U.S. patent application number 14/080296 was filed with the patent office on 2014-05-22 for methods of diagnosing atherosclerosis by measuring apoci.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY. The applicant listed for this patent is THE JOHNS HOPKINS UNIVERSITY. Invention is credited to Subroto Chatterjee, Peter O. Kwiterovich.
Application Number | 20140141017 14/080296 |
Document ID | / |
Family ID | 33567506 |
Filed Date | 2014-05-22 |
United States Patent
Application |
20140141017 |
Kind Code |
A1 |
Chatterjee; Subroto ; et
al. |
May 22, 2014 |
METHODS OF DIAGNOSING ATHEROSCLEROSIS BY MEASURING APOCI
Abstract
The present invention provides methods and compositions for
identifying compounds which inhibit ApoCI and which are useful in
the treatment or prevention of atherosclerosis, plaque rupture,
apoptosis, or myocardial infarction. The invention further provides
methods for treating subjects suffering from or at risk of
developing atherosclerosis, plaque rupture, apoptosis, or
myocardial infarction. The invention further provides methods for
diagnosing subjects at suffering from or at risk of developing
treatment or prevention of atherosclerosis, plaque rupture,
apoptosis, or myocardial infarction.
Inventors: |
Chatterjee; Subroto;
(Columbia, MD) ; Kwiterovich; Peter O.;
(Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY |
Baltimore |
MD |
US |
|
|
Assignee: |
THE JOHNS HOPKINS
UNIVERSITY
Baltimore
MD
|
Family ID: |
33567506 |
Appl. No.: |
14/080296 |
Filed: |
November 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10557283 |
Nov 30, 2006 |
8614064 |
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PCT/US04/16419 |
May 24, 2004 |
|
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14080296 |
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60473224 |
May 23, 2003 |
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60539769 |
Jan 28, 2004 |
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Current U.S.
Class: |
424/172.1 ;
435/18; 435/29; 435/6.12; 435/6.13; 435/7.1; 435/7.92; 436/501;
514/44R |
Current CPC
Class: |
G01N 2500/04 20130101;
G01N 2800/323 20130101; G01N 33/92 20130101; G01N 33/6893 20130101;
G01N 2500/00 20130101; G01N 2800/324 20130101 |
Class at
Publication: |
424/172.1 ;
436/501; 435/18; 435/6.12; 435/29; 435/7.1; 435/6.13; 435/7.92;
514/44.R |
International
Class: |
G01N 33/92 20060101
G01N033/92 |
Claims
1-16. (canceled)
17. A method of identifying a compound useful for the treatment or
prevention of atherosclerosis, plaque rupture, apoptosis, or
myocardial infarction comprising: a) contacting ApoCI polypeptide
with a test compound; and b) determining whether the test compound
binds to ApoCI, wherein a test compound that binds to ApoCI is
identified as a compound useful for the treatment or prevention of
atherosclerosis, plaque rupture, apoptosis, or myocardial
infarction.
18. A method of identifying a compound useful for the treatment or
prevention of atherosclerosis, plaque rupture, apoptosis, or
myocardial infarction comprising: a) contacting ApoCI polypeptide
with a test compound; and b) determining whether the test compound
inhibits ApoCI activity, wherein a test compound that inhibits
ApoCI activity is identified as a compound useful for the treatment
or prevention of atherosclerosis, plaque rupture, apoptosis, or
myocardial infarction.
19. The method of claim 18, wherein ApoCI activity is measured by
measuring the ability of ApoCI to activate N-SMase activity.
20. The method of claim 18, wherein ApoCI activity is measured by
measuring the ability of ApoCI to inhibit cell surface expression
of SR-BI or ABCA1.
21. The method of claim 18, wherein ApoCI activity is measured by
measuring the ability of ApoCI to induce apoptosis in a cell.
22. The method of claim 21, wherein the cell is selected from the
group consisting of: a vascular smooth muscle cell, an endothelial
cell, a macrophage, an epithelial cell, a fibroblast, and a T
lymphocyte.
23. The method of claim 22, wherein the cell is an aortic smooth
muscle cell.
24. The method of claim 21, wherein apoptosis is measured using a
DNA-laddering assay.
25. The method of claim 21, wherein apoptosis is measured using
fluorescence microscopy.
26. The method of claim 21, wherein apoptosis is measured by
measuring cytochrome c release.
