U.S. patent application number 10/430770 was filed with the patent office on 2004-01-08 for prevention or treatment of abnormal lipoprotein, atherosclerosis and cholestasis.
This patent application is currently assigned to Massachusetts Institute of Technology. Invention is credited to Braun-Egles, Anne, Krieger, Monty, Miettinen, Helena E..
Application Number | 20040006129 10/430770 |
Document ID | / |
Family ID | 29548317 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040006129 |
Kind Code |
A1 |
Krieger, Monty ; et
al. |
January 8, 2004 |
Prevention or treatment of abnormal lipoprotein, atherosclerosis
and cholestasis
Abstract
Using an animal model, transgenic animals that do not express
functional SR-BI and apoE which develop severe atherosclerosis, by
age four weeks in transgenic mice, a class of drugs, PROBUCOL
(4,4'-(isopropylidenedithio) bis(2,6-di-tert-butylphenol)) and
monoesters of PROBUCOL, and BO 653,
2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-benzofuran,
has been discovered which is useful in normalizing abnormal
lipoprotein levels and/or characteristics, such as those found in
lipoprotein X-associated disease. These animals are good models for
screening of drugs useful in the treatment and/or prevention of
cardiac fibrosis, myocardial infarction, defects in electrical
conductance, atherosclerosis, unstable plaque, and stroke, as well
as for lipoprotein disorders such as cholestasis and lipoprotein X
associated disorders. Studies demonstrate normalization of
lipoprotein levels and structure, as well as significant decreases
in atherosclerosis and prevention of heart attack, even when
administered after disease onset.
Inventors: |
Krieger, Monty; (Needham,
MA) ; Braun-Egles, Anne; (Strasbourg, FR) ;
Miettinen, Helena E.; (Helsinki, FI) |
Correspondence
Address: |
PATREA L. PABST
HOLLAND & KNIGHT LLP
SUITE 2000, ONE ATLANTIC CENTER
1201 WEST PEACHTREE STREET, N.E.
ATLANTA
GA
30309-3400
US
|
Assignee: |
Massachusetts Institute of
Technology
|
Family ID: |
29548317 |
Appl. No.: |
10/430770 |
Filed: |
May 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10430770 |
May 5, 2003 |
|
|
|
10147651 |
May 16, 2002 |
|
|
|
Current U.S.
Class: |
514/458 ;
514/469; 514/474; 514/546; 514/707 |
Current CPC
Class: |
C07K 14/775 20130101;
A01K 2267/0375 20130101; C07K 14/705 20130101; A01K 2217/075
20130101; A61K 31/10 20130101; A61K 49/0008 20130101; A01K 2267/03
20130101; A61P 9/10 20180101; A61K 31/35 20130101; A01K 2227/105
20130101; Y10S 514/824 20130101; A61P 1/16 20180101; C12N 15/8509
20130101; A01K 67/0276 20130101; A61K 31/34 20130101; A61P 3/06
20180101; A61P 9/04 20180101 |
Class at
Publication: |
514/458 ;
514/474; 514/469; 514/546; 514/707 |
International
Class: |
A61K 031/375; A61K
031/355; A61K 031/343; A61K 031/105; A61K 031/22 |
Goverment Interests
[0002] The U.S. government has certain rights to this invention by
virtue of Grants HL41484, HI-52212, and HL20948 from the National
Institutes of Health-National Heart, Lung and Blood Institute to
Monty Kreiger and HL63609 and HL53793 to M. Simons and M. J. P.
from the National Institutes of Health.
Claims
We claim:
1. A method for treating or preventing a disorder or disease
characterized by abnormal lipoprotein or cholesterol metabolism
comprising administering to an individual in need thereof a
compound selected from the group consisting of
4,4'-(isopropylidenedithio) bis(2,6-di-tert-butylphenol),
monoesters and other derivatives thereof,
2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-benzofuran or
a derivative thereof, vitamin E and vitamin C, wherein the compound
is administered in an amount effective to decrease lipoprotein
levels or normalize lipoprotein structure or reduce abnormal
cholesterol metabolism.
2. The method of claim 1 wherein the compound is
4,4'-(isopropylidenedithi- o) bis(2,6-di-tert-butylphenol),
monoesters and other derivatives thereof.
3. The method of claim 1 wherein the compound is
2,3-Dihydro-5-hydroxy-2,2- -dipentyl-4,6-di-tert-butyl-benzofuran
or a derivative thereof.
4. The method of claim 1 wherein the compounds are selected from
the group consisting of vitamin E and vitamin C.
5. The method of claim 1 wherein the compound is administered
orally.
6. The method of claim 1 wherein the compound is administered to an
individual with cholestasis.
7. The method of claim 1 wherein the compound is administered to an
individual with atherosclerosis.
8. The method of claim 1 wherein the compound is administered to an
individual with lipoprotein X.
9. The method of claim 1 wherein the compound is administered to an
individual with a disease due to abnormalities in cholesterol
metabolism.
10. The method of claim 9 wherein the disease is Niemann-Pick Type
C or Tangier diseases.
11. A formulation for use in any of the methods of claims 1-10.
Description
[0001] This application claims priority to U.S. Ser. No. 10/147,651
filed May 16, 2002, entitled "Screening of Compounds for Treatment
of Atherosclerosis and Heart Attack" by Monty Krieger, Anne
Braun-Egles and Helena Miettinen.
BACKGROUND OF THE INVENTION
[0003] The present invention is generally in the area of methods of
prevention or treatment of abnormal lipoprotein disorders,
atherosclerosis, and heart attack.
[0004] Atherosclerosis is the leading cause of death in western
industrialized countries. Atherosclerosis is the process in which
deposits of fatty substances, cholesterol, cellular waste products,
calcium and other substances build up in the inner lining of an
artery. This buildup is called plaque. It usually affects large and
medium-sized arteries. Some hardening of arteries often occurs when
people grow older. Plaques can grow large enough to significantly
reduce the blood's flow through an artery, and it is thought that
much damage occurs when they become fragile and rupture. Plaques
that rupture cause blood clots to form that can block blood flow or
break off and travel to another part of the body. If either happens
(occlusive plaques with or without occlusive blood clots) and
blocks a blood vessel that feeds the heart, it causes a heart
attack. If it blocks a blood vessel that feeds the brain, it causes
a stroke.
[0005] Atherosclerosis is a slow, complex disease that typically
starts in childhood and often progresses when people grow older. In
some people it progresses rapidly, even in their third decade. Many
scientists think it begins with inflammation or damage to the
artery, including its innermost layer of cells called the
endothelium. Causes of damage to the arterial wall include elevated
levels of cholesterol and triglyceride in the blood, high blood
pressure, tobacco smoke, and diabetes. The risk of developing
atherosclerosis is directly related to plasma levels of LDL
cholesterol and inversely related to HDL cholesterol levels.
Because of the inflammation and/or damage to the endothelium, fats,
cholesterol, platelets, cellular waste products, calcium and other
substances are deposited in the artery wall. These may stimulate
artery wall cells to produce other substances that result in
further buildup of cells. These cells and surrounding material
thicken the artery wall significantly. The artery's luminal
diameter shrinks and blood flow decreases, reducing the oxygen and
other nutrients supply to downstream tissue. A blood clot can form
near this plaque and blocks the artery, stopping the blood flow.
Alternatively, the plaque can grow large enough to effectively
occlude the vessel. Research also suggests that inflammation may
play an important role in triggering heart attacks and strokes.
Inflammation is the body's response to infection and injury, and
blood clotting is often part of that response.
[0006] Atherosclerosis often shows no symptoms until flow within a
blood vessel has become seriously compromised. Typical symptoms of
atherosclerosis include chest pain when a coronary artery is
involved, or leg pain when a leg artery is involved. Sometimes
symptoms occur only with exertion. In some people, however, they
may occur at rest.
[0007] Cholestasis is any condition in which bile excretion from
the liver is blocked, which can occur either in the liver or in the
bile ducts. There are many causes of cholestasis. Extrahepatic
cholestasis (which occurs outside the liver) can be caused by bile
duct tumors, strictures, cysts, diverticula, and other damage.
