U.S. patent application number 09/951936 was filed with the patent office on 2002-08-08 for sr-bi and apo e knockout animals and use thereof as models for atherosclerosis and heart attack.
Invention is credited to Edelberg, Jay M., Krieger, Monty, Trigatti, Bernardo.
Application Number | 20020108131 09/951936 |
Document ID | / |
Family ID | 46203882 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020108131 |
Kind Code |
A1 |
Krieger, Monty ; et
al. |
August 8, 2002 |
SR-BI and apo E knockout animals and use thereof as models for
atherosclerosis and heart attack
Abstract
Transgenic animals that do not express functional SR-BI and ApoE
develop severe atherosclerosis, by age four weeks in transgenic
mice. Moreover, these animals exhibit progressive heart block by
age four weeks, and die by age nine weeks. Pathology shows
extensive fibrosis of the heart and occlusion of coronary arteries.
The occlusion appears to be due to clotting, 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 fibrosis, myocardial
infarction, defects in electrical conductance, atherosclerosis,
unstable plaque, and stroke. In contrast to other known models for
atherosclerosis, these animals do not have to be fed extreme diets
for long periods before developing atherosclerosis. No other known
model for heart attacks and stroke is known.
Inventors: |
Krieger, Monty; (Needham,
MA) ; Edelberg, Jay M.; (New York, NY) ;
Trigatti, Bernardo; (Hamilton, CA) |
Correspondence
Address: |
PATREA L. PABST
HOLLAND & KNIGHT LLP
SUITE 2000, ONE ATLANTIC CENTER
1201 WEST PEACHTREE STREET, N.E.
ATLANTA
GA
30309-3400
US
|
Family ID: |
46203882 |
Appl. No.: |
09/951936 |
Filed: |
September 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09951936 |
Sep 13, 2001 |
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09606324 |
Jun 28, 2000 |
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60164679 |
Nov 10, 1999 |
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60141361 |
Jun 28, 1999 |
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Current U.S.
Class: |
800/3 ; 424/9.2;
800/14; 800/18; 800/9 |
Current CPC
Class: |
A61K 31/10 20130101;
C07K 14/775 20130101; A61K 31/34 20130101; C12N 15/8509 20130101;
A01K 2267/03 20130101; C07K 14/705 20130101; A01K 2267/0375
20130101; A61K 49/0008 20130101; A01K 67/0276 20130101; A01K
2227/105 20130101; A01K 2217/075 20130101 |
Class at
Publication: |
800/3 ; 800/14;
800/18; 800/9; 424/9.2 |
International
Class: |
A01K 067/027; A61K
049/00 |
Goverment Interests
[0001] 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.
Claims
We claim:
1. A method for screening for compounds having an effect on
disorders selected from the group consisting of cardiac fibrosis,
myocardial infarction, defects in electrical conductance,
atherosclerosis, unstable plaque, stroke and diseases associated
with abnormal cardiac structure or function or elevated cholesterol
or lipoprotein levels comprising administering the compound to an
animal which is deficient in active SR-BI and apolipoprotein and
determining the effect on the animals relative to control animals
not treated with compound.
2. The method of claim 1 wherein the apolipoprotein is Apo E.
3. The method of claim 1 wherein the animal does not express
SR-BI.
4. The method of claim 1 wherein the animal does not express active
SR-BI.
5. The method of claim 2 wherein the animal is an SR-BI and Spo E
knockout.
6. The method of claim 1 wherein the animal is a rodent.
7. The method of claim 6 wherein the animal is a mouse, rat,
hamster or gerbil.
8. The method of claim 1 wherein the animal is treated with a
compound which lowers the level of SR-BI.
9. The method of claim 1 wherein the animal is treated with a
compound which lowers the level of apolipoprotein.
10. The method of claim 1 wherein the animals are screened for
alterations in levels of cholesterol or lipoproteins.
11. A transgenic animal which is deficient in active SR-BI and
apolipoprotein.
12. The animal of claim 11 wherein the apolipoprotein is Apo E.
13. The animal of claim 11 which is a rodent.
14. The animal of claim 13 which is a mouse, rat, hamster or
gerbil.
15. The animal of claim 11 wherein the animal is an SR-BI and Apo E
knockout.
Description
BACKGROUND OF THE INVENTION
[0002] The present invention is generally in the area of transgenic
animal models of atherosclerosis and methods for screening for
inhibitors acting via interaction with the SR-BI scavenger
receptor.
[0003] The intercellular transport of lipids through the
circulatory system requires the packaging of these hydrophobic
molecules into water-soluble carriers, called lipoproteins, and the
regulated targeting of these lipoproteins to appropriate tissues by
receptor-mediated pathways. The most well characterized lipoprotein
receptor is the LDL receptor, which binds to apolipoproteins B-100
(apoB-100) and E (apoE), which are constituents of low density
lipoprotein (LDL), the principal cholesteryl-ester transporter in
human plasma, very low-density lipoprotein (VLDL), a
triglyceride-rich carrier synthesized by the liver,
intermediate-density lipoprotein (IDL), and catabolized
chylomicrons (dietary triglyceride-rich carriers).
[0004] All members of the LDL receptor gene family consist of the
same basic structural motifs. Ligand-binding (complement-type)
cysteine-rich repeats of approximately 40 amino acids are arranged
in clusters (ligand-binding domains) that contain between two and
eleven repeats. Ligand-binding domains are always followed by
EGF-precursor homologous domains. In these domains, two EGF-like
repeats are separated from a third EGF-repeat by a spacer region
containing the YWTD motif. In LRP and gp330, EGF-precursor
homologous domains are either followed by another ligand-binding
domain or by a spacer region. The EGF-precursor homology domain,
which precedes the plasma membrane, is separated from the single
membrane-spanning segment either by an O-linked sugar domain (in
the LDL receptor and VLDL receptor) or by one (in C. elegans and
gp330) or six EGF-repeats (in LRP). The cytoplasmic tails contain
between one and three "NPXY" internalization signals required for
clustering of the receptors in coated pits. In a later compartment
of the secretory pathway, LRP is cleaved within the eighth
EGF-precursor homology domain. The two subunits LRP-515 and LRP-85
(indicated by the brackets) remain tightly and non-covalently
associated. Only partial amino acid sequence of the vitellogenin
receptor and of gp330 are available.
[0005] LDL receptors and most other mammalian cell-surface
receptors that mediate binding and, in some cases, the endocytosis,
adhesion, or signaling exhibit two common ligand-binding
characteristics: high affinity and narrow specificity. However, two
additional lipoprotein receptors have been identified which are
characterized by high affinity and broad specificity: the
macrophage scavenger receptors class A type I and type II.
[0006] Scavenger receptors mediate the endocytosis of chemically
modified lipoproteins, such as acetylated LDL (AcLDL) and oxidized
LDL (OxLDL), and have been implicated in the pathogenesis of
atherosclerosis (Krieger and Herz, 1994 Annu. Rev. Biochem. 63,
601-637; Brown and Goldstein, 1983 Annu. Rev. Biochem. 52, 223-261;
Steinberg et al., 1989 N. Engl. J. Med. 320, 915-924). Macrophage
scavenger receptors exhibit complex binding properties, including
inhibition by a wide variety of polyanions, such as maleylated BSA
(M-BSA) and certain polynucleotides and polysaccharides, as well as
unusual ligand-cross competition (Freeman et al., 1991 Proc. Natl.
Acad. Sci. U.S.A. 88, 4931-4935, Krieger and Herz, 1994). Several
investigators have suggested that there may be at least three
different classes of such receptors expressed on mammalian
macrophages, including receptors which recognize either AcLDL or
OxLDL, or both of these ligands (Sparrow et al., 1989. J. Biol.
Chem 264, 2599-2604; Arai et al., 1989 Biochem. Biophys. Res.
Commun. 159, 1375-1382; Nagelkerke et al., 1983 J. Biol. Chem. 258,
12221-12227).
[0007] The first macrophage scavenger receptors to be purified and
cloned were the mammalian class A type I and II receptors. These
are trimeric integral membrane glycoproteins whose extracellular
domains have been predicted to include .alpha.-helical coiled-coil,
collagenous and globular structures (Kodama et al., 1990 Nature
343, 531-535; Rohrer et al., 1990 Nature 343, 570-572; Krieger and
Herz, 1994). The collagenous domain, shared by the class A type I
and type II receptors, apparently mediates the binding of
polyanionic ligands (Acton et al., 1993 J. Biol. Chem. 268,
3530-3537; Doi et al., 1993 J. Biol. Chem. 268, 2126-2133). The
class A type I and type II molecules, which are the products of
alternative splicing of a single gene, are hereafter designated
class A scavenger receptors (SR-AI and SR-AII). The class A
receptors, which bind both AcLDL and OxLDL (Freeman et al., 1991),
have been proposed to be involved in host defense and cell
adhesion, as well as atherogenesis (Freeman et al., 1991; Krieger,
1992 Trends Biochem. Sci. 17, 141-146; Fraser et al., 1993 Nature
364, 343-346; Krieger and Herz, 1994).
[0008] Based on models of the predicted quaternary structures of
the class A type I and type II macrophage scavenger receptors, both
contain six domains, of which the first five are identical: the
N-terminal cytoplasmic region, the transmembrane region, spacer,
.alpha.-helical coil, and collagen-like domains. The C-terminal
sixth domain of the type I receptor is composed of an eight-residue
spacer followed by a 102-amino acid cysteine-rich domain (SRCR),
while the sixth domain of the type II receptor is only a short
oligopeptide.