27. The method of claim 21, wherein apoptosis is measured by
measuring caspase activation.
28. The method of claim 27, wherein the caspase is caspase-3.
29. A method of identifying a compound useful for the treatment or
prevention of atherosclerosis, plaque rupture, apoptosis, or
myocardial infarction comprising: a) contacting a cell that
expresses ApoCI with a test compound; and b) determining whether
the test compound inhibits ApoCI expression, wherein a test
compound that inhibits ApoCI expression is identified as a compound
useful for the treatment or prevention of atherosclerosis, plaque
rupture, apoptosis, or myocardial infarction.
30. The method of claim 29, wherein ApoCI expression is measured by
measuring the level of ApoCI mRNA.
31. The method of claim 30, wherein ApoCI mRNA is measured using a
method selected from the group consisting of: Northern blotting,
primer extension, nuclease protection, and RT-PCR.
32. The method of claim 29, wherein ApoCI expression is measured by
measuring the level of ApoCI polypeptide.
33. The method of claim 32, wherein the ApoCI polypeptide is
secreted into the culture medium.
34. The method of claim 29, wherein the cell is a liver cell.
35. The method of claim 32, wherein the ApoCI polypeptide is
measured using a method selected from the group consisting of:
Western blotting, ELISA, RIA, and MALDI-TOF.
36. The method of claim 18, wherein the compound increases HDL
metabolism.
37. A method of treating a subject suffering from or at risk for
developing atherosclerosis, plaque rupture, apoptosis, or
myocardial infarction comprising administering to the subject a
therapeutically effective amount of an ApoCI inhibitor.
38. A method of increasing HDL metabolism in a subject, comprising
administering to the subject a therapeutically effective amount of
an ApoCI inhibitor.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
10/557,283, filed on Nov. 30, 2006, which is a 371 National Stage
Application of PCT/US04/016419, filed May 24, 2004, which claims
the benefit of U.S. Provisional Application Ser. No. 60/539,769,
filed on Jan. 28, 2004, and U.S. Provisional Application Ser. No.
60/473,224, filed on May 23, 2003. The entire contents of each of
these applications is incorporated herein by this reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention features methods and compositions for
identifying compounds which inhibit ApoCI and which are useful in
the treatment or prevention of atherosclerosis, plaque rupture,
apoptosis, or myocardial infarction. The invention further features
methods for treating subjects suffering from or at risk of
developing atherosclerosis, plaque rupture, apoptosis, or
myocardial infarction. The invention further features methods for
diagnosing subjects at suffering from or at risk of developing
treatment or prevention of atherosclerosis, plaque rupture,
apoptosis, or myocardial infarction.
[0004] 2. Background
[0005] Over the last decade, considerable body of evidence
indicates that premature plaque rupture due to apoptotic death of
aortic smooth muscle cells (ASMC) in the fibrous cap is a major
contributor to the pathological sequelae of atherosclerosis, i.e.
arterial thrombosis (Godfrey K M and Barker D J P (2000) Am. J.
Clin. Nutr. 71(Suppl):13445-352S; Kwiterovich, Jr., P O. et al.
(2002) Am. J. Card. 90(Suppl 8A):1i-10i; Kwiterovich Jr P O. et al.
(In press, 2004) Ethn. Dis.) leading to myocardial infarctions
(Diaz M. et al. (1989) Metabolism. 38:435-8) or stroke (Kaser S. et
al. (2001) Metabolism. 50:723-8). Other studies based upon the use
of carotid artery biopsies and immunohistochemical techniques
reveal that macrophages and T lymphocytes are the major cell type
found closely associated with the sites of plaque rupture in human
subjects (Diaz M. et al. (1989) Metabolism. 38:435-8). Caspases are
cystein-aspartate specific proteases and contribute critically in
the final phase of apoptosis i.e. executing the cleavage of DNA, an
irreversible process in apoptosis. Both caspase-1 and caspase-3
have been implicated to contribute to the execution phase of
apoptosis in vivo in human atherosclerotic plaques (Merzouk H. et
al. (1997) Acta. Paediatr. 86:528-32; Radunovic N. et al. (2000) J.
Clin. Endocrin. Metab. 85:85-88).