Other potential causes for this type include stones in the common
bile duct, pancreatitis, pancreatic tumor or pseudocyst, primary
sclerosing cholangitis, and compression due to a mass or tumor on a
nearby organ. Intrahepatic cholestasis (which occurs inside the
liver) can be caused by sepsis (generalized infection), cacterial
abscess, drugs, total parenteral nutrition (being fed
intravenously), lymphoma, tuberculosis, sarcoidosis and
amyloidosis. Other causes of this form of the disorder include
primary biliary cirrhosis, primary sclerosing cholangitis, viral
hepatitis (A, B, C, etc.), alcoholic liver disease, pregnancy,
Sjogren's syndrome and others.
[0008] The cellular mechanisms of cholestasis have as yet to be
completely understood. Diagnosis can be made using history,
physical examination, a few laboratory parameters, serology and
also ERCP. Treatment depends largely on the cause of the
cholestasis. Normally, bile is produced in the liver, moved to the
gallbladder and excreted into the gut through the biliary tract, to
aid in the digestion of fats and the excretion of small molecules
from the body. Flow from the liver to the gallbladder and
ultimately to the gut can be slowed or stopped by certain drugs.
When the flow of bile is inhibited, an individual may become
jaundiced. Drugs such as ursodesoxycholic acid and
immunosuppressants are causes of the leading disorders such as PBC
and PSB. Many drugs can cause cholestasis. Some more common
culprits include: gold salts, nitrofurantoin, anabolic steroids,
oral contraceptives, chlorpromazine, prochlorperazine, sulindac,
cimetidine, erythromycin, tobutamide, imipramine, ampicillin and
other penicillin-based antibiotics. This list is not comprehensive,
as other medications can also unexpectedly cause cholestasis in
some individuals.
[0009] The disturbance of lipid metabolism is seen in some
inherited diseases and also in patients with some kinds of
underlying diseases. The presence of its disturbance can be
detected by measuring the concentrations of cholesterol and
triglyceride in serum. Although hyperlipidemia or hypolipidemia can
be the result of abnormal lipid metabolism, hyperlipidemia is of
more concern to physicians because of the close association with
atherosclerosis and kidney disease. Responsible genes for some
primary (or hereditary) hyperlipidemic diseases have been confirmed
as follows; LPL or apo C-II for primary chylomicronemia, LDL
receptor for familial hypercholesterolemia and apo B-100 for
familial defective apo B-100. However, the responsible gene remains
controversial for familial combined hyperlipidemia, though
AI/CIII/AIV cluster is one of the possible candidate genes.
Secondary hyperlipidemia is caused by various diseases such as
diabetes mellitus, renal diseases and cholestasis. This type of
hyperlipidemia is improved by therapy for the underlying diseases.
Hyperlipidemia with a marked increase of low-density lipoprotein
(LDL) and high-density lipoprotein (HDL) cholesterol levels is a
common feature in patients with chronic cholestatic liver disease.
Excess morbidity and mortality from cardiovascular disease has not
been reported in these patients. This may be due to the particular
lipoprotein pattern observed during chronic cholestasis,
characterized by elevated serum HDL cholesterol, which may have a
cardioprotective effect. However, in a subgroup of patients with
chronic cholestasis, hyperlipidemia is characterized by markedly
elevated LDL levels with normal or low HDL levels, probably
reflecting hypercholesterolemia with coexisting familial and
nutritional origins.
[0010] Lipoprotein-X (Lp-X) is an abnormal low-density lipoprotein
frequently found in liver disease and in patients with familial
lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes.
It is regarded as the most sensitive and specific biochemical
parameter for the diagnosis of intra- and extrahepatic cholestasis.
Moreover, Lp-X is supposed to contribute to the development of
hypercholesterolemia in cholestatic liver disease, because it fails
to inhibit de novo cholesterol synthesis. Compared to other
lipoproteins, Lp-X contains a high content of unesterified
cholesterol (30%, w/w) to phosphatidylcholine (60%, w/w). Lp-X
isolated from sera of patients with obstructive jaundice has a high
content of unesterified cholesterol and phosphatidylcholine and
contained apolipoprotein E, apoCs, and albumin.
[0011] It is an object of the present invention to provide methods
and drugs for treating or preventing abnormal lipoproteins, for use
in disorders such as cholestasis and lipoprotein X and for treating
diseases due to abnormalities in cholesterol metabolism (e.g.,
Niemann-Pick Type C and Tangier diseases).
[0012] It is another object of the present invention to provide
methods and compounds for the treatment of atherosclerosis.
SUMMARY OF THE INVENTION
[0013] Using an animal model, transgenic animals that do not
express functional SR-BI and apoE which develop severe
atherosclerosis by age four-five weeks i, a class of drugs,
PROBUCOL (4,4'-(isopropylidenedithio- )
bis(2,6-di-tert-butylphenol)) and monoesters of PROBUCOL, and BO
653,
2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-benzofuran,
has been discovered which is useful in normalizing abnormal
lipoprotein levels and/or characteristics. These animals exhibit
progressive heart dysfunction starting by age four-six weeks, and
die by age eight weeks (between 5 and eight weeks). Pathology shows
extensive fibrosis of the heart and occlusion of coronary arteries.
The occlusion appears to be due to atherosclerosis, since fat
deposition is in the walls. These animals are good models for the
following diseases, and for screening of drugs useful in the
treatment and/or prevention of these disorders: cardiac ischemia,
cardiac fibrosis, myocardial infarction, defects in EKGs, including
electrical conductance, heart failure, cardiac dysfunction (e.g.,
reduced ejection fraction), cardiac hypertrophy/dialation, and
occlusive coronary artery disease (e.g., atherosclerosis). These
animals can also be used to screen for drugs for use in treating
lipoprotein disorders such as cholestasis and lipoprotein X.
[0014] Animals (apoE -/- SR-BI +/-) were fed PROBUCOL beginning at
the time of mating. Offspring were weaned at three weeks and fed
PROBUCOL. Survival is shown in FIG. 1A. In contrast to animals not
fed PROBUCOL, 50% of whom are dead at six weeks of age, all animals
on PROBUCOL have a normal phenotype (MRI of heart function, ECG,
echocardiogram, histology) at six weeks. At seven to eight months,
there is evidence of atherosclerosis and some myocardial infarction
in the treated animals. This demonstrates that the compound has a
preventative action. Animals who are taken off of the PROBUCOL at
weaning all die within ten to twelve weeks.
[0015] In another study, the majority of animals whose parents were
not fed PROBUCOL, but who received the PROBUCOL beginning before or
after five weeks of age, survived for a few months (See FIG. 1B),
demonstrating that the compound also has a therapeutic benefit. The
earlier the treatment with PROBUCOL, the longer the survival of the
animals. These studies showed that PROBUCOL treatment could extend
the lives of these animals even when it is administered after the
development of a significant level of heart disease. Further
studies demonstrate that these animals have abnormal lipid
structures similar to those present in cases resulting in the
appearance of lipoprotein X, which are normalized by treatment with
PROBUCOL.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1a and 1b are graphs of percent survival versus age
for double knockout dKO mice compared to mice fed PROBUCOL.
[0017] FIG. 1a, dKO mice, n=13, fed normal chow, dashed line (death
by eight weeks); dKO mice, n=10, 0.5% PROBUCOL diet from conception
solid line (latest death at 60 weeks); and dKO-Pww mice, n=9,
PROBUCOL diet from conception until weaning (death at about 18
weeks). FIG. 1b, dKO-P<5 mice (PROBUCOL diet administered only
after weaning but prior to 5 weeks of age, n=9; and dKO-P>5,
PROBUCOL diet administered immediately after 5 weeks of age,
n=7.
[0018] FIG. 2 is a graph of the cholesterol lipoprotein profile
(lipoproteins fractionated by size using FPLC) showing total
cholesterol (mg/dl) for VLDL, IDL/LDL, and HDL for dKO mice
untreated (black circles) or fed 0.5% PROBUCOL from conception
(open circles).