[0009] Using a murine macrophage cDNA library and a COS cell
expression cloning technique, Endemann, Stanton and colleagues,
(Endemann, et al. 1993 J. Biol. Chem. 268, 11811-11816; Stanton, et
al. J. Biol. Chem. 267, 22446-22451), reported the cloning of cDNAs
encoding two additional proteins that can bind OxLDL. The binding
of OxLDL to these proteins was not inhibited by AcLDL. These
proteins are FcgRII-B2 (an Fc receptor) (Stanton et al., 1992) and
CD36 (Endemann et al., 1993). The significance of the binding of
OxLDL to FcgRII-B2 in transfected COS cells is unclear because
FcgRII-B2 in macrophages apparently does not contribute
significantly to OxLDL binding (Stanton et al., 1992). However,
CD36 may play a quantitatively significant role in OxLDL binding by
macrophages (Endemann et al., 1993). In addition to binding
oxidized LDL, CD36 binds thrombospondin (Asch et al., 1987 J. Clin.
Invest. 79, 1054-1061), collagen (Tandon et al., 1989 J. Biol.
Chem. 264, 7576-7583), long-chain fatty acids (Abumrad et al., 1993
J. Biol. Chem. 268, 17665-17668) and Plasmodium falciparum infected
erythrocytes (Oquendo et al., 1989 Cell 58, 95-101). CD36 is
expressed in a variety of tissues, including adipose, and in
macrophages, epithelial cells, monocytes, endothelial cells,
platelets, and a wide variety of cultured lines (Abumrad et al.,
1993; and see Greenwalt et al., 1992 Blood 80, 1105-1115 for
review). Although the physiologic functions of CD36 are not known,
it may serve as an adhesion molecule due to its collagen-binding
properties. It is also been proposed to be a long-chain fatty acid
transporter (Abumrad et al., 1993) and a signal transduction
molecule (Ockenhouse et al., 1989 J. Clin. Invest. 84, 468-475;
Huang et al., 1991 Proc. Natl. Acad. Sci. U.S.A. 88, 7844-7848),
and may serve as a receptor on macrophages for senescent
neutrophils (Savill et al., 1991 Chest 99, 7 (suppl)).
[0010] Modified lipoprotein scavenger receptor activity has also
been observed in endothelial cells (Arai et al., 1989; Nagelkerke
et al., 1983; Brown and Goldstein, 1983; Goldstein et al., 1979
Proc. Natl. Acad. Sci. U.S.A. 76, 333-337). At least some of the
endothelial cell activity apparently is not mediated by the class A
scavenger receptors (Bickel et al., 1992 J. Clin. Invest. 90,
1450-1457; Arai et al., 1989; Nagelkerke et al., 1983; Via et al.,
1992 The Faseb J. 6, A371), which are often expressed by
macrophages (Naito et al., 1991 Am. J. Pathol. 139, 1411-1423;
Krieger and Herz, 1994). In vivo and in vitro studies suggest that
there may be scavenger receptor genes expressed in endothelial
cells and macrophages which differ from both the class A scavenger
receptors and CD36 (Haberland et al., 1986 J. Clin. Inves. 77,
681-689; Via et al., 1992; Sparrow et al., 1989; Horiuchi et al.,
1985 J. Biol. Chem. 259, 53-56; Arai et al., 1989; and see below).
Via, Dressel and colleagues (Ottnad et al., 1992 Biochem J. 281,
745-751) and Schnitzer et al. 1992 J. Biol. Chem. 267, 24544-24553)
have detected scavenger receptor-like binding by relatively small
membrane associated proteins of 15-86 kD. In addition, the LDL
receptor related protein (LRP) has been shown to bind lipoprotein
remnant particles and a wide variety of other macromolecules. Both
the mRNA encoding LRP and the LRP protein are found in many tissues
and cell types (Herz, et al., 1988 EMBO J. 7:4119-4127; Moestrup,
et al., 1992 Cell Tissue Res. 269:375-382), primarily the liver,
the brain and the placenta. The predicted protein sequence of the
LRP consists of a series of distinctive domains or structural
motifs, which are also found in the LDL receptor.
[0011] As described by Kreiger, et al., in PCT/US95/07721 "Class
Bland CI Scavenger Receptors" Massachusetts Institute of Technology
("Krieger, et al."), two distinct scavenger receptor type proteins
having high affinity for modified lipoproteins and other ligands
have been isolated, characterized and cloned. Hamster and murine
homologs of SR-BI, an AcLDL and LDL binding scavenger receptor,
which is distinct from the class A type I and type II macrophage
scavenger receptors, has been isolated and characterized. In
addition, DNA encoding the receptor cloned from a variant of
Chinese Hamster Ovary Cells, designated Var-261, has been isolated
and cloned. dSR-CI, a non-mammalian AcLDL binding scavenger
receptor having high ligand affinity and broad specificity, was
isolated from Drosophila melanogaster.
[0012] It was reported by Kreiger, et al. that the SR-BI receptor
is expressed principally in steroidogenic tissues and liver and
appears to mediate HDL-transfer and uptake of cholesterol.
Competitive binding studies show that SR-BI binds LDL, modified
LDL, negatively charged phospholipid, and HDL. Direct binding
studies show that SR-BI expressed in mammalian cells (for example,
a varient of CHO cells) binds HDL, without cellular degradation of
the HDL-apoprotein, and lipid is accumulated within cells
expressing the receptor. These studies indicate that SR-BI might
play a major role in transfer of cholesterol from peripheral
tissues, via HDL, into the liver and steroidogenic tissues, and
that increased or decreased expression in the liver or other
tissues may be useful in regulating uptake of cholesterol by cells
expressing SR-BI, thereby decreasing levels in foam cells and
deposition at sites involved in atherogenesis.
[0013] Atherosclerosis is the leading cause of death in western
industrialized countries. The risk of developing atherosclerosis is
directly related to plasma levels of LDL cholesterol and inversely
related to HDL cholesterol levels. Over 20 years ago, the pivotal
role of the LDL receptor in LDL metabolism was elucidated by
Goldstein, et al., in the Metabolic and Molecular Bases of
Inherited Disease, Scriver, et al. (McGraw-Hill, NY 1995), pp.
1981-2030. In contrast, the cellular mechanisms responsible for HDL
metabolism are still not well defined. It is generally accepted
that HDL is involved in the transport of cholesterol from
extrahepatic tissues to the liver, a process known as reverse
cholesterol transport, as described by Pieters, et al., Biochim.
Biophys. Acta 1225, 125 (1994), and mediates the transport of
cholesteryl ester to steroidogenic tissues for hormone synthesis,
as described by Andersen and Dietschy, J. Biol. Chem. 256, 7362
(1981). The mechanism by which HDL cholesterol is delivered to
target cells differs from that of LDL. The receptor-mediated
metabolism of LDL has been thoroughly described and involves
cellular uptake and degradation of the entire particle. In
contrast, the receptor-mediated HDL metabolism has not been
understood as well. Unlike LDL, the protein components of HDL are
not degraded in the process of transporting cholesterol to cells.
Despite numerous attempts by many investigators, the cell-surface
protein(s) that participate in the delivery of cholesterol from HDL
to cells had not been identified before the discovery that SR-BI
was an HDL receptor.
[0014] It is an object of the present invention to provide methods
and reagents for designing drugs that can stimulate or inhibit the
binding to and lipid movements mediated by SR-BI and redirect
uptake and metabolism of lipids and cholesterol by cells.
SUMMARY OF THE INVENTION
[0015] Transgenic animals that do not express functional SR-BI and
ApoE develop severe atherosclerosis, by age four weeks in
transgenic mice. Moreover, these animals exhibit progressive heart
block by age four weeks, and die by age nine weeks. Pathology shows
extensive fibrosis of the heart and occlusion of coronary arteries.
The occlusion appears to be due to clotting, since fat deposition
is in the walls. Equivalent animals can be produced using single
knockout animals with an inhibitor, for example, an inhibitor of
SR-BI administered to an ApoE knockout, or verse versa. 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 fibrosis, myocardial infarction, defects
in electrical conductance, atherosclerosis, unstable plaque, and
stroke. In contrast to other known models for atherosclerosis,
these animals do not have to be fed extreme diets for long periods
before developing atherosclerosis. No other known model for heart
attacks and stroke is known.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The role of SR-BI has now been confirmed as the principle
mediator of cholesteryl ester transport from peripheral tissues to
the liver and other steroidogenic tissues, including the adrenal
gland, testes and ovaries. The studies described herein demonstrate
that animals which are deficient in both SR-BI and ApoE are not
only excellent models for atheroslerosis but also myocardial
infarction and stroke, since the animals develope progressive heart
block and coronary artery occlusions characterized by clots
resembling those in heart attack patients.
[0017] These animals can be used to screen for drugs that are
effective as therapeutics or diagnostics of heart disease.
[0018] Pharmaceutical Compositions
[0019] Compounds are preferably administered in a pharmaceutically
acceptable vehicle. 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. The compounds can also be administered locally by topical
application of a solution, cream, gel, or polymeric material (for
example, a Pluronic.TM., BASF).
[0020] 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. No. 4,906,474, 4,925,673, and 3,625,214.
[0021] The pharmaceutical compositions are administered in an
effective amount effective to modify or treat the disorder. These
are readily determined by measuring blood, urine and/or tissue
samples using clinically available tests. The exact dosages can be
determined based on the use of animal models which are accepted as
predictive of the effects of drugs on steroid levels, for example,
of contraceptives or cortisone.
