronal influences (bilateral disruption of the vagal
nerves did not eliminate the HR differences, not shown).
Although reduced HR might have contributed to reduced
blood pressure and contractility, and might complicate interpretation of differences in dP/dt values, it is unlikely that
these relatively small baseline differences caused the large
changes in both ⫹dP/dt and ⫺dP/dt. Furthermore, values for
the products of pressures (P) with either ⫹dP/dt or ⫺dP/dt
showed the same trends, indicating a minimal or insignificant
influence of pressure on dP/dt values (not shown). The
decreased aortic blood pressures and abnormal contractility
and relaxation in these dKO mice are consistent with primary
cardiac dysfunction. We also measured carotid arterial blood
pressure in dKO mice (n⫽3) and control littermates (apoE
KO mice with a heterozygous null mutation in SR-BI, n⫽3)
at 3 (chloral hydrate anesthesia, 0.2 mg/g) and again at 4
(urethane anesthesia, 1 mg/g) weeks of age. We observed no
blood pressure differences at 3 weeks and only a slight
relative reduction in the dKO mice at 4 weeks (data not
shown). Thus, it is unlikely that hypertension was responsible
for the ventricular hypertrophy or other cardiac defects
exhibited by dKO animals.
MRI images (Figure 4A) at end-diastole or end-systole show
that, whereas the LVEDVs were similar (left panels, black
arrows), the LV end systolic volumes (LVESVs) were substantially higher in dKO hearts than in the controls (right panels,
black arrows). Consequently, the ejection fractions of the dKO
hearts, a critical measure of heart function, were substantially
lower (⬇50%) than those of controls (Figure 4B).
In unanesthetized, conscious mice, normal ECG patterns
were seen in controls (eg, apoE KO: Figure 4C, top; other
controls not shown), whereas striking abnormalities were
observed in 6 of 12 dKO mice. One exhibited an ST elevation
of unclear etiology (not shown), and 5 showed severe ST
depression (Figure 4C, bottom), indicating subendocardial
ischemia.14,16 In 5 of 8 dKO mice, but not in any controls,
Figure 4. MRI and ECG analyses. A, Representative long-axis
MRI images of hearts from apoE KO (top) and dKO (bottom)
mice at end diastole (left) and end systole (right). Blood-filled
heart chambers appear white. Black arrows indicate lumens of
the left ventricle (LV). Also indicated in the top left panel are the
lumens of the right atrium (RA, white arrowhead), left atrium (LA,
black arrowhead), and right ventricle (RV, white arrow). B, MRIbased average ejection fractions from control apoE KO (n⫽5)
and dKO (n⫽4) mice. **P⫽0.0002 by ANOVA. C, ECGs from
nonanesthetized, conscious apoE KO (top panel) or dKO (bottom) mice obtained using footpad-leads. Brackets indicate ST
regions. Heart rates (mean bpm⫾ standard deviation) of the
conscious mice measured from the ECGs were 761⫾54 (apoE
KO, n⫽9); 652⫾82 (dKO, n⫽12); 698⫾70 (SR-BI KO, n⫽6);
646⫾80 (SR-BI-het, n⫽4); P⫽0.01, ANOVA (apoE KO greater
than others). D, ECGs from avertin-anesthetized apoE KO (top)
and dKO (middle and bottom) mice obtained using standard
limb-leads. Brackets indicate widened QRS complexes and
arrow shows an escaped QRS complex. In the bottom panel,
arrows indicate a P wave and a P wave–independent QRS
complex.
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Circulation Research
February 22, 2002
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Figure 5. Angiographic and histological analysis of coronary arteries. A, Ex vivo angiograms of wild-type (left) and dKO (right) hearts.
Arrows indicate left anterior descending (LAD) coronary artery; arrowheads (right), multiple severe stenoses in coronary arteries branching from the LAD. B through I, Histological sections (D, paraffin section, all others frozen sections) of coronary arteries from different
mice were stained as follows: B, C, E, and G, Oil red O (“lipid”)/hematoxylin; D and F, trichrome; H, anti-fibrin antibody; I, control without primary antibody (“no ab”). The sections illustrate the following: B, lesion-free left main coronary artery of an apoE KO mouse; C
through I, coronary artery lesions of dKO mice. C and E, Nearly complete occlusions in the left main coronary artery from one mouse
either distal to (C) or at (E) the ostium. The complex occlusion in E (arrow) is adjacent to a typical lipid-rich atheromatous plaque
(arrowhead). D, Occluded major right coronary artery of a different mouse with perivascular fibrosis and inflammation. F through I, Serial
cross-sections through a septal coronary artery of another dKO mouse. F and G, Cholesterol clefts (arrow) are seen within the lipid-rich
core of the lesion. H, Red-brown intense immunostaining of fibrin deposits within the plaque. I, No antibody control for panel H. Scale
bars: 40 ␮m.
avertin anesthesia induced or uncovered cardiac conductance
defects (Figure 4D, widened QRS, middle panel), which in
some cases included escaped QRSs (middle panel) and
progressed to complete AV blocks and bradycardic death
(Figure 4D, bottom). These conductance defects were not
observed in unanesthetized mice. Further studies will be
required to determine if development of heart blocks might
contribute to the spontaneous death of unanesthetized dKO
mice. The gravimetric, hemodynamic, MRI, and ECG findings unequivocally demonstrate impaired heart function in
dKO mice, possibly because of extensive myocardial fibrosis.