DETAILED DESCRIPTION OF THE INVENTION
[0019] I. Pharmaceutical Compositions
[0020] Pharmacologically Active Compounds
[0021] The double knockout (dKO) mouse for apolipoprotein E and the
HDL receptor (SR-BI) was used to identify compounds that can
normalize abnormal lipoprotein levels and/or structure. SR-BI
controls HDL structure and metabolism and appears to be important
for reverse cholesterol transport. These mice display
characteristics of coronary artery disease such as high serum
cholesterol levels, atherosclerosis, patchy myocardial infarct,
cardiac enlargement and premature death. They also are
characterized by abnormal lipid levels and structure.
[0022] As demonstrated by the examples, PROBUCOL dramatically
increases the life expectancy of the dKO mice. Untreated mice have
a median lifespan of 6 weeks while PROBUCOL-treated mice can live
up to 419 days (mean of 36 weeks). Histological study of the
cardiac tissue at 6 weeks of age demonstrates that PROBUCOL also
eliminated most of the functional and morphological indicators of
occlusive atherosclerotic coronary artery disease, myocardial
infarct and cardiac dysfunction. No fibrosis due to myocardial
infarct, atherosclerotic plaques or atherosclerosis in the root of
the aortic arch is seen in treated mice at this age. Serum
cholesterol levels are decreased two fold in PROBUCOL-treated mice
and PROBUCOL also corrects the defective red blood cell maturation
seen in dKO mice.
[0023] Based on these studies, a number of compounds are useful in
altering lipid levels and cholesterol metabolism. A preferred class
of compounds are PROBUCOL (4,4'-(isopropylidenedithio)
bis(2,6-di-tert-butylphenol)) and monoesters of PROBUCOL, for
example, as described in U.S. Pat. No. 6,121,319 to Somers and
other derivatives as described by FR 2168137, FR 2140771, FR
2140769, FR 2134810, FR 2133024, and FR 2130975, including the
derivatives developed by Atherogenics Corporation. These compounds
have potent antioxidant properties and block oxidative modification
of LDL. Another useful compound available from Chugai of Japan is
BO 653, 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-
-butyl-benzofuran, an antioxident. Noguchi, et al., Arch. Biochem.
Biophys. 1:347 (1997).
[0024] Based on the PROBUCOL data, other compounds that will be
effective include vitamin E and vitamin C, both hypocholesterolemic
and antioxident compounds, both as fertility enhancing agents as
well as for treatment and/or prevention of cardiovascular disease
or atherosclerosis. The preferred compounds have both
activities.
[0025] Pharmaceutical Carriers
[0026] Compounds are preferably administered in a pharmaceutically
acceptable vehicle for oral administration. Suitable pharmaceutical
vehicles are known to those skilled in the art. For parenteral
administration, the compound will usually be dissolved or suspended
in sterile water or saline. For enteral administration, the
compound will be incorporated into an inert carrier in tablet,
liquid, or capsular form. Suitable carriers may be starches or
sugars and include lubricants, flavorings, binders, and other
materials of the same nature.
[0027] Alternatively, the compound may be administered in liposomes
or microspheres (or microparticles). Methods for preparing
liposomes and microspheres for administration to a patient are
known to those skilled in the art. U.S. Pat. No. 4,789,734 describe
methods for encapsulating biological materials in liposomes.
Essentially, the material is dissolved in an aqueous solution, the
appropriate phospholipids and lipids added, along with surfactants
if required, and the material dialyzed or sonicated, as necessary.
A review of known methods is by G. Gregoriadis, Chapter 14.
"Liposomes", Drug Carriers in Biology and Medicine pp. 287-341
(Academic Press, 1979). Microspheres formed of polymers or proteins
are well known to those skilled in the art, and can be tailored for
passage through the gastrointestinal tract directly into the
bloodstream. Alternatively, the compound can be incorporated and
the microspheres, or composite of microspheres, implanted for slow
release over a period of time, ranging from days to months. See,
for example, U.S. Pat. Nos. 4,906,474, 4,925,673, and
3,625,214.
[0028] II. Treatment of Disorders or Diseases
[0029] Disorders which may be treated include cholestasis,
lipoprotein x, and disorders caused by LCAT deficiencies. Other
disorders include atherosclerosis and unstable plaque, where the
formulations are administered in an amount effective to normalize
the lipoproteins levels and/or structure, reduce plaque deposition,
or reduce the levels of other indicators. The pharmaceutical
compositions can also be administered in an effective amount to
modify or prevent the development of the disorder. Reduction in
symptoms are readily determined by measuring blood, urine and/or
tissue samples using clinically available tests, as demonstrated
below.
[0030] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLE 1
Analysis of CHD and Atherosclerosis in SR-BI/apoE Double Knockout
Mice
[0031] Mice with homozygous null mutations in the genes for both
the high density lipoprotein receptor SR-BI and apolipoprotein E
(SR-BI/apoE double KO, `dKO`, mice) express many features of CHD.
dKO mice fed a low-fat, low cholesterol diet were examined using a
variety of histologic, angiographic and functional
(electrocardiography, hemodynamics, MRI) methods. They
spontaneously exhibited the following characteristics:
hypercholesterolemia, atherosclerosis, extensive lipid-rich
coronary occlusions, multiple myocardial infarctions, cardiac
dysfunction (e.g., cardiomegaly, markedly reduced ejection fraction
(.about.50%) and contractility (.about.3-fold)) and death at
.about.6 weeks of age. Their coronary lesions were strikingly
similar to human atherosclerotic plaques. Many of their
fibroatheromatous plaques contained structures reminiscent of
cholesterol clefts and extensive deposition of fibrin, suggesting
the possibility of plaque rupture/bleeding into the plaque.
[0032] Materials and Methods
[0033] Animals:
[0034] Mice (mixed with C57BL/6.times.129 background) were housed
and fed a normal chow diet and genotypes were determined by PCR
analysis as described in the patent application. All the analyses
described were performed on 4-6 weeks old, male and/or female mice.
No significant differences were observed between male and female
animals. The study complied with all institutional and NIH
guidelines for the use of laboratory animals.
[0035] Histology:
[0036] Mice were euthanized and tissues were prepared for
cryosectioning. Tissue for paraffin sections were first fixed in
10% buffered formalin (J. T. Baker, NJ). In some instances, heparin
was administered (450 U/20 g, i.v.) prior to euthanasia to prevent
systemic coagulation. Tissues were sectioned and stained with
Masson's trichrome (Sigma), hematoxylin and eosin (reference to
protocol) or oil red O (lipid) and Mayer's hematoxylin.
Immunohistochemistry was performed with a mouse monoclonal
anti-fibrin antibody (NYB-T2G1, Accurate Chemical & Scientific
Corp., NY, diluted to 1:g/ml), using the M.O.M..TM. staining
procedure (as described in the Vector M.O.M..TM. Immunodetection
kit; Vector, CA) and peroxidase activity was revealed with
3-amino-9-ethylcarbazole (AEC) substrate (as described, Vector,
CA). Immunodetection of macrophages was performed using the
monoclonal rat anti-mouse F4/80 antibody (MCA 497, Serotec, NC,
diluted 1:10) and a biotinylated rabbit anti-rat, mouse adsorbed,
secondary antibody (Vector, CA). Staining was revealed using
avidin-biotinylated peroxidase complex (Vectastain Elite ABC kit,
Vector, CA) and diaminobenzidine substrate (Vector, CA) as
suggested by the manufacturer. Sections were counterstained with
Mayer's hematoxylin.
[0037] Gravimetric Analysis:
[0038] Mice were euthanized with a lethal dose of anesthetic
(avertin 2.5%; 0.8 ml/20 g) and immediately weighed. After
perfusion, hearts were excised, dissected free of connective tissue
and major systemic vessels, and weighed. The atria were then
removed, the right ventricular free wall was removed and weighed,
and the left ventricle+septum was opened, blotted to remove
internal liquid, and weighed.