[0022] Generation of Transgenic Animals for Screening
[0023] With the knowledge of the cDNA encoding SR-BI and regulatory
sequences regulating expression thereof, it is possible to generate
transgenic animals, especially rodents, for testing the compounds
which can alter SR-BI expression, translation or function in a
desired manner. This procedure for transient overexpression in
animals following infection with adenoviral vectors is described
below in the examples.
[0024] There are basically two types of animals which are useful:
those not expressing functional SR-BI, which are useful for testing
of drugs which may work better in combination with an inhibitor of
SR-BI to control levels of lipid, cholesterol, lipoprotein or
components thereof, and those which overexpress SR-BI, either in
those tissues which already express the protein or in those tissues
where only low levels are naturally expressed.
[0025] The animals in the first group are preferably made using
techniques that result in "knocking out" of the gene for SR-BI,
although in the preferred case this will be incomplete, either only
in certain tissues, or only to a reduced amount. These animals are
preferably made using a construct that includes complementary
nucleotide sequence to the SR-BI gene, but does not encode
functional SR-BI, and is most preferably used with embryonic stem
cells to create chimeras. Animals which are heterozygous for the
defective gene can also be obtained by breeding a homozygote normal
with an animal which is defective in production of SR-BI. These
animals can then be crossed with other transgenic or knockout
animals, as described in the following examples. Equivalent animals
can be produced using single knockout animals with an inhibitor,
for example, an inhibitor of SR-BI administered to an ApoE
knockout, or verse versa.
[0026] The animals in the second group are preferably made using a
construct that includes a tissue specific promoter, of which many
are available and described in the literature, or an unregulated
promoter or one which is modified to increase expression as
compared with the native promoter. The regulatory sequences for the
SR-BI gene can be obtained using standard techniques based on
screening of an appropriate library with the cDNA encoding SR-BI.
These animals are most preferably made using standard
microinjection techniques.
[0027] These manipulations are performed by insertion of cDNA or
genomic DNA into the embryo using microinjection or other
techniques known to those skilled in the art such as
electroporation, as described below. The DNA is selected on the
basis of the purpose for which it is intended: to inactivate the
gene encoding an SR-BI or to overexpress or express in a different
tissue the gene encoding SR-BI. The SR-BI encoding gene can be
modified by homologous recombination with a DNA for a defective
SR-BI, such as one containing within the coding sequence an
antibiotic marker, which can then be used for selection
purposes.
[0028] Animal Sources
[0029] Animals suitable for transgenic experiments can be obtained
from standard commercial sources. These include animals such as
mice and rats for testing of genetic manipulation procedures, as
well as larger animals such as pigs, cows, sheep, goats, and other
animals that have been genetically engineered using techniques
known to those skilled in the art. These techniques are briefly
summarized below based principally on manipulation of mice and
rats.
[0030] Microinjection Procedures
[0031] The procedures for manipulation of the embryo and for
microinjection of DNA are described in detail in Hogan et al.
Manipulating the mouse embryo, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1986), the teachings of which are incorporated
herein. These techniques are readily applicable to embryos of other
animal species, and, although the success rate is lower, it is
considered to be a routine practice to those skilled in this
art.
[0032] Transgenic Animals
[0033] Female animals are induced to superovulate using methodology
adapted from the standard techniques used with mice, that is, with
an injection of pregnant mare serum gonadotrophin (PMSG; Sigma)
followed 48 hours later by an injection of human chorionic
gonadotrophin (hCG; Sigma). Females are placed with males
immediately after hCG injection. Approximately one day after hCG,
the mated females are sacrificed and embryos are recovered from
excised oviducts and placed in Dulbecco's phosphate buffered saline
with 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus
cells are removed with hyaluronidase (1 mg/ml). Pronuclear embryos
are then washed and placed in Earle's balanced salt solution
containing 0.5% BSA (EBSS) in a 37.5.degree. C. incubator with a
humidified atmosphere at 5% CO.sub.2, 95% air until the time of
injection.
[0034] Randomly cycling adult females are mated with vasectomized
males to induce a false pregnancy, at the same time as donor
females. At the time of embryo transfer, the recipient females are
anesthetized and the oviducts are exposed by an incision through
the body wall directly over the oviduct. The ovarian bursa is
opened and the embryos to be transferred are inserted into the
infundibulum. After the transfer, the incision is closed by
suturing.
Embryonic Stem (ES) Cell Methods
Introduction of cDNA into ES cells:
[0035] Methods for the culturing of ES cells and the subsequent
production of transgenic animals, the introduction of DNA into ES
cells by a variety of methods such as electroporation, calcium
phosphate/DNA precipitation, and direct injection are described in
detail in Teratocarcinomas and embryonic stem cells, a practical
approach, ed. E. J. Robertson, (IRL Press 1987), the teachings of
which are incorporated herein. Selection of the desired clone of
transgene-containing ES cells is accomplished through one of
several means. In cases involving sequence specific gene
integration, a nucleic acid sequence for recombination with the
SR-BI gene or sequences for controlling expression thereof is
co-precipitated with a gene encoding a marker such as neomycin
resistance. Transfection is carried out by one of several methods
described in detail in Lovell-Badge, in Teratocarcinomas and
embryonic stem cells, a practical approach, ed. E. J. Robertson,
(IRL Press 1987) or in Potter et al Proc. Natl. Acad. Sci. U.S.A.
81, 7161(1984). Calcium phosphate/DNA precipitation, direct
injection, and electroporation are the preferred methods. In these
procedures, a number of ES cells, for example, 0.5.times.10.sup.6,
are plated into tissue culture dishes and transfected with a
mixture of the linearized nucleic acid sequence and 1 mg of pSV2neo
DNA (Southern and Berg, J. Mol. Appl. Gen. 1:327-341 (1982))
precipitated in the presence of 50 mg lipofectin in a final volume
of 100 .mu.l. The cells are fed with selection medium containing
10% fetal bovine serum in DMEM supplemented with an antibiotic such
as G418 (between 200 and 500 .mu.g/ml). Colonies of cells resistant
to G418 are isolated using cloning rings and expanded. DNA is
extracted from drug resistant clones and Southern blotting
experiments using the nucleic acid sequence as a probe are used to
identify those clones carrying the desired nucleic acid sequences.
In some experiments, PCR methods are used to identify the clones of
interest.
[0036] DNA molecules introduced into ES cells can also be
integrated into the chromosome through the process of homologous
recombination, described by Capecchi, (1989). Direct injection
results in a high efficiency of integration. Desired clones are
identified through PCR of DNA prepared from pools of injected ES
cells. Positive cells within the pools are identified by PCR
subsequent to cell cloning (Zimmer and Gruss, Nature 338, 150-153
(1989)). DNA introduction by electroporation is less efficient and
requires a selection step. Methods for positive selection of the
recombination event (i.e., neo resistance) and dual
positive-negative selection (i.e., neo resistance and ganciclovir
resistance) and the subsequent identification of the desired clones
by PCR have been described by Joyner et al., Nature 338, 153-156
(1989) and Capecchi, (1989), the teachings of which are
incorporated herein.
Embryo Recovery and ES cell Injection
[0037] Naturally cycling or superovulated females mated with males
are used to harvest embryos for the injection of ES cells. Embryos
of the appropriate age are recovered after successful mating.
Embryos are flushed from the uterine horns of mated females and
placed in Dulbecco's modified essential medium plus 10% calf serum
for injection with ES cells. Approximately 10-20 ES cells are
injected into blastocysts using a glass microneedle with an
internal diameter of approximately 20 .mu.m.
Transfer of Embryos to Pseudopregnant Females
[0038] Randomly cycling adult females are paired with vasectomized
males. Recipient females are mated such that they will be at 2.5 to
3.5 days post-mating (for mice, or later for larger animals) when
required for implantation with blastocysts containing ES cells. At
the time of embryo transfer, the recipient females are
anesthetized. The ovaries are exposed by making an incision in the
body wall directly over the oviduct and the ovary and uterus are
externalized. A hole is made in the uterine horn with a needle
through which the blastocysts are transferred. After the transfer,
the ovary and uterus are pushed back into the body and the incision
is closed by suturing. This procedure is repeated on the opposite
side if additional transfers are to be made.
Identification of Transgenic Animals.
[0039] Samples (1-2 cm of mouse tails) are removed from young
animals. For larger animals, blood or other tissue can be used. To
test for chimeras in the homologous recombination experiments,
i.e., to look for contribution of the targeted ES cells to the
animals, coat color has been used in mice, although blood could be
examined in larger animals. DNA is prepared and analyzed by both
Southern blot and PCR to detect transgenic founder (F.sub.0)
animals and their progeny (F.sub.1 and F.sub.2).
[0040] Once the transgenic animals are identified, lines are
established by conventional breeding and used as the donors for
tissue removal and implantation using standard techniques for
implantation into humans.
[0041] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLE 1: Production and Characterization of Transgenic Animals
which do not express SR-BI.
[0042] To determine directly if SR-BI normally plays an important
role in HDL metabolism in vivo and to establish an experimental
system to examine the role of SR-BI in pathologic states, mice
containing a targeted null mutation in the gene encoding SR-BI were
generated.
[0043] Materials and Methods
[0044] Generation of SR-BI mutant mice.