[0006] Apolipoprotein C-I (ApoCI), a 6.6-kDa single-chain plasma
protein of 57 amino acids, has a basic pI because of its high
content of lysine (16 mol %) and contains no histidine, tyrosine,
cysteine, or carbohydrate (Jong, M. C. et al. (1999) Arterioscler.
Thromb. Vasc. Biol. 19:472-484; Shachter, N. S. (2001) Curr. Opin.
Lipidol. 12:297-304). Residues 7 to 24 and 35 to 53 of ApoCI are
important for binding to plasma lipids (Jong, M. C. et al. (1999)
Arterioscler. Thromb. Vasc. Biol. 19:472-484; Shachter, N. S.
(2001) Curr. Opin. Lipidol. 12:297-304). ApoCI is a component of
very-low-density (VLDL), intermediate density, and high-density
lipoproteins (HDL). ApoCI displaces apolipoprotein E (apoE) from
VLDL and intermediate density, thereby decreasing their clearance
from plasma (Windier, E. E. and Havel R. J. (1985) J. Lipid Res.
26:556-565). ApoCI decreases the binding of (3-VLDL to a remnant
receptor, the low-density lipoprotein (LDL) receptor-related
protein (LRP) (Kowal R. C. et al. (1990) J. Biol. Chem.
265:10771-10779; Weisgraber, K. H. et al. (1990) J. Biol. Chem.
265:22453-22459), and apoE-mediated binding of VLDL and
intermediate density to the LDL receptor (LDLR) (Windier, E. E. et
al. (1980) J. Biol. Chem. 255:10464-10471; Sehayek, E. and
Eisenberg, S. (1991) J. Biol. Chem. 266:18259-18267). ApoCI
inhibits cholesterol ester transfer protein (Gautier, T. et al.
(2000) J. Biol. Chem. 275:37504-37509) and phospholipase A2
activity (Poensgen, J. (1990) Biochim. Biophys. Acta.
1042:188-192). ApoCI stimulates lecithin cholesterol acyl
transferase to .about.80% of that of apolipoprotein A-I (apoA-I)
(Soutar, A. K. et al. (1975) Biochemistry 14:3057-3064).
[0007] Human ApoCI-transgenic mice, with a wild-type background or
with a knockout background for the LDLR or apoE, manifest a marked
combined hyperlipidemia because of significantly delayed remnant
clearance (Shachter, N. S. et al. (1996) J. Clin. Invest.
98:846-855; Jong, M. C. et al. (1998) J. Clin. Invest. 101:145-152;
Jong, M. C. et al. (2001) Diabetes 50:2779-2785; Jong, M. C. et al.
(1996) J. Clin. Invest. 98:2259-2267; Jong, M. C. et al. (1999)
Biochem. J. 338:281-287; Jong, M. C. et al. (1996) Arterioscler.
Thromb. Vasc. Biol. 16:934-940; Conde-Knape, K. et al. (2002) J.
Lipid Res. 43:2136-2145). Free fatty acid levels are elevated
because of reduced fatty acid uptake in peripheral tissues, which
is an effect that is paradoxically associated with increased
sensitivity to insulin and protection from obesity (Jong, M. C. et
al. (1998) J. Clin. Invest. 101:145-152; Jong, M. C. et al. (2001)
Diabetes 50:2779-2785). Of particular interest here, Conde-Knape et
al (Conde-Knape, K. et al. (2002) J. Lipid Res. 43:2136-2145),
using a moderately expressing ApoCI transgenic on apoE-null
background to study the effect of ApoCI independent of apoE, found
a marked combined dyslipidemia that included an ApoCI-enriched HDL
and increased atherosclerosis. ApoCI-enriched HDL (but not VLDL)
had a marked inhibitory effect on hepatic lipase (Conde-Knape, K.
et al. (2002) J. Lipid Res. 43:2136-2145). ApoCI knockouts are
normolipidemic rather than hypolipidemic (van Ree, J. H. et al.
(1995) Biochem. J. 305:905-911). Cholesterol ester transfer
protein-transgenic/apoC knockout mice manifest a markedly increased
transfer of cholesteryl esters from HDL to VLDL (Gautier, T. et al.
(2002) J. Biol. Chem. 277:31354-31363).