[0039] Magnetic Resonance Imaging:
[0040] Mice were anesthetized with chloral hydrate (i.p. 200-320
mg/kg; Sigma, St. Louis, Mo.) and placed in a 2T small bore magnet
(Bruker) on a custom designed body coil that also contains ECG
electrode patches. Anesthesia was adjusted by 1-2% isoflurane to
keep the heart rates constant during the experiment. Image
acquisition was ECG gated. After a set of scout images, 6-7 images
corresponding to 1 mm thick cross-sectional slices (perpendicular
to the long axis of the heart as shown in FIG. 5, and spanning the
entire heart) were collected for each heart, both at end systole
and end diastole. For each slice, dimensions (cross-sectional
areas) were calculated for the left ventricular (LV) wall+septum
and the LV chamber. These were multiplied by the thickness of each
slice and summed for all of the appropriate slices to obtain total
LV tissue volumes as well as LV end diastolic and end systolic
luminal volumes (LVEDV and LVESV respectively). These values were
used to determine ejection fractions (EF
(%)=((LVEDV-LVESV)/LVEDV).times.100).
[0041] Hemodynamic Evaluation:
[0042] Heparin (1 U/10 g i.p.) was administered and mice were
anesthetized with chloral hydrate as described above. Mice were
intubated and ventilated (model 687, Harvard Apparatus Inc,
Holliston, Mass.) with room air at a rate of 130/min and a tidal
volume of 0.14 .mu.l/g. Local anesthesia (0.05 ml of 0.5% Lidocaine
HC1; Abbott Laboratories, North Chicago, Ill.) was administered at
the neck. The right carotid artery was exposed via a midline neck
incision through blunt dissection and cannulated with a 1.4 Fr
micromanometer catheter (SPR-671, Millar Instruments, Houston,
Tex.). The catheter was advanced into the aorta for aortic pressure
measurements, and then into the LV for direct measurement of
ventricular pressure. Measurements were performed before and after
cutting both right and left vagal nerves to determine the effects
of the vagal reflexes. Pressure data from the micromanometer were
recorded using a Windaq DI 220 converter and Windaq Pro software
(Dataq Instrument, Akron, Ohio). This software was also used to
calculate systolic and diastolic left ventricular pressures, heart
rate and maximum positive and negative dP/dt values.
[0043] Ex vivo Angiography:
[0044] After measurement of hemodynamic, each heart was exposed by
median sternotomy and the ascending aorta was cannulated with
polyethylene tubing (PE50: Becton Dickinson and Company, Sparks,
Md.). The right atrium was opened for drainage. The heart was
flushed with PBS (retrograde direction) until the effluent was
clear, harvested and then a barium sulfate suspension (E-Z-EM,
Inc., Westbury, N.Y.) was injected manually at a maximum pressure
of 80 mmHg. Coronary angiograms were obtained with a Micro 50 X-ray
(Micro 50, General Electric, Milwaukee, Wis., 20 kV, 20 sec
exposure).
[0045] Electrocardiography:
[0046] Electrocardiograms (ECG's) were recorded using two methods.
For mice anesthetized with avertin (reference) measurements were
made using standard limb leads. For conscious, non-anesthetized
mice, ECG's were recorded using AnonyMOUSE.RTM. ECG Screening Tools
(Mouse Specifics, Inc., Boston). The apparatus includes paw-sized
conductive pads embedded in a platform, and electronics with
solid-state gating programmed to record ECGs when three single pads
contact three paws of the unrestrained mouse. The signals were
acquired digitally (DI-220, DATAQ Instruments, Inc., Akron, Ohio)
at a sampling rate of 2500 samples/second. Data were recorded for
2-3 sec to provide a continuous record of 20-30 ECB signals. From
these signals, the shapes, QRS widths and heart rates were
determined. Average heart rates were 646.+-.80 for SR-BI
heterozygous null (n-4), 698.+-.70 for SR-BI JI (n=6), 761.+-.54
for apoE KO (n=9) and 652.+-.82 for dKO (n=12) mice (p=0.01). As a
control, ECGs of 3 avertin-anesthetized double KO mice were
recorded using the AnonyMOUSE.RTM. method. In 2 of the 3 animals,
patterns of abnormal conductance similar to those frequently
observed with the standard limb-leads method in similarly
anesthetized mice were observed.
[0047] Statistical Analysis:
[0048] Data were analyzed with StatView software, using either a
two-tailed, unpaired Student's t test, for comparison of two
groups, or an ANOVA test, for comparison of three or four
groups.
[0049] Results
[0050] When fed a normal low fat/low cholesterol standard chow
diet, dKO mice develop extensive, accelerated atherosclerosis in
their aortic sinuses by approximately 5 weeks of age, and die at an
early age. None of the control mice died during this period from
weaning (three weeks of age) to eight weeks of age. The controls
included homozygous apoE deficient (apoE KO) mice and apoE KO mice
with a heterozygous SR-BI null mutation as well as wild type and
homozygous SR-BI deficient (SR-BI KO) mice. In contrast, the dKO
mice died between 5 and 8 weeks of age, with 50% of the deaths
occurring by 6 weeks of age. Before they died, the dKO mice
exhibited a 1-2 day period of progressively reduced general
activity associated with a change in outward appearance (ruffled
fur, abnormal gait). A series of studies of the cardiac morphology,
physiology, and histology of 4-6 week old dKO mice were conducted
using wild type, SR-BI KO and apoE KO mice as controls.
[0051] Macroscopic Lesions and Extensive Cardiac Fibrosis in the
Myocardium of dKO Mice
[0052] Macroscopic examination of the hearts from control and dKO
mice revealed two striking abnormalities in the dKO hearts. First,
dKO hearts appeared enlarged relative to those from control wild
type or single KO mice. Furthermore, hearts from dKO mice exhibited
pale, discolored patched, which were not observed in hearts from
any of the control mice. These lesions suggested the presence of
extensive myocardial infarction and scarring. These lesions were
always present in the AV groove of the left ventricle and
frequently, but not always, present at various locations on the
right ventricular wall, the left ventricular wall and/or the
apex.
[0053] Histologic analysis of heart sections was performed to
characterize the nature of these lesions. A longitudinal section of
a dKO mouse heart was trichrome stained. Trichrome stains healthy
mycardium red and fibrotic tissue blue. Fibrotic areas were
invariably seen in the regions surrounding the mitral valves and
left ventricular outflow tract. The mitral valves themselves
appeared unaffected. Higher magnification of trichrome and H&E
stained sections show that these lesions were composed of fibrotic
connective tissue, very few remaining myocytes and large, dilated
cells, many of which appeared to be mononuclear inflammatory cells.
These were characterized by extensive fibrosis, inflammation, and
in some cases, diffuse necrosis and myocardial scarring, typical of
a healed infarct. These lesions appeared more well-organized and
contained fewer dilated cells than those in the outflow tract area.
Numerous macrophages were also detected in these lesions, as well
as in lesions in the papillary muscle. Thus, macroscopic and
microscopic observations indicate that multiple myocardial infarcts
developed in the dKO mice.
[0054] In hypercholesterolemic animals, macrophages can accumulate
extensive cytosolic lipid deposits (foam cells). Because the dKO
mice are hypercholesterolemic, outflow tract and papillary muscle
sections were stained with oil red O, which stains neutral lipid
droplets red. Lipid staining was particularly intense in the
macophage-rich regions exhibiting extensive fibrosis. In these
regions, oil red O staining appeared both in a concentrated,
intense, globular pattern, reminiscent of intracellular lipid and
in a puntate pattern, reminiscent of extracellular lipid. Some oil
red O staining was also detected in non-fibrotic tissue throughout
the heart, between, but not within, the myocardial fibers. This
staining was substantially more diffuse than that in fibrotic
regions. The coincident distribution of lipid and macrophages
suggested that at least some of the apparently intracellular lipid
was present in macrophage foam cells within the myocardium.
[0055] Heart Function in dKO Mice
[0056] To determine if the extensive lesions present in the double
KO hearts were associated with altered heart function, a series of
morphologic and functional analyses were performed using control
and dKO mice. These included an assessment of heart size
(gravimetry and MRI), measurements of physiological parameters
(hemodynamics and left ventricular ejection fractions) and
electrocardiographic (ECG) analysis.