[0045] SR-BI genomic DNA was isolated from a mouse strain 129 DNA
library (Genome Systems, St. Louis, Mo.), and screened by PCR
amplification using primer pairs corresponding to the 5' and 3'
ends of the mSR-BI cDNA. From one clone a 12 kb Xba I fragment
containing the first coding exon was identified. A replacement-type
targeting vector, containing 0.75 kb and 9 kb short and long
homology regions and the po12sneobpA and herpes simplex virus
thymidine kinase (TK) cassettes, was constructed using standard
methods. The vector was linearized and 100 .mu.g were transfected
by electroporation (240 V, 500 .mu.F) into 112.times.10.sup.6
murine D3 embryonic stem cells, which were then plated onto
irradiated mouse embryonic fibroblast feeder layers. After
G418/gancyclovir positive/negative selection for 7-8 days, 492 of
the 5800 surviving colonies were picked and screened by PCR
analysis using primers specific for the targeted allele (primer 1
5'-TGAAGGTGGTCTTCAAGAGCAGTCCT-3' (SEQ ID NO:5); and primer 3
5'-GATTGGGAAGACAATAGCAGGCATGC-3' (SEQ ID NO:6); all oligonucleotide
primers were synthesized by Research Genetics). The presence of the
targeted allele (amplification of a 1.4 kb band) was confirmed by
Southern blot analysis of Xba I digested genomic DNA using probes
that yielded either the predicted 12 kb fragment characteristic of
the wild-type allele or the predicted 2.5 kb and 9 kb fragments
from the targeted mutant allele. Bam HI digested genomic DNA was
also probed with a 0.9 kb fragment derived by Pst I digestion of
the neomycin resistance gene cassette to confirm the presence of a
single neo gene in the mutant cells. Embryonic stem cell clones
containing a disrupted SR-BI allele were injected into C57BL/6
blastocysts, which were implanted into recipient females. The
resulting chimeric mice were crossed to C57BL/6 female mice to
generate F1 wild-type (srbI.sup.+/+) and heterozygous
(srbI+/.sup.+-) mice on an identical 129 (agouti)/C57BL/6
background. FI heterozygotes were crossed to generate F2 wild-type
(srbI.sup.+/+), heterozygous mutant (srbI.sup.+/-) and homozygous
mutant (srbI.sup.+/-) progeny. The presence of the targeted or
wild-type SR-BI alleles in DNA extracted from tail biopsies was
detected by PCR amplification using primer 1 in combination with
either primer 3 (mutant specific) or primer 2 (wild-type specific;
5'-TATCCTCGGCAGACCTGAGTCGTGT-3' (SEQ ID NO:7)). Genotypes were
confirmed by Southern blot analysis. Mice were housed in
microisolator cages and were fed ad libitum a regular rodent chow
diet (Prolab 3000, PMI Feeds Inc., St. Louis, Mo.).
[0046] Analysis of animal tissues:
[0047] Samples were obtained from fasted (4-8 hrs) or non-fasted
mice that were approximately 8-12 weeks old (F1 generation) or 5-11
weeks old (F2 generation).
[0048] Immunoblot Analysis.
[0049] Animals were sacrificed and livers and adrenal glands were
removed and immediately frozen. Membranes from homogenates were
prepared. 50 .mu.g of protein per specimen were analyzed by
SDS-polyacrylamide (8%) gel electrophoresis and immunoblotting with
chemiluminescence detection as previously described using rabbit
antipeptide polyclonal antibodies which specifically recognize
either the approximately 82 kDa murine SR-BI protein
(anti-mSR-BI.sup.485) or the approximately 36 kDa .epsilon.-COP
control cytoplasmic protein (anti-.epsilon.COP).
[0050] Plasma and Adrenal Cholesterol Analysis.
[0051] Plasma total cholesterol (unesterified plus esterified,
mg/dl) was measured using an enzymatic kit (Sigma Chemicals, St.
Louis, Mo.). Adrenal glands were homogenized as described above.
Protein concentrations in the homogenates were measured using the
method of Lowry et al.. Duplicate samples of homogenates (30-70
.mu.l each) were extracted with 2 ml of hexane/isopropanol (2:1)
for 1 h at room temperature, back-washed with 1 ml of water, and
phases separated by centrifugation at 800.times.g for 5 min. The
upper organic phase was recovered and evaporated at 37.degree. C.
in a Speedvac concentrator and cholesterol was measured in the
dried pellet using an enzymatic kit (Sigma). Cholesterol values
were corrected based on the recovery of a [.sup.3H]cholesteryl
ester internal standard added prior to lipid extraction. Total
cholesterol content was expressed as .mu.g of cholesterol/mg total
protein.
[0052] Lipoprotein Analysis.
[0053] Pooled plasma (150 .mu.l total from 2-6 animals) was diluted
with an equal volume of elution buffer (154 mM NaCl 1 mM EDTA, pH
8) and subjected to FPLC using two Superose 6 columns (Pharmacia,
Piscataway, N.J.) connected in series. Proteins were eluted at 0.25
ml/min. Forty seven fractions (0.5 ml) were collected after the
first 14 ml were eluted and total cholesterol in each fraction was
determined as described above. Immunoblotting of the FPLC fractions
was performed with specific anti-apoA-I, anti-apoA-II or anti-apoE
antibodies on independent samples or by sequential labeling of a
single membrane to permit simultaneous visualization of all three
proteins.
[0054] Statistical Analysis.
[0055] Results are expressed as the arithmetic mean.+-.standard
deviation. The statistical significance of the differences of the
mean between groups was evaluated using the Student t test for
unpaired comparisons. The .chi..sup.2 test was used for genotype
distribution analysis. P values <0.05 are considered to be
statistically significant.
[0056] Results and Discussion
[0057] The SR-BI gene was inactivated in embryonic stem cells by
standard homologous recombination methods. The segments replaced in
the recombined mutant ("Targeted Allele") include the entire coding
region of the first coding exon (126 bp, 42 amino acids, containing
5' untranslated sequence, a short N-terminal cytoplasmic domain,
and a portion of the N-terminal putative transmembrance domain that
probably also functions as an uncleaved leader sequence for
insertion into the ER during biogenesis) and an additional 554
bases of the adjacent downstream intron. The mutated locus is
expected to encode a transcript which would not be translated or
would be translated into non-functional, non-membranous, and
presumably unstable, protein. The strategy for the targeted
disruption of the SR-BI locus in the mouse is shown in FIG. 3. FIG.
3 is a restriction map of the genomic DNA surrounding the first
coding exon of the murine gene encoding SR-BI. The targeting vector
and the predicted structure of the targeted (mutant) allele are
shown and described in the text. The locations of the sequences for
the PCR primers used to specifically detect either the wild-type
(primers 1 and 2) or targeted mutant (primers 1 and 3) alleles are
indicated along with the predicted PCR product lengths.
Abbreviations: TK, herpes simplex thymidine kinase; neo,
po12sneobpA expression cassette, X, Xba I; B, Bam, HI; S, Sac I;
"ATG", codon for the initiator methionine. Two sets of primer pairs
specific for the wild-type (primers 1 and 2) or targeted mutant
(primers 1 and 3) alleles were used to screen genomic DNA by PCR as
described in heterozygous and F2 homozygous mutant animals are
shown. Immunoblot analysis of hepatic membranes (50 .mu.g
protein/lane) from unfasted wild-type (F1 and F2 generations),
heterozygous (F1 and F2 generations) and homozygous mutant (F2
generation) male mice were performed using polyclonal antipeptide
antibodies to SR-BI (approximately 82 kDa, top) or the internal
control .epsilon.-COP (approximately 36 kDa). Essentially identical
results were obtained using specimens from female mice)
confirmation of the expected null mutation by PCR.
[0058] Three independently derived embryonic stem cell clones
containing the targeted allele were injected into C57BL/6
blastocysts and two produced 24 male chimeras, of which 11 gave
germ line transmission of the targeted SR-BI allele when crossed to
c57BL/6 females. F1 offspring were either homozygous (+/+) for the
wild type allele or heterozygous (+/-) with both mutant and
wild-type PCR products. F1 heterozygotes should be isogenic with
the F1 wild-type controls except at the SR-BI locus. Wild-type,
heterozygous and homozygous mutant F2 generation offspring, whose
phenotypes are subject to genetic background variability, were
generated from F1 intercrosses. In the F2 progeny analyzed to date
(n=317), the observed ratios of wild-type heterozygous mutant
homozygous mutant offspring were 1.0:1.7:0.5, values significantly
different from the expected Mendelian ratio of 1:2:1 (p=0.003).
Thus, there may be partially penetrant effects of the mutation
either on neonatal survival or on embryonic development, which
would be consistent with the distribution of SR-BI on the material
surfaces of cells in the placenta and yolk sac during embryonic
development.
[0059] All of the mutants looked normal (weight, general appearance
and behavior) and the males were fertile. No offspring from female
homozygous mutants have been obtained following multiple attempts
to do so, indicating a substantial, and possibly complete, decrease
in fertility in these females. Immunoblot analysis of liver
membranes from F1 (+/+,+/-) and F2 (+/+,+/-,-/-) mice using
anti-peptide antibodies which recognize the C-terminus of the SR-BI
protein (anti-mSR-BI.sup.495) or a segment of the putative
extracellular loop (anti-mSR-BI.sup.230), revealed that there was
about half as much mSR-BI protein in the heterozygous mutants as in
the wild-type controls and no detectable SR-BI in the homozygous
mutants. No fragment or other variants of the full-length protein
were detected in any of the samples. In contrast, no significant
differences were observed in the levels of the control protein,
.epsilon.-COP. Similar results were observed using adrenal tissue.