[0008] In humans, Bjorkegren et al reported a significant
enrichment of ApoCI in VLDL remnants in normolipidemic patients
with coronary artery disease and exaggerated postprandial
triglyceridemia (Bjorkegren, J. et al. (2000) Circulation
101:227-230) and in healthy, normolipidemic men with early
asymptomatic atherosclerosis (Bjorkegren, J. et al. (2002)
Arterioscler Thromb Vasc Biol. 22:1470-1474).
SUMMARY OF THE INVENTION
[0009] The present invention is based, at least in part, on the
discovery that treatment of aortic smooth muscles cells (ASMC) with
ApoCI or ApoCI-enriched HDL particles induces apoptosis that
accompanied the activation of the neutral sphingomyelinase signal
transduction cascade. Increased plasma levels of ApoCI, perhaps
associated with HDL, may contribute to the complications of
atherosclerosis by inducing apoptosis in ASMC, a biochemical
mechanism that contributes to plaque rupture and coronary artery
disease. This invention is further based, at least in part on the
discovery that a prominent ApoCI-enriched HDL peak is present in
the blood of low-birthweight infants.
[0010] Accordingly, in one embodiment, the invention provides
methods of determining whether a subject is at risk for developing
atherosclerosis-associated plaque rupture or myocardial infarction
comprising measuring the level of ApoCI protein in a biological
sample from the subject, and comparing the level of ApoCI protein
in the biological sample from the subject to the level of ApoCI
protein from a control, wherein an increased level of ApoCI protein
as compared to the control indicates that the subject is at risk
for developing atherosclerosis-associated plaque rupture or
myocardial infarction. The subject may or may not have been
previously diagnosed with atherosclerosis. In one embodiment, the
subject is an infant (e.g., a low-birthweight infant). In a
preferred embodiment, the biological sample is selected from blood,
serum, and plasma. In one embodiment, the biological sample is
taken from an infant's umbilical cord.
[0011] In one embodiment, the level of ApoCI protein is detected by
a method selected from the group consisting of Western blot, ELISA,
RIA, and/or MALDI-TOF. In another embodiment, the level of ApoCI
protein is detected by measuring ApoCI activity. In a preferred
embodiment, ApoCI activity is measured by measuring the ability of
ApoCI to activate N-SMase activity. In another embodiment, ApoCI
activity is measured by determining the ability of ApoCI to induce
apoptosis in a cell.
[0012] In another embodiment, the invention provides methods of
identifying a compound useful for the treatment or prevention of
atherosclerosis, plaque rupture, apoptosis, or myocardial
infarction comprising contacting ApoCI polypeptide with a test
compound, and determining whether the test compound binds to ApoCI,
wherein a test compound that binds to ApoCI is identified as a
compound useful for the treatment or prevention of atherosclerosis,
plaque rupture, apoptosis, or myocardial infarction.
[0013] In another embodiment, the invention provides methods of
identifying a compound useful for the treatment or prevention of
atherosclerosis, plaque rupture, apoptosis, or myocardial
infarction comprising contacting ApoCI polypeptide with a test
compound, and determining whether the test compound inhibits ApoCI
activity, wherein a test compound that inhibits ApoCI activity is
identified as a compound useful for the treatment or prevention of
atherosclerosis, plaque rupture, apoptosis, or myocardial
infarction. In a preferred embodiment, ApoCI activity is measured
by measuring the ability of ApoCI to activiate N-SMase activity. In
another embodiment, ApoCI activity is measured by measuring the
ability of ApoCI to inhibit expression of SR-BI or ABCA1. In still
another embodiment, ApoCI activity is measured by measuring the
ability of ApoCI to induce apoptosis in a cell. In preferred
embodiments, apoptosis may be measured using a DNA-laddering assay,
fluorescence microscopy, by measuring cytochrome c relase, by
measuring caspase activation (e.g., caspase-3 activation),
[0014] In a preferred embodiment, the cell is a vascular smooth
muscle cell (e.g., an aortic smooth muscle cell).
[0015] In another embodiment, the invention provides methods of
identifying a compound useful for the treatment or prevention of
atherosclerosis, plaque rupture, apoptosis, or myocardial
infarction comprising contacting a cell (e.g., a liver cell) that
expresses ApoCI with a test compound, and determining whether the
test compound inhibits ApoCI expression, wherein a test compound
that inhibits ApoCI expression is identified as a compound useful
for the treatment or prevention of atherosclerosis, plaque rupture,
apoptosis, or myocardial infarction.