Coronary Artery Disease: Angiography and Histology
To determine if occlusive coronary artery disease may have
contributed to cardiac dysfunction, we performed ex vivo
angiography (Figure 5A). No obvious defects were apparent
in control hearts (wild type, n⫽4, left panel; apoE KO, n⫽4,
and SR-BI KO, n⫽3, data not shown). Five of seven dKO
hearts examined showed stenoses and occlusions of branches
of the left coronary arteries (eg, right panel, arrowheads), and
there were 2 instances of apparent stenoses in the main
coronary arteries (not shown).
Histological analyses of dKO hearts revealed extensive
coronary artery disease (CAD) (Figures 5C through 5I).
There were complex occlusions of major arterial branches in
the LV free wall (9 of 10 mice analyzed), the septum (10 of
11), and the RV wall (11 of 12). No occlusions were seen in
age-matched controls (apoE KO, Figure 5B; SR-BI KO and
wild type not shown). Figure 5C shows a partially cellular,
lipid-rich lesion almost completely occluding the lumen of a
left coronary branching artery. Figure 5D shows fibrosis and
inflammatory cells surrounding an occluded artery in the RV
wall of another dKO mouse. Proximal lesions in coronary
ostia (Figure 5E, arrow; adjacent to a typical, lipid-rich
atheromatous plaque in the sinus, arrowhead) were also seen
in 7 of 10 dKO mice. These complex lesions are probably
responsible for the patchy MIs in the LV and RV. Figures 5F
through 5I show serial cross-sections through an occluded
coronary artery from another dKO mouse. Trichrome (Figure
5F) and lipid staining (Figure 5G) revealed numerous cholesterol clefts (arrows)15 within a lipid-rich, acellular, potentially ‘necrotic’ core.17,18 Frequently, a substantial portion of
the lesions appeared to be acellular, and some of these
amorphous regions stained blue with trichrome (eg, Figure
5F), suggesting the presence of collagen. Immunostaining
showed fibrin deposits in the core regions of 8 of 10 lesions
observed in 3 of 3 dKO mice (eg, Figures 5H and 5I) but not
in age-matched apoE KO controls (n⫽3, not shown). This
thrombosis may be a consequence of bleeding into these
complex lesions or perhaps plaque rupture.
Discussion
The severe occlusive, fibrin-containing coronary arterial lesions, probable ischemia, multiple MIs, enlarged hearts, and
cardiac dysfunction in very young (⬇5 weeks old), low-fat/
low-cholesterol fed SR-BI/apoE dKO mice provide a novel
model of CHD. Both SR-BI and apoE normally play critical
roles in lipoprotein metabolism and can protect mice from
atherosclerosis.2– 4,5,9,19 –22 ApoE apparently influences atherosclerosis by mechanisms both independent of (or only
subtly dependent on), as well as dependent on, its effects on
plasma lipoprotein structure and abundance (reviewed in
References 20 and 21). Hepatic expression of the HDL
receptor SR-BI controls HDL structure and metabolism and
plays an important role in reverse cholesterol transport
(RCT), the transport via HDL of cholesterol from peripheral
tissues (including atheromatous plaques) to the liver for
recycling or biliary excretion.9,10,19,22–24 SR-BI can also mediate cholesterol efflux from cells.25 SR-BI deficiency doubles plasma cholesterol levels and decreases biliary cholesterol secretion.9,10,24 Combined deficiencies of SR-BI and
apoE profoundly alter lipoprotein metabolism,9 resulting in
decreased biliary cholesterol and increased plasma choles-
Braun et al
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terol in VLDL-sized and in abnormally large HDL-like
particles.9
The molecular mechanisms responsible for the dramatically accelerated occlusive atherosclerotic disease in the dKO
mice relative to the apoE KO mice may include9 (1) changes
in plasma proatherogenic and antiatherogenic lipoproteins,
(2) altered cholesterol flux into or out of the artery wall, and
(3) decreased RCT. SR-BI has also recently been shown to
mediate HDL-dependent endothelial nitric oxide synthetase
activation in vascular endothelium26 and the cellular uptake
from lipoproteins of vitamin E,27–29 which can inhibit atherosclerosis in apoE KO mice.30 –32 Loss of these activities may
contribute to the accelerated atherosclerosis in dKO mice.