Thus, the mutated SR-BI gene is a functionally null allele.
[0060] To determine how decreased SR-BI protein expression
influenced lipoprotein metabolism, the plasma cholesterol levels in
male and female wild-type and mutant mice were compared. Because
there were no statistically significant differences between the
data from animals derived from the two independent embryonic stem
cell clones, data from these two independent sets of animals were
pooled. Relative to wild-type controls there were statistically
significant increases in the plasma total cholesterol
concentrations of approximately 30-40% in F1 and F2 heterozygotes
and 2.2-fold in F2 homozygous mutants. In contrast to the increased
plasma cholesterol in the mutants, there was no statistically
significant change in the levels of plasma apoA-I. These findings
are consistent with the suggestion that hepatic SR-BI plays a key
role in selective removal of cholesterol from circulating HDL-lower
levels of hepatic SR-BI were expected to increase plasma HDL
cholesterol but not directly alter apoA-I levels.
[0061] To determine if the elevated levels of plasma cholesterol in
the mutants were due to changes in HDL, pooled plasma samples from
F1 male and female and F2 male animals were subjected to FPLC and
the total cholesterol content as well as the relative amounts of
apoA-I, apoA-II and apoE in each fraction were measured. For
wild-type mice (srbI.sup.+/+) most of the cholesterol, apoA-I and
apoA-II were in the HDL fraction, with small or undetectable
amounts in the VLDL and IDL/LDL fractions. There was an apparently
low level of apoE which both co-migrated with the HDL and with a
small cholesterol peak in the IDL/LDL region. The cholesterol and
apolipoprotein profiles of the heterozygous mutants were similar to
those of the wild-type controls, except that there was an increase
in the amount of cholesterol in the HDL fractions and there was a
tendency of the HDL peak (cholesterol and/or apolipoproteins) to be
broader than that of wild-type and shifted slightly to the left,
which may represent large HDL particles. This suggested that there
might be a difference in the average sizes of the HDL particles due
to the inactivation of one of the SR-BI alleles; however, this
shift was not observed in all specimens. In the F2 homozygous
mutant animals (srbI.sup.-/-) the cholesterol was found in a large,
somewhat heterogeneous peak in the HDL range, but shifted to the
left (larger apparent size) of the wild-type HDL peak. The amount
of cholesterol in the IDL/LDL fraction varied between samples.
[0062] Combined immunoblot analysis of fractions 23-28 from the
chromatograms were performed with polyclonal antibodies to apoE,
apoA-I and apoAII. Additional analysis of these and independent
chromatograms established that there were no additional peaks
containing apoA-I in fractions containing larger lipoproteins
(fractions 1-22) and that the only other peak containing a small
amount of apoE was in fraction 6, which corresponds to VLDL. The
distributions of apoA-I and apoA-II were similar to that of
cholesterol, although, unlike the case for apoA-I there was a
notable reduction in the amount of apoA-II relative to that seen in
wild type and heterozygous mutant animals. Conversely, in the
homozygous mutants there was a substantial increase in the amount
of apoE, whose distribution profile (larger particles, centered
around fractions 26-28) differed from, but overlapped, those of
apoA-I and apoA-II.
[0063] These results with the mutant animals, in which the changes
in SR-BI expression are in the physiologic range, are complementary
to and consistent with the observation that transient
adenovirus-mediated hepatic SR-BI overexpression results in
dramatically decreased levels of HDL cholesterol and increased
delivery of HDL-associated lipid to hepatocytes and the bile. In
rodents, most of the plasma HDL cholesterol appears to be removed
by the liver via selective uptake and the liver appears to be the
site of the highest total amount of SR-BI protein expression. It
seems likely that buildup of large, cholesterol-enriched
lipoprotein particles in the circulation of SR-BI mutants was
primarily due to decreased hepatic selective HDL cholesterol
uptake. Thus, it appears that murine plasma HDL cholesterol levels
are particularly sensitive to physiologically relevant changes in
the levels of hepatic SR-BI protein expression (e.g., approximately
50% reduction in heterozygotes). The effect of the null mutation in
SR-BI on total plasma cholesterol levels was quantitatively similar
to that of a null mutation in the LDL receptor. For both sets of
mutants, total plasma cholesterol levels were approximately 36%
above wild-type controls for heterozygotes and approximately 114%
for homozygotes. It is important to emphasize that while the
magnitudes of the effects on total plasma cholesterol of these
distinct mutations (SR-BI vs. LDL receptor) are similar, the
mechanistic consequences on lipoprotein metabolism (e.g., effects
on the various lipoproteins) differ.
[0064] In addition to playing an important role in regulating
plasma HDL cholesterol, SR-BI has been implicated in the delivery
of HDL cholesterol to the adrenal gland and other steroidogenic
tissues, both for the accumulation of esterified cholesterol stores
and for steroid hormone synthesis. To examine this, the cholesterol
content of adrenal glands in mutant and wild-type mice was
measured. The results are shown in Table 1. As predicted,
cholesterol stores in the adrenal gland dropped substantially in
the heterozygous and homozygous mutants to 58% and 28% of control,
respectively. It was also noted that the color of intact adrenal
glands from homozygous mutants was brownish-red while that of
wild-type and heterozygous animals was light yellow and, in
preliminary studies, a dramatic decrease in oil red O staining of
the adrenal cortex was observed in the homozygous mutants relative
to the wild-type mice. Thus, the total cholesterol content, color
and oil red O staining characteristics of the adrenal glands in
SR-BI homozygous mutants resembled those in their
cholesterol-depleted counterparts in other murine mutants,
including null mutants in the SR-BI ligand apoA-I. This similarity
with apoA-I knockouts is consistent with the possibility that the
reduction in adrenal cholesterol in the SR-BI homozygotes is a
direct consequence of the loss of the key receptor for selective
lipid uptake. Recent antibody blocking experiments have provided
additional support for a major role of mSR-BI in delivering HDL
cholesterol to cultured adrenocortical cells for steroidogenesis.
Based on the tissue distribution and hormonal regulation of SR-BI
protein expression and the phenotypes of apoA-I knockouts, it seems
likely that there would also be reductions in cholesterol stores in
other steroidogenic tissues (e.g., ovary, testes) in SR-BI
homozygous mutants. Adrenal cholesterol deficiency in both the
apoA-I and SR-BI homozygous mutants also suggests that LDL
receptors in the mouse, in which there normally is little LDL in
the plasma, do not normally contribute significantly to murine
adrenal cholesterol accumulation.
1TABLE 1 EFFECTS OF DISRUPTION OF THE GENE ENCODING SR-BI ON PLASMA
TOTAL CHOLESTEROL AND APO A-I CONCENTRATIONS, AND ADRENAL GLAND
TOTAL CHOLESTEROL CONTENT IN WILD-TYPE (srbI.sup.+/+), AND
HETEROZYGOUS (srbI.sup.+/-), AND HOMOZYGOUS (srbI.sup.-/-) MUTANT
MICE. F1 Generation F2 Generation.sup..xi. Plasma Total Plasma
Total Plasma Adrenal Gland Cholesterol Cholesterol ApoA-I Total
Cholesterol srbI % of % of % of % of genotype gender mg/dl control
mg/dl control mg/dl control .mu./mg protein control +/+ male 93
.+-. 8 (29) 100 99 .+-. 12 (18) 100 -- -- -- -- female 80 .+-. 7
(13) 100 94 .+-. 20 (27) 100 -- -- Both 89 .+-. 10 (42) 100 96 .+-.
17 (45) 100 25 .+-. 3 (10) 100 128 .+-. 28 (5) 100 +/- male 126
.+-. 10 (21) 100 137 .+-. 21 (29) 100 -- -- -- -- female 112 .+-. 9
(23) 140 118 .+-. 9 (49) 112 -- -- -- -- Both 126 .+-. 12 (44) 134
126 .+-. 22 (78) 131 28 .+-. 2 (12) 112 74 .+-. 18 (6) 58 -/- male
-- -- 220 .+-. 41 (10) 222 -- -- -- -- female -- -- 209 .+-. 32 (7)
222 -- -- -- -- Both -- -- 216 .+-. 37 (17) 225 27 .+-. 3 (11) 36
.+-. 7 (5) 28 Values for F1 generation represent mean .+-. standard
deviation. Values for F2 generation in parenthesis represent the
numbers of animals analyzed. Values for plasma total cholesterol
determined with an Autoanalyzer and human apoA-I standards. F1
generation animals were not fasted. F2 generation animals were not
fasted prior to analysis of adrenal gland cholesterol levels but
were fasted for 4-8 h prior to analysis of plasma.
EXAMPLE 2: SR-BI/Apo E Double Knockout Mice.
[0065] To study the effects of a lack of expression of the gene
encoding the Scavenger Receptor, class B type I (SR-BI) on
atherosclerosis, mice deficient in SR-BI (SR-BI KO mice) were
crossed to mice deficient in apolipoprotein E (apo E KO mice). Mice
deficient in both SR-BI and apo E (SR-BI/apo E double KO mice) did
not survive beyond 8-9 weeks of age. Analysis of atherosclerosis in
these mice revealed extensive atherosclerotic plaque in the aortic
sinuses of SR-BI/apo E double KO mice at 5-7 weeks of age, at which
time, no atherosclerotic plaque formation was detectable in mice
deficient in either SR-BI or apo E alone. Further analysis of
SR-BI/apo E double KO mice revealed that the animals died as the
result of progressive heart block (major cardiac conduction
defects), as revealed by changes in electrocardiograms and
extensive cardiac fibrosis. These were accompanied by coronary
artery atherosclerosis. Complete occlusion of coronary arteries
with a lipid-poor material which appears to represent the formation
of occlusive fibrin/platelet clots, strongly suggests that the mice
die of myocardial infarctions due to atherosclerosis/thrombosis,
just like humans.