[0016] In one embodiment, ApoCI expression is measured by measuring
the level of ApoCI mRNA (e.g., by Northern blotting, primer
extension, nuclease protection, and/or RT-PCR). In another
embodiment, ApoCI expression is measured by measuring the level of
ApoCI polypeptide (e.g., by Western blotting, ELISA, RIA, and/or
MALDI-TOF). In a preferred embodiment, the ApoCI polypeptide is
secreted into the culture medium.
[0017] In a preferred embodiment, the compounds identified using
the methods of the invention increase HDL metabolism.
[0018] In still another embodiment, the invention provides methods
of treating a subject suffering from or at risk for developing
atherosclerosis, plaque rupture, apoptosis, or myocardial
infarction comprising administering to the subject a
therapeutically effective amount of an ApoCI inhibitor.
[0019] In yet another embodiment, the invention provides methods of
increasing HDL metabolism in a subject, comprising administering to
the subject a therapeutically effective amount of an ApoCI
inhibitor.
[0020] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A-1C depict the effect of apolipoproteins on
apoptosis in human aortic smooth muscles cells (ASMC). ASMC were
grown on sterilized glass cover slips and incubated for 24 hours
with media alone (control), ApoCIII (negative control, 2.5 .mu.g/mL
medium), ApoCI (2.5 .mu.g/mL medium), ApoCI-enriched HDL (1 .mu.g
apoA-I/mL medium), or ApoCI-poor HDL (5 .mu.g apoA-I/mL medium) for
24 hours. Cells were then fixed with ethanol-acetic acid (3:1
volume/volume) and washed 3 times with phosphate-buffered saline.
The cells were then stained with DAPI reagent, mounted on a glass
cover slide, and subjected to fluorescence microscopy. FIG. 1A: The
nucleus of normal cells stains blue and the nucleus of apoptotic
cells stains is fragmented white. FIG. 1B: 300 to 500 cells in each
sample were counted and the percent of apoptotic cells was plotted.
Values are mean.+-.SD; **P<0.01, ***P<0.001. FIG. 1C: DNA
ladder assay. Confluent cultures of ASMC were incubated with
control medium alone, ApoCI (2.5 .mu.g/mL medium), ApoCIII
(negative control, 2.5 .mu.g/mL medium), and TNF-.alpha. (positive
control, 20 ng/mL medium). Genomic DNA was extracted and 10 .mu.g
DNA was subjected to agarose gel electrophoresis. The gels were
calibrated with commercially available DNA standards. After
electrophoresis, the gels were stained with ethidium bromide and
then photographed.
[0022] FIGS. 2A-2B depict the effect of ApoCI, the N-SMase
inhibitor, GW4869, and C-2 ceramide on apoptosis in cultured human
ASMC. FIG. 2A: ASMC (1.times.10.sup.3) were seeded on sterilized
glass cover slips and grown in tissue culture medium with 10% fetal
calf serum for 48 hours. Next, fresh serum-free medium, with and
without GW4869 (20 .mu.mol/L in DMSO), was added to some dishes.
Thirty minutes later, vehicle DMSO, ApoCI (2.5 .mu.g/mL), or
C2-ceramide (10 .mu.mol/L) were added to dishes, and the incubation
continued for 24 hours. The cells were then fixed with
ethanol-acetic acid (3:1 v/v), stained with DAPI reagent, assessed
by fluorescent microscopy, and photographed. Top to bottom: control
cells; cells incubated with ApoCI.+-.the N-SMase inhibitor, GW4869;
and cells incubated with ApoCI, GW4869, and C2-ceramide. FIG. 2B:
The DAPI-stained ASMC from the four experimental conditions
described was analyzed for quantitative apoptosis. Mean (SD) from 4
experiments. *P<0.05.