[0057] Cardiomegaly
[0058] Macroscopically, hearts from dKO mice appeared larger than
those from age-matched control mice. Gravimetric and MRI analyses
were conducted to quantitatively characterize the sizes of the
intact hearts and their chambers. Gravimetric analysis revealed a
strong linear correlation (r.sup.2=0.74, p<0.0001) between heart
and body weights for all control mice. The hearts of dKO mice,
however, were disproportionately large, both because the dKO mice
were smaller than age-matched controls and because their absolute
heart sizes were larger. The mean heart-to-body weight ratio for
dKO mice (9.4 mg/g.+-.2.3, n=9) was 1.6-fold greater than that for
apoE KO mice (5.9.+-.0.5 mg/g, n=9, P=0.002) and 1.8-fold greater
than those for SR-BI KO and wild-type mice (5.3 mg/g.+-.0.3, n=8
for each, P=0.001). The increase in the heart weights of dKO mice
included an increase in left ventricular plus septum and right
ventricular tissue mass (normalized to body weight). This was
confirmed by MRI analysis of LV+S tissue volume to body weight
ratio. In contrast, the body weight corrected LV end diastolic
chamber volumes were only slightly higher for the dKO hearts,
suggesting only minor dilation. Thus, the increased size of dKO
hearts was due primarily to increased ventricular tissue mass, a
finding consistent with compensating cardiomegaly due to an
underlying abnormality in heart activity.
[0059] Hemodynamic and MRI Analysis
[0060] Cardiac function was evaluated by hemodynamic analysis and
MRI. Average aortic diastolic and systolic blood pressures and
heart rates (HR) were significantly lower in dKO than in wild type
or single KO control mice. A substantial decrease in left
ventricular systolic pressure (LVSP) and contractility (positive
dP/dt) was observed in the dKO mice, indicating left ventricular
systolic dysfunction. There was a similarly significant (3-fold)
reduction in negative dP/dt, suggesting that left ventricular
relaxation was also impaired. The moderately elevated LVEDP was not
statistically significant. Average HR of dKO mice remained low
relative to controls after bilateral disruption of the vagal
nerves, indicating that this difference in HR was not due to
extracardiac neuronal influences. Although reduced HR might have
contributed to low blood pressure and low contractility (dP/dt),
and small differences in LVEDP and HR complicate interpretation of
dP/dt differences between the strains, it is unlikely that these
relatively small baseline differences lead to the large changes in
+dP/dt and -dP/dt. Therefore, the results are consistent with a
primary cardiac dysfunction, including decreased aortic blood
pressures and abnormalities in both contractility and
relaxation.
[0061] MRI was used to determine cardiac ejection fractions (EF), a
critical measure of heart function, of control apoE KO and dKO
mice. MRI images at, end-diastole or -systole show that, while the
LVEDVs were similar, the LV end systolic volumes (LVESVs) were
higher in dKO hearts than in the controls. Consequently, the EFs of
the dKO hearts were substantially lower (.about.50%) than those of
the controls.
[0062] Electrocardiograms (ECG) were performed on unanesthetized,
conscious mice. While the ECGs of the controls were normal,
striking abnormalities were observed in the ECGs of six of 12
unanesthethized dKO mice. One exhibited an ST elevation of unclear
etiology and five showed severe ST depression, indicating
subendocardial ischemia. When ECGs were performed on
avertin-anesthetized mice, in 5 of 8 dKO mice, but not in any
controls, that anesthesia induced or uncovered cardiac conductance
defects, which in some cases included escaped QRSs and progressed
to complete AV blocks and bradycardic death. These results,
together with the gravimetric, hemodynamic and MRI findings,
unequivocally demonstrate impaired heart function in dKO mice,
possibly because of extensive myocardial fibrosis.
[0063] Coronary Artery Disease: Angiography and Histology
[0064] To determine if occlusive coronary disease may have
contributed to cardiac dysfunction, ex vivo angiography was
performed. No obvious defects were apparent in control hearts. In
contrast, out of 7 dKO hearts examined, 5 clearly showed stenoses
and occlusions of branches of the left coronary arteries. Two
instances of apparent stenoses were also observed in the main
coronary arteries.
[0065] Histologic analyses of hearts revealed extensive coronary
artery disease (CAD) in dKO mice. There were complex occlusions of
major arterial branches in the LV free wall (9 of 10 mice
analyzed), the septum (10 of 11) and/or the RV wall (11 of 12). No
occlusions were seen in age-matched controls. A partially cellular,
lipid-rich lesion almost completely occluding the lumen of a left
coronary branching artery was observed. Fibrosis and inflammatory
cells surrounding an occluded artery in the RV wall of another dKO
mouse was also observed. Proximal lesions in coronary ostia were
also seen in 7 of 10 dKO mice. These lesions are probably
responsible for the patchy MIs in the LV and RV. Serial
cross-sections through an occluded coronary artery from a different
dKO mouse were stained with trichrome and lipid staining, which
revealed numerous structures reminiscent of cholesterol clefts
within a lipid-rich core. Immunostaining showed fibrin deposits in
the core regions of eight of ten lesions observed in 3 of 3 dKO
mice but not in any age-matched apoE KO control mice (n=3). The
presence of fibrin within the plaques occluding coronary arteries
in dKO mice indicates thrombosis, probably resulting from rupture
of or bleeding into these complex lesions.
EXAMPLE 2
Analysis of the Abnormal Lipoproteins in SR-BI/ApoE Double Knockout
Mice
[0066] Materials and Methods
[0067] Animals are the same as analyzed in Example 1 for
atherosclerosis and heart disease.
[0068] Plasma and Bile Analysis
[0069] Blood was collected in a heparinized syringe by cardiac
puncture from mice fasted overnight. Plasma was subjected to FPLC
analysis, either immediately after isolation or after storage at
4.degree. C. Total cholesterol was assayed. Cholesterol from
non-apoB containing lipoproteins was determined either using the EZ
HDL kit (Sigma, based on an antibody which blocks detection of
cholesterol in non-HDL lipoproteins, and validated by us using
human or mouse lipoproteins, not shown) or after precipitation with
magnesium/dextran sulfate (Sigma). Plasma (0.4 .mu.l) and FPLC
fractions or pools were analyzed by SDS-polyacrylamide or agarose
gel electrophoresis and immunoblotting with chemiluminescence
detection using primary anti-apolipoprotein antibodies (Sigma, or
gifts from J. Herz and H. Hobbs) and corresponding horseradish
peroxidase coupled secondary antibodies (Jackson Immuno Research or
Amersham). The Attophos chemifluorescence kit (Amersham) and an
alkaline phosphatase coupled goat anti-rabbit secondary antibody
(gift from D. Housman) were used with a Storm Fluorimager
(Molecular Dynamics) for quantitative analysis. Plasma progesterone
concentrations were determined by radioimmunoassay (Diagnostics
Products Corp, Los Angeles, Calif.). Cholesterol was extracted from
gallbladder bile and assayed.
[0070] Histology and Immunofluorescence Microscopy:
[0071] Mice anesthetized with 2.5% avertin were perfused through
the left ventricle with 20 ml of ice cold PBS containing 5 mM EDTA.
Hearts were collected directly, or the mice were perfused (5 ml)
with paraformaldehyde and the hearts collected and treated. Hearts
and ovaries were frozen in Tissue Tek OCT (Sakura, Torrance,
Calif.). Serial cross sections (10 .mu.m thickness through aortic
sinuses, 5 .mu.m for ovaries, Reichert-Jung cryostat) were stained
with oil red O and Meyer's hematoxylin. Images were captured for
morphometric analysis using a computer assisted microscopy imaging
system and lesion size was quantified as the sum of the
cross-sectional areas of each oil red O staining atherosclerotic
plaque in a section using NIH Image software. Immunohistochemistry
with a monoclonal anti-.alpha. smooth muscle actin antibody (Sigma,
gift from R. Hynes) was performed). Cumulus/oocyte complexes,
isolated from the oviducts of superovulated females or denuded
oocytes (zona pellucida removed) were immunostained with polyclonal
rabbit anti-murine SR-BI antibodies gift from K. Kozarsky) and
Cy3-labeled donkey anti-rabbit secondary antibodies (gift from R.
Rosenberg).