[0066] These animals should prove useful as a model for human
coronary artery disease and myocardial infarctions, and probably
stroke. This animal system should prove to be amenable to the rapid
testing of potential drugs (since the mice succumb to MI's very
rapidly - - - within weeks). These results also suggest that under
certain circumstances, manipulation of mice deficient in either
SR-BI or apo E alone (for example interventions to alter
lipoprotein metabolism, altered steroidogenesis etc.) might give
rise to similarly severe coronary artery disease and myocardial
infarctions, giving rise to equally useful models of human coronary
artery disease.
[0067] The HDL receptor SR-BI mediates the selective uptake of
plasma HDL cholesterol by the liver and steroidogenic tissues. As a
consequence, SR-BI can influence plasma HDL cholesterol levels, HDL
structure, biliary cholesterol concentrations, and the uptake,
storage and utilization of cholesterol by steroid hormone producing
cells. Here we used homozygous null SR-BI knockout mice to show
that SR-BI is required for maintaining normal biliary cholesterol
levels, oocyte development and female fertility. We also used
SR-BI/apoE double homozygous knockout mice to show that SR-BI can
protect against early onset atherosclerosis. Although the
mechanisms underlying the effects of SR-BI loss on reproduction and
atherosclerosis have not been established, potential causes include
changes in: i) plasma lipoprotein levels and/or structure, ii)
cholesterol flux into or out of peripheral tissues (ovary, aortic
wall), and iii) reverse cholesterol transport, as indicated by the
significant reduction of gallbladder bile cholesterol levels in
SR-BI and SR-BI/apoE double knockout mice relative to controls. If
SR-BI has similar activities in humans, it may become an attractive
target for therapeutic intervention in a variety of diseases.
INTRODUCTION
[0068] High density lipoprotein (HDL)-cholesterol levels are
inversely proportional to the risk for atherosclerosis Gordon et
al., N. Engl. J Med. 321, 1311-1316 (1989). This may partly be due
to "reverse cholesterol transport" (RCT), in which HDL is proposed
to remove excess cholesterol from cells, including those in the
artery wall Johnson, et al., Biochim. Biophys. Acta, 1085, 273-298
(1991), Tall, A. R. J Lipid Res. 34, 1255-1274 (1993), Pieters, et
al., Biochim. Biophys. Acta 1225, 125-134 (1994), Fielding, et al.,
J. Lipid. Res. 36, 211-228 (1995), Oram, et al., J. Lipid Res. 37,
2473-2491 (1996), Breslow, J. L. In The Metabolic and Molecular
Bases of Inherited Diseases. eds. Scriver, C. R., Beaudet, A. L.,
Sly, W. S., & Valle, D. (McGraw-Hill, New York), pp. 2031 -2052
(1995), and transport it, either indirectly or directly Glass, et
al., Proc. Natl. Acad. Sci. U.S.A. 80, 5435-5439 (1983) and Glass,
et al., J. Biol. Chem. 260, 744-750 (1985), to the liver for
biliary secretion. HDL can also directly deliver cholesterol to
steroidogenic tissues (adrenal gland, testis, ovary) for storage in
cytoplasmic cholesteryl ester droplets and for steroid hormone
synthesis, Gwynne, et al., Endocr. Rev. 3, 299-329 (1982), Kovanen,
et al., J. Biol. Chem. 254, 5498-5505 (1979), and Plump, et al., J.
Clin. Invest. 97, 2660-2671 (1996). Thus, HDL may influence a
variety of endocrine functions, including reproduction. A key
mechanism of receptor-mediated direct delivery of HDL cholesteryl
esters to the liver and steroidogenic tissues is selective
cholesterol uptake, in which only the cholesteryl esters of the HDL
particles (not the apolipoproteins) are efficiently transferred to
cells, Glass, et al., (1983), and Glass, et al., (1985).
[0069] The class B type I scavenger receptor, SR-BI, is a cell
surface HDL receptor which mediates selective lipid uptake, Acton,
et al., Science 271, 518-520 (1996), Babitt, et al., J. Biol. Chem.
272, 13242-13249 (1997), Gu, et al., J. Biol. Chem. 273,
26338-26348 (1998), Temel, R. E., et al., Proc. Natl. Acad. Sci.
U.S.A. 94, 13600-13605 (1997), Kozarsky, K. F., et al., Nature 387,
414-417 (1997), Rigotti A., et al., Proc. Natl. Acad. Sci. U.S.A.
94, 12610-12615 (1997), Varban, M. L. et al.,. Proc. Natl. Acad.
Sci. U.S.A. 95,4619-4624 (1998), Wang, N., et al., J. Biol. Chem.
273, 32920-32926 (1998), Ueda, Y., et al., J. Biol. Chem. 274,
7165-7171 (1999), reviewed in Rigotti, A., et al., Curr. Opin.
Lipidol. 8, 181-188 (1997), and Krieger, M Ann. Rev. Biochem. 68,
523-558 (1999). It is most highly expressed in the liver and
steroidogenic tissues, in which its activity is regulated by
trophic hormones, Acton, (1996), Rigotti, A., et al., J. Biol.
Chem. 271, 33545-33549 (1996), Wang, N., et al., J. Biol. Chem.
271, 21001-21004 (1996), Landschulz, K. T., et al., J. Clin.
Invest. 98, 984-995 (1996), Mizutani, T., et al., Biochem. Biophys.
Res. Commun. 234, 499-505 (1997), Li, X., et al., Endocrinology
139, 3043-3049 (1998), Reaven, E., et al., Endocrinology 139,
2847-2856 (1998), Rajapaksha, W. R., et al., Mol. Cell. Endocrinol.
134, 59-67 (1997), and Azhar, S., et al., J. Lipid Res. 39,
1616-1628 (1998). As a consequence, SR-BI is a key regulator of HDL
cholesterol levels, Kozarsky, (1997), Rigotti A., et al., (1997),
Varban, M. L. et al., (1998), Wang, N., et al., (1998), and Ueda,
Y., et al., (1999), and adrenal cholesterol stores, Rigotti A., et
al., (1997). The finding that hepatic SR-BI overexpression leads to
significant increases in biliary cholesterol content, Kozarsky, K.
F., et al., (1997), and Sehayek, E., et al., Proc. Natl. Acad. Sci.
U.S.A. 95, 10194-10199 (1998), is consistent with gene targeting
studies Rigotti A., et al., (1997), and Varban, M. L. et al.,
(1998), which suggest an important role for SR-BI in RCT. In
addition to HDL, SR-BI can bind other ligands, including
lipoproteins (LDL, modified LDL, VLDL) and apolipoproteins, Acton,
S. L., et al., J. Biol. Chem. 269, 21003-21009 (1994), Murao, K.,
et al., J. Biol. Chem. 272, 17551-17557 (1997), Calvo, D., et al..
Arterioscler. Thromb. Vasc. Biol. 17, 2341-2349 (1997), Rigotti,
A., et al., J. Biol. Chem. 270, 16221-16224 (1995), Xu, S., et al.,
J. Lipid Res. 38, 1289-1298 (1997), and can mediate efflux of
unesterified cholesterol from cells to HDL, Ji, Y., et al., J.
Biol. Chem. 272, 20982-20985 (1997), and Stangl, H., et al., J.
Biol. Chem. 273, 31002-31008 (1998).
[0070] Because inactivation of SR-BI is associated with both
decreased RCT, Rigotti A., et al., (1997), and Varban, M. L. et
al., (1998), and increased plasma HDL cholesterol (albeit in
abnormally large particles containing apolipoproteins AI (apoA-I)
and E (apoE) Rigotti A., et al., (1997), a key question has arisen:
Do decreases in SR-BI expression inhibit or promote
atherosclerosis? Here we addressed this question by studying
crosses between apoE KO mice, which on a chow diet spontaneously
develop atherosclerosis at around 3 months of age, Zhang, S. H., et
al., Science 258, 468-471 (1992), Zhang, S.H., et al., J. Clin.
Invest. 94, 937-945 (1994), and Plump, A. S., et al., Cell 71,
343-353 (1992), and SR-BI KO mice. The results clearly show that
genetically suppressing SR-BI activity in apoE KO mice dramatically
accelerates the onset of atherosclerosis. We also report that
female mice deficient in SR-BI alone are infertile, apparently due
in part to abnormalities in the viability and developmental
potential of their oocytes. Thus, genetic ablation of SR-BI has
profound effects on both cardiovascular and reproductive
pathophysiology in mice.