[0023] FIGS. 3A-3C depict the effects of ApoCI, ApoCIII,
TNF-.alpha., ApoCI-enriched HDL, and ApoCI-poor HDL on the activity
of N-SMase in ASMC. Confluent cultures of ASMC were preincubated
for 30 minutes with serum-free (control) medium, then ApoCI (2.5
.mu.g/mL medium), ApoCIII (2.5 .mu.g/mL medium), TNF-.alpha. (20
ng/mL medium) (FIG. 3A, top), or ApoCI-enriched HDL (1 .mu.g
apoA-I/mL medium), or ApoCI-poor HDL (5 .mu.g apoA-I/mL medium)
(FIG. 3B, middle) were added individually to cells. The cells were
then incubated and harvested at the indicated time points, and the
activity was of N-SMase determined using [.sup.14C]sphingomyelin as
substrate. The N-SMase activity in control cells was 22 nmol/h per
milligram cell protein, and this value was considered as 100%
activity. In a separate experiment (FIG. 3C bottom), cells
(0.5.times.10.sup.5) were seeded in P60 Petri dishes and grown in
DMEM supplemented with 10% fetal bovine serum for 5 days. After
preincubation of cells for 30 minutes in serum-free medium,
increasing amounts of ApoCI were added. After incubation for 10
minutes, cells were harvested, and N-SMase activity was
measured.
[0024] FIGS. 4A-4D depict the effect of ApoCI, ApoCI-enriched HDL,
and ApoCI-poor HDL on the level of ceramide in ASMC. Confluent ASMC
were incubated with ApoCI (2.5 .mu.g/mL) with time (FIG. 4A, upper
left) or with ApoCI-enriched HDL (1 .mu.g apoA-I/mL medium) or
ApoCI-poor HDL (5 .mu.g apoA-I/mL medium) (FIG. 4B, upper right)
for 30 minutes. FIG. 4C: The cells were incubated with ApoCI alone
(2.5 .mu.mol/L) for 5 minutes or preincubated with GW4869 (20
.mu.mol/L) for 30 minutes. After addition of ApoCI (2.5 .mu.g/mL),
incubation was continued for 6 hours, 12 hours, and 24 hours. After
incubation, cells were harvested and the levels of ceramide
determined using the diacylglycerol kinase assay. The level of
phosphate was measured and the data (mean.+-.SD) were expressed as
percentage of the total ceramide present in untreated cells
(vehicle DMSO). *P<0.05. In a separate experiment (FIG. 4D,
right), confluent cells were grown on cover slips and pretreated
with serum-free medium for 30 minutes. The cells were then
incubated with vehicle DMSO alone (control), ApoCI (2.5 .mu.g/mL),
or ApoCI (2.5 .mu.g/mL) plus the N-SMase inhibitor GW4869 (20
.mu.mol/L) for 24 hours. The cells were then washed, treated with a
primary anticeramide antibody, washed with phosphate-buffered
saline, and treated with a fluorescein isothiocyanate-conjugated
anti-mouse IgG (FIG. 4C, lower panel).
[0025] FIGS. 5A-5C depict the effect of ApoCI, ApoCIII,
ApoCI-enriched HDL, and GW4869 on cytochrome c release in ASMC.
After treatment of confluent cells for 24 hours with ApoCI (2.5
.mu.g/mL medium), ApoCIII (2.5 .mu.g/mL medium), or ApoCI-enriched
HDL (1 .mu.g apoA-I/mL medium), the cells were lysed. The cell
lysate was used as the source of cytochrome c, and the pellet was
used as a source of caspase-3. The cell lysates were separated by
SDS-polyacrylamide gel electrophoresis, blotted onto
nitrocellulose, and cytochrome c detected using a polyclonal
antibody against cytochrome c (FIG. 5A, top panel). The gels were
then subjected to densitometric scanning (FIG. 5B, middle panel).
In a separate experiment (FIG. 5C, right), confluent cells were
grown on cover slips and pretreated with serum-free medium for 30
minutes. The cells were then incubated with control medium, ApoCI
(2.5 .mu.g/mL medium), TNF-.alpha. (20 ng/mL), or ApoCI (2.5
.mu.g/mL)+GW4869 (20 .mu.mol/L) for 24 hours. The cells were then
washed, treated with a primary anti-cytochrome c antibody, washed
with phosphate-buffered saline, and treated with a fluorescein
isothiocyanate-conjugated anti-rabbit IgG (FIG. 5C, lower
panel).
[0026] FIG. 6 depicts representative 3D FSE MRI aortic vessel wall
images for three animal groups. MRI vessel wall images of group
I-III on three comparable anatomic levels (A-C) of the sub-renal
aorta (dashed arrows) demonstrate excellent image quality. Focal
and general vessel wall thickening can be documented in all groups
at all levels. Note the variable anatomic position of the inferior
vena cava (solid arrows) and the variable venouos wall thickening
due to slow flowing blood artifacts.