Few of the current models of CHD exhibiting cardiac fibrosis,
hypertrophy, heart dysfunction, and in some cases, premature
death33– 40 involve primary defects in lipoprotein metabolism
(see Introduction).5,6 The SR-BI/apoE dKO mice are distinct
because they have extensive coronary artery lesions with
fibrin deposition and spontaneously develop extensive MIs
on a standard chow diet at a very young age (5 weeks). Severe
cardiac dysfunction and repetitive MIs (with associated risk
of arrhythmias) due to occlusive CAD are likely to contribute
to their premature deaths. The possible roles of associated
myocardial inflammation and lipid accumulation in the myocardium, and of other metabolic/organ systems in the premature death of the dKO mice, remain to be determined (eg, we
have observed a reduced hematocrit and reticulocytosis in
dKO mice41 that might have resulted in increased susceptibility to myocardial ischemia). Nevertheless, the sudden
bradycardic death that occasionally accompanied avertin
anesthesia (Figure 4D) was almost certainly a consequence of
the induction of cardiac conductance defects and complete
AV block.
The occlusive lesions in coronary arteries of SR-BI/apoE
dKO mice were highly complex, containing cholesterol clefts
and fibrin deposits. Although there are previous reports of
spontaneous atherosclerotic lesions in older apoE KO mice
that stain positively with anti-fibrinogen antibodies and/or
show evidence of necrotic zones, abundant cholesterol clefts,
and hemorrhage,17,42– 46 we are not aware of other reports of
direct immunohistochemical detection of fibrin in murine
atherosclerotic plaque. Additional studies are required to
determine the mechanism of fibrin deposition (eg, hemorrhage due to plaque fission or rupture,47 erosion of small
vessels supplying the plaques,48 or some other mechanism).
In the dKO mice, fibrin deposition in plaque might either
contribute to or be a consequence of occlusive lesion growth,
or both. Although not required for atherosclerotic plaque
formation in apoE KO mice,44 fibrin deposition in the context
of repetitive vascular injury might stimulate the growth of
plaques.43
The occlusive lesions in SR-BI/apoE dKO mice apparently
result in ischemia (see Figure 4C) and the formation of
multiple patchy MIs with variable sizes and locations. Future
studies will be required to determine if thrombosis plays a
role in this pathology. In humans, multiple infarcts lead to a
gradual decline in systolic function, first manifest under stress
and later seen under resting conditions. It is striking that the
young dKO mice (5 to 6 weeks old) at rest exhibit systolic
Murine Coronary Heart Disease
275
dysfunction (hemodynamic and EF abnormalities). This and
an abnormally high heart-to-body weight ratio49 indicate
severe cardiac dysfunction. Furthermore, in humans with
heart disease50,51 and SR-BI/apoE dKO mice, anesthesia can
induce substantial conductance abnormalities (eg, brady-arrhythmias and AV blocks). Thus, these dKO mice may prove
to be a useful model to investigate the mechanisms underlying the development of complex CAD and MI. They may also
be useful for preclinical testing of potential genetic and/or
pharmacological therapies for CHD.
Acknowledgments
This work was supported by grants HL41484, HL64737, HL66105,
and HL52212 to M.K., and HL63609 and HL53793 to M. Simons
and M.J.P. from the US National Institutes of Health and grant EIG
9940074 from the American Heart Association to M. Simons. A.B.
was a Human Frontiers Science Program postdoctoral fellow. B.L.T.
was a Medical Research Council of Canada postdoctoral fellow. We
thank J. Bao, A. Bashir, D. Burstein, J. Chen, P. Christie, E.
Edelman, C. Egles, T. Hampton, R. Hynes, M. Josephson, H. Kanji,
J. M. Kwiecien, F. Mamuya, H. Miettinen, J. Munasinghe, R.
Oppenheimer, M. Penman, F. Schoen, R. Thiro Wirasinghe, and M.
Viñals for technical assistance, advice, and/or helpful discussions.
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Loss of SR-BI Expression Leads to the Early Onset of Occlusive Atherosclerotic Coronary
Artery Disease, Spontaneous Myocardial Infarctions, Severe Cardiac Dysfunction, and P
remature Death in Apolipoprotein E−Deficient Mice
Anne Braun, Bernardo L. Trigatti, Mark J. Post, Kaori Sato, Michael Simons, Jay M. Edelberg,
Robert D. Rosenberg, Mark Schrenzel and Monty Krieger
Circ Res. 2002;90:270-276; originally published online January 3, 2002;
doi: 10.1161/hh0302.104462
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