MATERIALS AND METHODS
[0071] Animals: Mice (mixed C57BL/6.times.129 background) were
housed and fed a normal chow diet as described in Rigotti A., et
al., (1997). SR-BI.sup.-/- mice Rigotti A., (1997), and
apoE.sup.-/- mice (The Jackson Laboratory, Zhang, S. H., et al.,
(1992), and Zhang, S. H., et al., (1994)), were mated and the
double heterozygous offspring were intercrossed. The resulting
SR-BI.sup.+/-ApoE.sup.-/- offspring were mated to produce single
apoE KO and double SR-BI/apoE KO animals. Genotypes were determined
by PCR analysis (Rigotti A., et al., (1997), also see The Jackson
Laboratory web site). Estrus cycles were followed by vaginal
cytology, Nelson, J. F., et al., Biol. Reprod. 27, 327-339 (1982),
and external appearance, Hogan, B., et al., Manipulating the Mouse
Genome (Cold Spring Harbor Laboratory Press, Plainview, N.Y.)
Second edition. p. 129-191 (1994). Superovulation was induced by
intraperitoneal injection of 5 IU each of pregnant mare's serum
(Calbiochem) and human chorionic gonadotropin (Organon) as
described in Hogan, B., et al., (1994). Pseudopregnancy was induced
by mating (confirmed by detection of vaginal seminal plug) with
vasectomized males (Taconic) Hogan, B., et al., (1994). Ovaries
were harvested and prepared for sectioning as described below, and
oocytes and preimplantation embryos were harvested as described
Hogan, B., et al., (1994) and cultured in KSOM medium with amino
acids (Specialty Media).
[0072] Plasma and bile analysis: Blood was collected in a
heparinized syringe by cardiac puncture from mice fasted overnight.
Plasma was subjected to FPLC analysis, Rigotti A., et al., (1997),
either immediately after isolation or after storage at 4.degree. C.
Total cholesterol was assayed as described in Rigotti A., et al.,
(1997). 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; Zhang,
S. H., et al., (1992), and Plump, A. S., et al., J. Lipid Res. 38,
1033-1047 (1997). Plasma (0.4 .mu.l) and FPLC fractions or pools
were analyzed by SDS-polyacrylamide, Rigotti A., et al., (1997), or
agarose gel electrophoresis, Fielding, C. J. et al., Methods
Enzymol. 263, 251-259 (1996), and immunoblotting, Towbin, H., et
al., Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354 (1979), and
Ishida, B. Y., et al., J. Lipid Res. 31, 227-236 (1990), with
chemiluminescence detection as previously described Rigotti A., et
al., (1997), 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 as described in Puglielli, L., et al.,
Biochem. J 317, 681-687 (1996).
[0073] Histology and immunofluorescence microscopy: 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 as described
Bourassa, P.-A.K. et al., J. Histotechnology 21, 33-38 (1998).
Hearts and ovaries were frozen in Tissue Tek OCT (Sakura, Torrance,
Calif.). Serial cross sections (10 .mu.m thickness through aortic
sinuses Zhang, S. H., et al., (1994), Paigen, B., et al.,
Atherosclerosis 68, 231-240 (1987), and Suzuki, H. et al., Nature
386, 292-296 (1997), 5 .mu.m for ovaries, Reichert-Jung cryostat)
were stained with oil red O and Meyer's hematoxylin, R. E. Coalson
in Staining Procedures, G. Clark, Ed. (Williams and Wilkins,
Baltimore) pp217-253 (1981). 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
Paigen, B., et al., (1987), using NIH Image software.
Immunohistochemistry with a monoclonal anti-.alpha. smooth muscle
actin antibody (Sigma, gift from R. Hynes) was performed as
described in Rigotti, A., et al., (1996). Cumulus/oocyte complexes,
isolated from the oviducts of superovulated females as described in
Hogan, B., et al., (1994), or denuded oocytes (zona pellucida
removed as in Hogan, B., et al., (1994)) were immunostained with
polyclonal rabbit anti-murine SR-BI antibodies (Acton, et al.,
(1996), or a gift from K. Kozarsky) and Cy3-labeled donkey
anti-rabbit secondary antibodies (gift from R. Rosenberg) as
described in Babitt, et al., (1997) and Hatzapoulos A. K., et al.,
J. Lipid Res. 39, 495-508 (1998).
[0074] Statistical Analysis: 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.
RESULTS AND DISCUSSION
[0075] Reproductive Pathophysiology: Homozygous SR-BI knockout (KO)
males exhibit normal fertility, Rigotti A., et al., (1997). In
contrast, homozygous KO females are infertile. In a two month
pairing of either homozygous KO or heterozygous females with
homozygous SR-BI KO males (n=8 for each), heterozygous females
produced 19 litters and 82 healthy offspring, whereas the
homozygous females produced no healthy offspring. Although two pups
from two homozygous SR-BI KO females were born, they died soon
after.
[0076] There were no obvious gross morphological abnormalities in
SR-BI KO ovaries. Six week old female mice were superovulated and
were mated to males of the other genotype (i.e., SR-BI.sup.+/+
females mated to SR-BI.sup.-/- males and vice versa) to generate
embryos with heterozygous mutant genotypes. Ovaries and
preimplantation embryos were harvested the following morning (day
0). . Typical oil red O staining of lipids in ovaries from
SR-BI.sup.+/+ or SR-BI.sup.-/- animals was performed. Phase
contrast microscopy of preimplantation embryos (cultured for one
day) from SR-BI.sup.+/+ or SR-BI.sup.-/- females mated to males of
the opposite genotype was also performed. Similar results were
observed when SR-BI.sup.-/- males were mated to SR-BI.sup.-/-
females. Plasma progesterone concentrations from pseudopregnant
females (6 days postmating, age 6-10 weeks, weight 19-25 g, n=8.)
(P=0.08). Percent of preimplantation embryos from SR-BI.sup.+/+ or
SR-BL.sup.-/- females with normal morphology during 3 days of
culture were calculated. The values represent the averages from 5
animals of each genotype. Total number of embryos: SR-BI.sup.+/+,
131; SR-BI.sup.-/-, 167. Histochemical analysis of ovaries from
superovulated females showed reduced oil red O-staining of lipids
in the ovarian corpora lutea of SR-BI KO relative to those of
wild-type animals. This suggests there was reduced cholesteryl
ester storage, as previously observed in adrenal glands from SR-BI
KO mice Rigotti A., et al., (1997). This raised the possibility
that there might have been insufficient amounts of cholesterol
substrate in the corpora lutea to sustain adequate steroid hormone
production for pregnancy. However, plasma progesterone levels
between pseudopregnant control and KO females 6 days after mating,
either without or with superovulation were not significantly
different. Furthermore, several other murine homozygous knockout
mutants (e.g. LCAT, ACAT, and apoA-I) exhibit similar lipid
depletion in steroidogenic tissues Plump, et al., (1996), Meiner,
V. L., et al., Proc. Natl. Acad. Sci U.S.A. 93, 14041-14046 (1996),
and Ng, D. S., et al., J. Biol. Chem. 272, 15777-15781 (1997),
without apparent female infertility. Thus, normal lipid stores are
not required for synthesis of adequate amounts of steroid hormones
for female fertility.
[0077] Although KO females were infertile, they exhibited no
obvious defects in their estrus cycles or numbers of oocytes
ovulated, either during normal estrus or after superovulation (wild
type (n=4), 52.+-.5 oocytes; SR-BI KO (n=3), 41.+-.8, P=0.2).
Because the estrus cycle and ovulation depend on estrogen (e.g.,
for follicular development and induction of LH receptors) and
progesterone (e.g., for follicular rupture), Elvin, J. A. et al.,
Reviews of Reproduction 3, 183-195 (1998), KO females apparently
synthesize adequate levels of intra- and extraovarian steroids for
at least some, if not all, ovarian functions.
[0078] Because the extent of ovulation by the KO mice appeared
normal, we compared the viability and development of heterozygous
(SR-BI.sup.+/-) preimplantation (1-cell) embryos placed into
culture the morning (day 0) after mating with males. Almost all
embryos from wild-type females had normal morphologies and
developed into morulas or blastocysts after 3 days in culture. In
contrast, the majority of embryos from KO females at harvesting had
an abnormal, non-refractile morphology, reminiscent of that seen in
embryos mechanically damaged during pronuclear injection, Hogan,
B., et al., (1994). The abnormal (presumably dead) embryos did not
develop further. All of the other embryos from SR-BI KO females
which appeared normal on day 0 eventually adopted the abnormal
morphology and arrested (most at the 1- or 2-cell stages) in
culture. We also observed a similar abnormal morphology in oocytes
from wild-type females that had been treated in culture with 50
.mu.g/ml of nystatin or filipin, cholesterol binding drugs which
can perturb membrane structure, Bolard J. Biochim. Biophys. Acta
864, 257-304 (1986).
[0079] The same abnormal morphology was seen in newly harvested
unfertilized oocytes from SR-BI KO (n=6), but not wild-type (n=7),
superovulated females, although at a lower frequency (31.+-.22%)
than in fertilized preimplantation embryos (69.+-.19%, P=0.02).
Therefore, some of the oocyte abnormalities apparently are
fertilization and cell division independent. Using immunostaining
with anti-SR-BI antibodies, we did not detect a signal for SR-BI in
wild-type oocytes, either denuded (zona pellucida removed) or in
cumulus complexes, above the background seen in oocytes from KO
animals, suggesting that after ovulation murine oocytes do not
express high levels of SR-BI (also see Reaven, E., et al., (1998)).
In contrast, substantial expression of SR-BI was detected in the
expanded cumulus cells surrounding ovulated oocytes from wild-type,
but not SR-BI KO, mice. These cells are derived from follicular
granulosa cells and are believed to play a key role in oocyte
development, Meiner, V. L., et al., (1996). SR-BI expression has
been reported to be induced in follicular granulosa cells soon
after a luteinizing pulse of human chorionic gonadotropin Mizutani,
T., et al., (1997), Li, X., et al., (1998), Reaven, E., et al.,
(1998), and Rajapaksha, W. R., et al., (1997).