[0072] Statistical Analysis
[0073] Data were analyzed using either a two-tailed, unpaired
Student t-test (total or EZ HDL cholesterol from plasma, bile or
FPLC fractions, progesterone and apoA-I levels) or an unpaired
nonparametric Kruskall-Wallis test (atherosclerotic plaque lesion
sizes) (Statview and Microsoft Excel). Values are presented as
means.+-.standard deviations.
[0074] Results and Discussion
[0075] To analyze the effects of SR-BI on atherosclerosis, SR-BI KO
and apoE KO (spontaneously atherosclerotic) mice were crossed and
the lipoprotein profiles and development of atherosclerosis in the
single and double homozygous KO females at 4-7 weeks of age
compared. Results for males were similar, except as noted. The
results are shown in FIG. 2. Plasma total cholesterol in the single
SR-BI KOs was increased relative to controls, because of an
increase in large, apoE-enriched HDL particles, while the even
greater relative plasma cholesterol increase in the single apoE KOs
was a consequence of a dramatic increase in cholesterol in VLDL and
IDL/LDL size particles. There was increased plasma cholesterol in
the double KOs relative to the single apoE KOs, mainly in VLDL size
particles. This might have occurred if SR-BI, which can bind apoB
containing lipoproteins, directly or indirectly contributes to the
clearance of the cholesterol in VLDL size particles in single apoE
KO mice (reduced clearance in its absence).
[0076] The normal size HDL cholesterol peak seen in the single apoE
KOs virtually disappeared in the double KOs. However, no
statistically significant differences (P=0.1) in plasma levels of
HDL's major apolipoprotein, apoA-I, were detected. Based on the
analysis of lipoproteins in the single SR-BI KO mice, abnormally
large HDL-like particles were expected to appear in the double KOs.
Indeed, the loss of normal sized HDL cholesterol and apoA-I in the
double KOs was accompanied by a shift of the apoA-I into the VLDL
and IDL/LDL size fractions. Furthermore, analysis of HDL-like
cholesterol in the FPLC fractions using the EZ HDL assay provides
evidence for the presence of abnormally large HDL-like particles in
the double KO mice. In the single apoE KO males, most of this
cholesterol was in particles with the size of normal HDL, while in
their double KO counterparts almost all of this cholesterol was in
abnormally large particles. In addition, there was approximately
3.7-fold more of this HDL-like cholesterol in the double (133.+-.24
mg/dl) than in the single (36.+-.16 mg/dl, P=0.005) KO mice. These
increases in the amounts and sizes of HDL-like cholesterol by
inactivation of the SR-BI gene in an apoE KO background were
reminiscent of those seen in a wild-type background (approximately
2.2-fold increase in cholesterol, although the HDL-like particles
in the double KO mice were much larger and more heterogeneous than
those in the SR-BI single KO mice. A similar trend was seen for
female mice, except that there were increased levels of abnormally
large HDL-like cholesterol in the single apoE KO females relative
to males. Preliminary cholesterol measurements using
magnesium/dextran sulfate precipitation of lipoproteins support the
EZ HDL findings of large HDL in the double KO animals.
[0077] Additional evidence for abnormally large HDL-like particles
in the IDL/LDL size range from both males and females was obtained
using agarose gel electrophoresis and immunoblotting. There was a
significant reduction in the amount of immunodetectable apoB
present in the IDL/LDL-sized particles from the double KOs relative
to the single apoE KOs, even though there was as much or more total
cholesterol in these fractions in the double KOs. In addition,
there was significantly greater heterogeneity in the
electrophoretic mobilities of apoA-I containing IDL/LDL-sized
particles. This was in part due to the presence of novel apoA-I
containing, apoB-free, HDL-like particles. In contrast, most of the
apoA-I in the single apoE KOs appeared to comigrate with apoB.
Thus, it appears that normal size HDL in the single apoE KO animals
was replaced by very large (VLDL/IDL/LDL-size) HDL-like particles
in the double KO animals. It is possible that normal size HDL is
converted into these large HDL-like particles in the absence of
both apoE and SR-BI because of substantially reduced selective
(SR-BI, mediated) and apoE-mediated uptake or transfer of
cholesterol from HDL particles.
[0078] In addition to examining plasma cholesterol, biliary
cholesterol was measured in the mice. Cholesterol levels in
gallbladder bile were significantly reduced in SR-BI single KO
(30%, P<0.005) and SR-BI/apoE double KO (47%, P<0.0005) mice
relative to their SR-BI.sup.+/+ controls. This is consistent with
the previous finding that hepatic overexpression of SR-BI increases
biliary cholesterol levels and indicates that SR-BI may normally
play an important role in the last stage of reverse cholesterol
transport--transfer of plasma HDL cholesterol into bile. The data
also suggest that apoE expression can regulate biliary cholesterol
content in a SR-BI KO, but not SR-BI.sup.+/+, background.
[0079] Atherosclerosis in the animals was further assessed by
analyzing plaque areas in aortic sinuses and the effects of SR-BI
gene disruption on plasma lipoproteins in apoE KO mice. Mice were
4-7 weeks old. Plasma apoA-I levels (mean.+-.SD, expressed as
relative units) were determined by SDS-polyacrylamide (15%) gel
electrophoresis followed by quantitative immunoblotting for
apoE.sup.-/- (n=7) and SR-BI.sup.-/-apoE.sup.-/- females (n=5)
(P=0.1). Lipoprotein cholesterol profiles: Plasma lipoproteins from
individual apoE.sup.-/- or SR-BI.sup.-/- apoE.sup.-/- females were
separated based on size (SUPEROSE 6-FPLC) and total cholesterol in
each fraction (expressed as mg/dl of plasma) was measured. Pooled
Superose 6-FPLC fractions (approximately 21 .mu.l per pool) from
females in an independent experiment were analyzed by
SDS-polyacrylamide gradient (3-15%) gel electrophoresis and
immunoblotting with an anti-apoA-I antibody. Each pool contained 3
fractions and lanes are labeled with the number of the middle
fraction in each pool. Average EZ HDL cholesterol FPLC profiles for
apoE.sup.-/- or SR-BI.sup.-/- apoE.sup.-/- males (n=3) or females
(n=3). Agarose gel electrophoresis and immunoblotting: Pooled
fractions (11-21, 3.5 .mu.l) from the IDL/LDL region of the
lipoprotein profile from individual apoE.sup.-/- or SR-BI.sup.-/-
apoE.sup.-/- females were analyzed using either anti-apoA-I or
anti-apoB antibodies. Migration was upward from negative to
positive. Gallbladder biliary cholesterol (mean.+-.SD): Total
gallbladder biliary cholesterol from both male and female mice of
the indicated genotypes (n=10 or 11 per genotype) was measured.
Except for the wild-type and apoE.sup.-/-values, all pairwise
differences were statistically significant (P<0.025-0.0005).
[0080] To determine the effects of SR-BI gene disruption on
atherosclerosis in apoE KO mice, atherosclerosis in SR-BI.sup.-/-
(n=8, 4-6 weeks old), apoE.sup.-/- (n=8, 5-7 weeks old), or
SR-BI.sup.-/- apoE.sup.-/- (n=7, 5-6 weeks old) female mice was
analyzed in cryosections of aortic sinuses stained with oil red O
and Meyer's hematoxylin as described in Methods. Representative
sections through the aortic root region and cross-sectional areas
of oil red O stained lesions in the aortic root region, showed
average lesion areas (mm.sup.2.+-.SD) for
SR-BI.sup.-/-apoE.sup.-/-, apoE.sup.-/- or SR-BI.sup.-/- mice,
respectively, were as follows 0.10.+-.0.07, 0.002.+-.0.002, and
0.001.+-.0.002 (P=0.0005). There were virtually no detectable
lesions in the single KO animals at this relatively young age (4-7
weeks). However, there was substantial, statistically significant,
lesion development in the double KOs in the aortic root region,
elsewhere in the aortic sinus, and in coronary arteries. The
lipid-rich lesions were cellular (hematoxylin stained nuclei were
seen at high magnification) and in some cases had a cellular cap
which stained with antibodies to smooth muscle actin. Thus, the
atherosclerotic plaques were relatively advanced.