[0027] FIGS. 7A-7B depict SNR and vessel wall thickness correlation
between histology and MRI. FIG. 2A: SNR measurements can detect
different stages of the atherosclerotic process in the vessel wall
since SNR values for group 2 animals are significantly lower than
group 3 (p=0.048) and have a trend for significantly lower SNR
(p=0.06) when compared to group 1 animals. FIG. 7B: 3D Black blood
Fast Spin Echo MRI vessel wall measurements correlated well with
histology in animal groups with plaque rupture (r=0.8).
[0028] FIGS. 8A-8F depict aortic wall imaging with plaque rupture
and corresponding histological flap. Sub-renal sagittal rabbit
aortic MR angiography with corresponding axial FSE-vessel wall
imaging (Aorta=sub-renal aorta, IVC=inferior vena cava) before
(FIGS. 8A and 8B) and after (FIGS. 8D and 8E) snake venom and
histamine administration. Note the high in-plane vessel wall image
resolution of 130-250 .mu.m (FIGS. 8B and 8D). The neo-intimal
dissection visualised by MR in vivo (FIG. 8E) correlated well with
histopathologic detection of ruptured atherosclerotic plaque (FIG.
8F). FIG. 8C shows the inverse correlation between serum
cholesterol levels (mg/dL) and vessel wall MRI signal in group 2
and 3 animals (r=0.76).
[0029] FIGS. 9A-9P depict immunohistochemical analysis of apoptotic
markers in atherosclerotic lesions post venom. Representative
immunohistochemical stainings from animals of group 1 (FIGS. 9A-9D
and 9I-9L) and group 2 with a ruptured plaque (FIGS. 9E-9H and
9M-9P). Note the strong positive immunohistochemical staining in
the group 2 animals adjacent to the ruptured plaque shown in FIG. 8
for ApoCI (FIG. 9F), ceramide (FIG. 9G), caspase-1 (FIG. 9H),
caspase-3 (FIG. 9M). See spots indicated by arrows (FIGS. 9N and
9O) documenting macrophages, ASMCs and a few apoptotic cells in the
group 2 animals adjacent to the ruptured plaque.
[0030] FIGS. 10A-10B depict representative immunoblot assay of
caspase-3 in rabbit aorta sections. Proteins (50 .mu.g per line)
from homogenate of aorta sections were separated using 4-15%
SDS-poly-acrylamide gel electrophoresis and transferred onto
nitrocellulose membrane. Western assay was performed using rabbit
anti-human caspase-3 antibody. FIG. 10A-control (animal group 1)
and FIG. 10B-ruptured plaque (group 2).
[0031] FIG. 11 depicts lipoprotein profiles from cord blood
obtained after sucrose density gradient ultracentrifugation. The
profile on the left is from a group 3 infant while that on the
right is from a group 0 infant.
[0032] FIGS. 12A-D depict MALDI-TOF MS of ApoCI-enriched HDL and
normal HDL. ApoCI-enriched HDL (panels A and B) and normal HDL
(panels C and D) were isolated from plasma of a group 3 infant
(panels A and C) and group 0 infant (panels B and D) and prepared
for MALDI-TOF MS.
[0033] FIG. 13 depicts gradient gel electrophoresis of HDL. Plasma
lipoproteins were isolated by ultracentrifugation at d>1.21 g/ml
and prepared for GGE. Following GGE, gels were stained for protein
and densitometric scans performed. Scans of HDL from a group 3
infant are depicted by the solid line and from a group 0 infant by
the broken line. Sizes of the HDL subclasses are shown in nm.
[0034] FIG. 14 depicts measured mean (1SD) plasma levels of lipids,
lipoprotein cholesterols, apolipoproteins, lipoprotein subclasses,
and lipoprotein sizes were determined by nuclear magnetic resonance
(NMR) spectroscopy (Otvos, J D. (2000) In: Rifai N. et al., Eds.
Handbook of Lipoprotein Testing. Washington, D.C.: AACC Press,
609-623) in cord blood from group 0, 1, 2 and 3 infants. The p
value given for each variable was corrected for the influence of
gestational age by linear regression.
[0035] FIG. 15 depicts gestational age in group 0, 1, 2, and 3
infants. The median and 25.sup.th and 75.sup.th percentiles (box)
and 5.sup.th and 95.sup.th percentiles (whiskers) for gestational
age are shown. The circles represent outliers.