[0080] Infertility in SR-BI KO females may be due to inadequate
delivery of HDL-cholesterol for membrane synthesis or
steroidogenesis, inadequate delivery of non-steroidal HDL lipids
(e.g., lipid soluble vitamins), or deficiencies in SR-BI functions
other than selective cholesterol uptake (lipid efflux, binding of
non-HDL ligands). The abnormal structure of plasma HDL in the KO
animals (large, apoE-rich, Rigotti A., et al., (1997)) may also
contribute to the infertility. Oocyte abnormalities may arise due
to the inability of cumulus cells to express SR-BI, before or after
ovulation, because SR-BI may be needed by these cells to properly
nourish the oocyte and/or support its development. SR-BI expression
may also be needed in ovarian interstitial and thecal cells
surrounding follicles Landschulz, K. T., et al., (1996), Mizutani,
T., et al., (1997), Li, X., et al., (1998), and Reaven, E., et al.,
(1998). during oocyte maturation or in the oviduct environment (at
least up to the one-cell stage). SR-BI might also play a role at
other stages of reproduction and development. For example, the
pattern of expression of SR-BI during later stages of pregnancy
Hatzapoulos A. K., et al., (1998), and Wyne, K. L. et al., J.
Lipid. Res. 39, 518-530 (1998), and the non-Mendelian (reduced)
yield of homozygous mutant offspring from heterozygous mothers,
Rigotti A., et al., (1997), suggest it participates in the normal
function of the decidua, yolk sac and/or placenta for nutrient
transport and steroid hormone synthesis. Although additional
mechanistic studies are necessary, the current data unequivocally
establish that SR-BI is important for normal oocyte maturation,
embryonic development and female fertility in mice.
[0081] Cardiovascular Pathophysiology: To analyze the effects of
SR-BI on atherosclerosis, we crossed SR-BI KO and apoE KO
(spontaneously atherosclerotic, Zhang, S. H., et al., (1992),
Zhang, S. H., et al., (1994), and Plump, A. S., et al., (1992)),
mice and compared the lipoprotein profiles and development of
atherosclerosis in the single and double homozygous KO females at
4-7 weeks of age. Results for males were similar, except as noted.
As reported in example 1, plasma total cholesterol in the single
SR-BI KOs was increased relative to controls, because of an
increase in large, apoE-enriched HDL particles, Rigotti A., et al.,
(1997), 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, Acton, S. L.,
et al., (1994), Murao, K., et al., (1997), Calvo, D., et al.,
(1997), directly or indirectly contributes to the clearance of the
cholesterol in VLDL size particles in single apoE KO mice (reduced
clearance in its absence), Wang, N., et al., (1998), and Ueda, Y.,
et al., (1999).
[0082] 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 Rigotti A., et
al., (1997), 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 .about.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 (.about.2.2-fold increase in cholesterol
Rigotti A., et al., (1997), also see FIG. 2A), 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 Rigotti A., et
al., (1997). 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 (40,45) support the EZ HDL
findings of large HDL in the double KO animals.
[0083] 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.
[0084] In addition to examining plasma cholesterol, we measured
biliary cholesterol 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 Kozarsky, K. F., et al., (1997) and
Sehayek, E., et al., (1998), 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.
[0085] Atherosclerosis in the animals was 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 (right, 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 (.about.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, Rigotti A., et al., (1997). 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 (Kovanen, et al., (1979), Plump, et al., (1996),
Acton, et al., (1996), Babitt, et al., (1997), Gu, et al., (1998),
Temel, R. E., et al., (1997), Kozarsky, K. F., et al., (1997),
Rigotti A., et al., (1997), Varban, M. L. et al., (1998), Wang, N.,
et al., (1998), and Ueda, Y., et al., (1999),, 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).
[0086] 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). Also see Table II. High magnification
views of serial sections of plaque from the aortic sinus of a 7
week old SR-BI/apoE double KO male, stained either with oil red O
and Meyer's hematoxylin or with an anti-.alpha. actin antibody
which recognizes smooth muscle cells. The lumen is to the left of
the plaque. The smooth muscle wall and cellular cap are indicated.
(Table II quantitative analysis of females; qualitative analysis of
a smaller sample of males gave similar results. There were
virtually no detectable lesions in the single KO animals at this
relatively young age (4-7 weeks, Zhang, S. H., et al., (1992),
Zhang, S. H., et al., (1994), Plump, A. S., et al., (1992).
However, there was substantial, statistically significant, lesion
development in the double KOs in the aortic root region, elsewhere
in the aortic sinus (Table II), 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.
[0087] Potential causes of the dramatically accelerated
atherosclerosis in the double KOs include: i) changes in relative
amounts of cholesterol in proatherogenic (e.g., increased VLDL
sized or abnormally large HDL-like particles) and antiatherogenic
(e.g., loss of normal HDL) lipoproteins, ii) altered flux of
cholesterol into or out of the aortic wall, perhaps directly due to
SR-BI-mediated efflux, Kozarsky, K. F., et al., (1997), Ji, Y., et
al., (1997), and Stangl, H., et al., (1998), iii) decreases in RCT,
suggested by the generation of abnormally large, HDL-like particles
and decreased biliary cholesterol levels due to absence of hepatic
SR-BI activity, and iv) changes in other metabolic/organ systems
which might influence the cardiovascular system. For example, there
was significant accumulation of oil red O staining lipids in other
tissues, including the myocardium, in the double, but not single,
KO animals (FIG. 3 and not shown). In addition, at 5-6 weeks of age
when the double KOs were studied, they were somewhat smaller
(.about.20 % lower weight) than single apoE KO controls.
[0088] While most did not exhibit overt signs of illness at that
time, they all died suddenly around 8-9 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.
[0089] The anti-atherosclerotic effect of SR-BI expression in apoE
KO mice is consistent with the recent reports that adenovirus- or
transgene- Arai, T., et al., J. Biol. Chem. 274, 2366-2371 (1999),
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 antiatherogenic,
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 Zhang, S. H., et al., (1994), and Plump, A. S., et al.,
(1992); although those mice have far higher total plasma
cholesterol levels (1800-4000 vs. .about.600 mg/dl). Perhaps 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, Fluiter, K., et al., J. Biol. Chem.
273, 8434-8438 (1998).
[0090] Taken together with earlier work Krieger, M (1999), the
current study provides compelling evidence for the proposal that,
at least in rodents, SR-BI is an HDL receptor which mediates
physiologically relevant selective cholesterol transport and plays
a key role in controlling plasma lipoprotein and biliary
cholesterol concentrations and RCT. It also influences HDL's
structure, cholesterol's delivery to and utilization by cells
(including those in steroidogenic tissues), reproductive and
cardiovascular physiology and possibly other aspects of lipid
metabolism, Hauser, H., et al., Biochemistry 37, 17843-17850
(1998). Because the in vitro activity, tissue distribution and
regulation of human SR-BI, Murao, K., et al., (1997), Cao, G., et
al., J. Biol. Chem. 272, 33068-33076 (1997), Calvo, D. et al., J.
Biol. Chem. 268, 18929-18935 (1993), and Liu, J., et al., J. Clin.
Endocrinol. Metab. 82, 2522-2527 (1997), resemble those of the
mouse, SR-BI may become an attractive target for prevention of or
therapeutic intervention in a variety of human diseases Acton, et
al., (1996), Kozarsky, K. F., et al., (1997), Rigotti A., et al.,
(1997), Rigotti, A., et al., (1997), and Krieger, M., (1999).
2TABLE II Average lesion sizes in the aortic sinuses of mice
deficient in SR-BI, apoE, or both. Mean lesion size (mm.sup.2)*
Valve Attachment Genotype Aortic Root Partial Valve Cusps Sites
Proximal Aorta Overall Mean.dagger-dbl. SR-BI.sup.-/- 0.001 .+-.
0.002 (8) 0.0003 .+-. 0.0008 (8) 0 .+-. 0 (8) 0 .+-. 0 (6) 0.0004
.+-. 0.001 (6) apoE.sup.-/- 0.002 .+-. 0.002 (9) 0.0006 .+-. 0.0009
(9) 0.001 .+-. 0.002 (9) 0.0002 .+-. 0.0003 (9) 0.001 .+-. 0.002
(9) SR-BI.sup.-/- 0.10 .+-. 0.07 (7) 0.07 .+-. 0.07 (7) 0.02 .+-.
0.01 (6) 0.02 .+-. 0.02 (6) 0.04 .+-. 0.04 (6) apoE.sup.-/- P
value.sup..dagger. 0.0005 0.006 0.002 0.003 0.001 *Values are the
means .+-. SD (number of animals indicated in parentheses).
.dagger-dbl.Means of combined values from the regions of the aortic
root partial valve cusps, valve attachment sites and proximal
aorta. .sup..dagger.Lesion sizes in each region were compared using
the Kruskall-Wallis test 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.
[0091]
Sequence CWU 1
1
3 1 26 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 tgaaggtggt cttcaagagc agtcct 26 2 26 DNA Artificial
Sequence Description of Artificial Sequence Primer 2 gattgggaag
acaatagcag gcatgc 26 3 25 DNA Artificial Sequence Description of
Artificial Sequence Primer 3 tatcctcggc agacctgagt cgtgt 25
* * * * *