[0081] While most did not exhibit overt signs of illness at that
time, they all died suddenly around 6 weeks of age.
Electrocardiographic studies indicated that premature death of the
double KOs was due to progressive heart block (cardiac conduction
defects) and histology revealed extensive cardiac fibrosis and
narrowing or occlusion of the coronary arteries, suggesting
myocardial infarction (MI) due to advanced atherosclerotic disease.
The anti-atherosclerotic effect of SR-BI expression in apoE KO mice
is consistent with the reports that adenovirus- or
transgene-mediated hepatic overexpression of SR-BI in the
cholesterol and fat-fed LDLR KO mouse reduces atherosclerosis.
Thus, pharmacologic stimulation of endogenous SR-BI activity may be
anti-atherogenic, possibly because of its importance for RCT. The
accelerated atherogenesis and loss of normal size HDL cholesterol
in the double KOs resembles that reported for high-fat diet fed
single apoE KO mice; although those mice have far higher total
plasma cholesterol levels (1800-4000 vs. approximately 600 mg/dl).
It is thought the similarities arise in part because the very high
levels of large lipoproteins in the fat-fed single apoE KO might
block the ability of SR-BI to interact with HDL and other ligands
(functional SR-BI deficiency due to competition), or because of
dietary suppression of hepatic SR-BI expression.
[0082] Results
[0083] The endogenous substrate assay showed that LCAT activity in
SR-BI KO plasma has 20% of wild-type activity. This is consistent
with the possibility that reduced substrate activity of the
abnormal lipoproteins in SR-BI KO mice or reduced activity of the
LCAT enzyme is responsible for the increased fraction of the
cholesterol in the plasma that is in the unesterified form (see
Example 3).
EXAMPLE 3
Prevention or Treatment of Atherosclerosis and Normalization of
Lipid Levels and Structure in SR-BI Knockout Mice
[0084] The animals described in the preceeding examples are useful
to screen for compounds that are effective for the prevention or
treatment of atherosclerosis and heart disease and treatment of
diseases that lead to the accumulation of lipoprotein X in the
plasma (liver diseases (cholestasis), LCAT dificiency) and other
diseases that influence cholesterol metabolism (NPC1, Tangier
disease). Several studies were conducted to demonstrate this.
[0085] Materials and Methods
[0086] The same animals were used as in the preceding examples.
[0087] Animals.
[0088] Mice were housed and fed a normal chow diet or chow (Teklad
7001) supplemented with 0.5% (wt/wt)
4,4'-(isopropylidene-dithio)-bis-(2,6-di-t- ertbutylphenol
(PROBUCOL; Sigma Chemical Co., St. Louis, Mo., USA). Mouse strains
(genetic backgrounds) were: wild-type and SR-BI KO (both 1:1 mixed
C57BL/6.times.129 backgrounds), apoA-I KO (C57BL/6; The Jackson
Laboratory, Bar Harbor, Me., USA). Double SR-BI/apoA-I KO mice were
produced by (a) mating SR-BI KO males with apoA-I KO females, (b)
transferring the resulting embryos into Swiss Webster recipients,
and (c) intercrossing the double heterozygous offspring. Colonies
were maintained by crossing double-KO males with females
heterozygous for the SR-BI null mutation and homozygous for the
apoA-I mutation to optimize the low yield of SR-BI homozygotes.
[0089] These animals were fed PROBUCOL at various times prior to or
after birth, as described in results.
[0090] Results
[0091] Effects on Survival of Genetic Disruption of the ApoA-I Gene
or PROBUCOL Treatment on the Fertility of Female SR-BI KO Mice.
[0092] Wild-type males were mated with female SR-BI KO (n=13,
average litter size=1, 2- to 6-month mating), SR-BI/apoA-I KO
(n=17, dark gray bars, average litter size=2.2, 4-month mating),
PROBUCOL-fed SR-BI KO (n=14s, average litter size=5.7, 1- to
2-month mating), and PROBUCOL-fed wild-type (n=9, white bars,
average litter size=5.3, 1- to 2-month mating) mice.
[0093] In the first study, PROBUCOL was fed to a mating pair (ApoE
-/- and SR-BI +/-). The offspring, who have had PROBUCOL since
conception, were weaned at three weeks of age. They continued to
receive PROBUCOL in the chow. At 6 weeks, when typically 50% of the
animals are dead in the absence of treatment, there is no abnormal
phenotype, as measured by MRI of heart function, ECG,
echocardiogram, and histology. There is no evidence of
atherosclerosis.
[0094] At 7-8 months, many of the animals receiving PROBUCOL are
still alive. However, they do have substantial atherosclerosis and
some myocardial infarctions. In contrast, normal wild-type mice
show no evidence of heart disease or atherosclerosis.
[0095] In a second study, animals receiving PROBUCOL were taken off
of the treatment at weaning (approximately three weeks of age). All
of the animals die within 10-12 weeks.
[0096] In a third study, the animals were not treated with PROBUCOL
until after weaning, either prior to 5 weeks of age (dKO-P<5;
mean age, 4.1 weeks, range 3.9-4.6 weeks) or later (dKO-P>5;
mean age, 5.2 weeks, range 5.1-5.3 weeks). The majority of those
fed PROBUCOL before 5 weeks of age survive a few months.
[0097] The survival of animals receiving PROBUCOL from conception
is shown in FIG. 1A; the survival of animals receiving PROBUCOL
from prior to or after five weeks of age is shown in FIG. 1B.
[0098] These results demonstrate that drugs can be used as
preventatives as well as therapeutics.
[0099] Effects of PROBUCOL Treatment on Lipoproteins and Red Blood
Cell Maturation
[0100] PROBUCOL treatment lowered by about two-fold the levels of
plasma total cholesterol (Table 1) and phospholipids (Table 2) in
dKO mice compared to those in untreated apoE KO mice. Treatment did
not lower the plasma triglyceride level of dKO mice that was
somewhat lower than that of apoE KO mice (Table 2). The abnormally
large HDL particles in dKO mice are found in the VDL/IDL/LDL, but
not the HDL, size ranges, because of the loss of SR-BI-mediated
selective lipid uptake. Unexpectedly, PROBUCOL treatment (open
circles) induced the appearance of a peak of cholesterol at the
position of normal sized HDL (fractions 29-34) that contained
apoA-I that is also seen in untreated apoE KO mice. In contrast,
PROBUCOL decreases the normal size HDL peak in apoE KO mice. The
shapes of the lipoprotein unesterified cholesterol and phospholipid
FPLC profiles were similar to that of total cholesterol. The ratios
of the relative amounts of unesterified cholesterol (UC) to total
cholesterol (TC) and phospholipid (PL) to TC in the major FPLC
lipoprotein fractions are shown in Tables 1 and/or 2.
[0101] There was a surprisingly high ratio of unesterified to total
cholesterol in both dKO and SR-BI KO mice compared to SR-BI
positive controls (Table 1). As a consequence in dKO mice the ratio
of surface/polar (UC and PL) to core/nonpolar (esterified
cholesterol and triglycerides) lipids was about 6-fold larger than
in apoE KO controls (Table 2). This high ratio suggested that the
structures of the lipoproteins in dKO mice would be abnormal.
Electron micrographs of negatively stained total plasma and major
lipoprotein fractions from dKO mice showed the presence of numerous
lamellar/vesicular, and stacked discoidal particles with abnormal
morphologies, reminiscent of those (e.g., lipoprotein-X) seen in
LCAT deficiency, cholestasis and other cases of abnormally high UC.
The large PROBUCOL-induced decrease (.about.4.8-fold) in plasma
unesterified cholesterol lowered the abnormally high ratios of
UC:TC in both dKO and SR-BI KO mice to the normal values in
SR-BI-positive controls (Table 1) and lowered the surface to core
lipid ratio in dKO mice to that in apoE KO controls (Table 2). As a
consequence, the morphologies of the major lipoprotein fractions in
dKO-P mice were restored to those in apoE KO controls.
[0102] There is a profound, reversible defect in RBC maturation in
dKO mice due to excessive accumulation in precursor reticulocytes
of unesterified cholesterol (visualized by staining with the
cholesterol-binding, fluorescent dye filipin). See Table 3.
Compared to wild-type and apoE KO mice, the untreated dKO mice were
anemic and exhibited profound reticulocytosis (irregularly shaped,
precursor reticulocytes, no biconcave mature erythrocytes). Their
large, macrocytic red cells (elevated mean copuscular volume)
differed morphologically from classic abnormal `target` cells seen
in LCAT-deficient patients and contained big, cholesterol-rich
autophagolysosomal inclusion bodies. Almost all of these
abnormalities, except the somewhat irregular shape, were corrected
by PROBUCOL treatment. Correction of the reticulocytosis in dKO
mice by PROBUCOL may contribute to its suppression of their CHD,
e.g., by improving tissue oxygenation.
[0103] Mice with homozygous null mutations in the HDL receptor
SR-BI and apoE genes (dKO mice) on a low fat diet rapidly develop
many cardinal features of human CHD, including
hypercholesterolemia, atherosclerosis in the aortic sinus,
occlusive coronary plaques, patchy MIs, cardiac enlargement and
dysfunction (reduced systolic function and ejection fraction, ECG
abnormalities), and premature death (50% mortality at .about.6
weeks) (1,16). They also exhibit a block in RBC maturation. Their
abnormally high ratio of lipoprotein surface (unesterified
cholesterol (UC), phospholipids)-to-core (esterified cholesterol,
triglycerides) lipids led to abnormal lipoprotein morphology
(lamellar/vesicular and stacked discoidal particles reminiscent of
those in other cases of high UC, e.g. LCAT deficiency, cholestasis
and apoE/hepatic lipase (HL) deficiency. The mechanism causing
excess UC in SR-BI null (dKO, SR-BI KO) mice is unknown, but may
involve the activities of LCAT, HL or other
lipoprotein-metabolizing proteins.
[0104] Treatment of dKO mice with the lipid-lowering- and
anti-oxidation-drug PROBUCOL extended their lives (from 6 to 36
weeks) and reversed essentially all early onset CHD-associated
anatomical, functional, cellular and biochemical pathology. A
substantial portion of the PROBUCOL-sensitive pathology apparently
occurred between 3 and 5 weeks of age.
1TABLE 1 Effects of Probucol on Plasma Unesterified and Total
Cholesterol Unesterified Total Genotype Cholesterol Cholesterol
Ratio (Unesterified/ drug treatment (mg/dL) (mg/dL) Total
Cholesterol) DKO none (n = 11) 781 .+-. 65*.dagger. 970 .+-.
83*.dagger. 0.806 .+-. 0.007*.dagger. Probucol (n = 10) 151 .+-. 21
456 .+-. 45 0.318 .+-. 0.013 SR-BI.sup.+/-apoE KO none (n = 5) 124
.+-. 11.paragraph. 421 .+-. 31** 0.296 .+-. 0.012.sctn. Probucol (n
= 5) 57 .+-. 3 244 .+-. 20 0.238 .+-. 0.015 apoE KO none (n = 5)
126 .+-. 14.paragraph., 433 .+-. 66** 0.291 .+-. 0.02.Yen. Probucol
(n = 3) 69 .+-. 6 249 .+-. 8 0.277 .+-. 0.02 SR-BI KO# none (n =
13) 108 .+-. 5 211 .+-. 6 0.515 .+-. .027 Probucol (n = 13) 24 .+-.
2 110 .+-. 6 0.218 .+-. .012 Wild-type control# none (n = 7) 33
.+-. 2 103 .+-. 4 0.315 .+-. .011 Probucol (n '2 7) 8 .+-. 2 34
.+-. 4 0.222 .+-. .033 Data are represented as mean .+-. standard
error, *P < 0.0001, compared with all the other groups with
ANOVA test. P values for t-test comparison with or without probucol
treatment: .dagger., <0.0001; .sctn., <0.020; .paragraph.,
<0.0003; **, <0.0031; .Yen., <0.035. # Blood samples from
4-5 week old animals fed a low fat chow diet were drawn, the same
animals were fed a probucol supplemented diet for an additional
7-25 days and a second set of samples were drawn (total cholesterol
data are from Pfeuffer, et al., (1992) Arterioscler. Thromb. Vasc.
Biol. 12, 870-878).
[0105]
2TABLE 2 Effects of Probucol on Plasma Phospholipid, Triglyceride
and lipid ratios dKO dKO + probucol apoE KO Plasma Phospholipid 678
.+-. 96 246 .+-. 95 (n = 3) 307 .+-. 51 (n '2 9) (mg/dL) (n = 8)
Plasma Triglyceride 53 .+-. 16 60 .+-. 44 (n = 3) 89 .+-. 28 (n =
6) (mg/dL) (n = 4) UC*100 Plasma.sctn. 86:60 43:61 30:68 TC
VLDL.sctn. 83:55 41:52 35:56 to IDL/LDL 79:60 37:51 36:62 PL*100
.sctn. TC HDL.sctn. -- 49:70 nd ratio Plasma Surface:Core 6.0 1.1
1.1 Lipid Ratio.dagger. Average of 3 (dKO ad dKO-P) or 2 (apoE)
independent determination from individual mice. The amounts of the
indicated lipids in each of the major lipoproteins (VLDL,fractions
3-9; IDL/LDL, fractions 10-26; HDL fractions 30-37) from the FPLC
profiles were added together and the averages were used to
calculated the relative amounts of unesterified cholesterol (UC) to
total cholesterol (TC) (UC*100/TC) and phospholipid (PL) to TC
(PL*100/TC). .dagger.(PL+UC)/(esterified cholesterol +
triglycerides); nd, not determined.
[0106]
3TABLE 3 Effects of Probucol on Red Blood Cell Parameters in apoE
KO and dKO mice Genotype Hematocrit Mean corpuscular Retinculocytes
(% Drug treatment (%) volume (fL) of total RHCs) apoE KO none* (n =
8) 52.5 .+-. 2.1.sctn. 57.6 .+-. 0.5 2.8 .+-. 1.2 Probucol (n = 3)
49.8 .+-. 1.5 54.2 .+-. 2.1 4.1 .+-. 0.8 dKO none*.dagger. (n = 6)
34.2 .+-. 1.7 84.1 .+-. 4.1 100 .+-. 10 Probucol (n = 3) 50.8 .+-.
2.4 54.2 .+-. 2.3 4.1 .+-. 0.5 All animals were 4-6 weeks old,
except one each of probucol-treated dKO and apoE KO that were 6
months old. .sctn.All values are means .+-. standard deviations.
.dagger.All of the values from the untreated dKO mice were
significantly different from those from the other three groups (P
< 0.0001 for mean corpuscular volume and % reticulocytes; P =
0.001 for hematocrit). Normal mouse reticulocyte counts range from
2% to 6%. *Data for untreated animals are from Mashima, et al.,
(2001) Curr. Opin. Lipidol. 12, 411-418.
EXAMPLE 4
Measurement of LCAT in SR-BI KO Mice
[0107] Decreased levels of the enzyme LCAT are found in certain
disorders, including lipoprotein X. Therefore, LCAT levels in the
DKO animals were measured.
[0108] Materials and Methods
[0109] To assay LCAT activity with endogenous substrate, tracer
[.sup.3H]cholesterol is added to the plasma overnight at 4.degree.
C., then the plasma warmed up and ester formation measured.
[0110] This assay was performed on plasma from control (wt) and
SR-BI KO mice.
[0111] Results
[0112] The endogenous substrate assay showed that LCAT activity in
SR-BI KO plasma has 20% of wild-type activity. This is consistent
with the possibility that reduced substrate activity of the
abnormal lipoproteins in SR-BI KO mice or reduced activity of the
LCAT enzyme is responsible for the increased fraction of the
cholesterol in the plasma that is in the unesterified form (see
Example 3 above).
[0113] Modifications and variations of the methods and materials
described herein will be obvious to those skilled in the art and
are intended to be encompassed by the following claims. The
teachings of the references cited herein are specifically
incorporated herein.
* * * * *