[0036] FIG. 16 depicts plots of gestational age versus large (L3)
LDL cholesterol (left) and largest (H5) HDL cholesterol (right) in
group 3 (solid circles) and group 0 (open circles) infants.
Regression lines are depicted for each group.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention is based, at least in part, on the
discovery that treatment of aortic smooth muscles cells (ASMC) with
ApoCI or ApoCI-enriched HDL particles induces apoptosis that
accompanied the activation of the neutral sphingomyelinase signal
transduction cascade. Increased plasma levels of ApoCI, perhaps
associated with HDL, may contribute to the complications of
atherosclerosis by inducing apoptosis in ASMC, a biochemical
mechanism that contributes to plaque rupture and coronary artery
disease. This invention is further based, at least in part on the
discovery that a prominent ApoCI-enriched HDL peak is present in
the blood of low-birthweight infants. Accordingly, the invention
provides methods for identifying subjects at risk for developing
plaque rupture and/or myocardial infarction based on measuring the
subject's plasma levels of ApoCI. In another embodiment, the
invention provides methods for identifying at birth subjects who
may later be at risk for atherosclerosis, plaque rupture, and/or
myocardial infarction. In still another embodiment, the invention
provides methods for identifying compounds which can inhibit ApoCI
expression and/or activity.
[0038] The data presented herein set for the initial discovery that
human large high density lipoproteins (HDL) enriched in
apolipoprotein C-1 (ApoCI), but not control HDL, isolated from
umbilical cord blood, induced apoptosis in human cultured arterial
smooth muscle cells (ASMC). Highly purified ApoCI, but not ApoCIII,
also induced apoptosis in aortic smooth muscle cells (ASMC). This
effect was mediated by the activity of neutral sphingomyelinase
(N-SMase). ApoCI stimulated Src kinase in addition to N-SMAse,
indicating that a second messenger pathway was involved. ApoCI
stimulated N-SMase, cleaving sphingomyelin to generate ceramide. In
turn, ceramide stimulated the release of cytochrome-C from the
mitochondria and contributed to the activation of caspases,
resulting in DNA fragmentation and apoptotic cell death of the
ASMC. This signal transduction cascade was abrogated by
preincuabtion of cells with an antibody against N-SMase. Additional
data showed that ApoCI transiently and significantly decreased the
cellular level of the scavenger receptor binding protein (SR-BI)
and the adenosine triphosphate (ATP) binding cassette (ABC-A1)
protein, proteins involved in the cellular uptake of cholesteryl
esters from HDL and in the egress of cholesterol from cells onto
nascent HDL, respectively. ApoCI did not alter the level of
caveolin. Thus, ApoCI also has a role in cellular metabolism of
cholesterol via the SRI and ABCA-1 pathways.
[0039] ApoCI is a small apolipoprotein secreted from human liver
into blood on the triglyceride-rich lipoprotein, very low density
lipoprotein (VLDL). ApoCI has several effects in blood. It inhibits
the uptake of VLDL by the VLDL receptor, and in transgenic mice
promotes hypertriglyceridemia and hypercholesterolemia by
decreasing the catabolism and uptake of VLDL. Transgenic ApoCI
mice, on a background of LDL receptor deficiency appear to be more
propone to atherosclerosis than LDL receptor deficient mice alone
but the mechanism of the atherosclerosis is not understood. In
addition to its role in VLDL metabolism, ApoCI can be transferred
from VLDL up to HDL as a result of lipolysis of triglyceride on
VLDL. On HDL, ApoCI can inhibit cholesterol ester transfer protein
(CETP), decreasing the transfer of cholesteryl esters from HDL up
to VLDL. ApoCI can stimulate lecithin cholesterol ester transferase
(LCAT), facilitating the formation of cholesteryl esters on HDL.
Thus, ApoCI can also impact HDL metabolism in blood. Despite this
literature, the cellular effects of ApoCI, particularly on the
cells of the vascular wall, were unknown. As a result of the
discoveries detailed herein, ApoCI promotes apoptosis of ASMC.
Since ASMC are essential to maintain the integrity of the fibrous
cap of the atherosclerotic lesion, apoptosis of the ASCM can
promote the rupture of the fibrous cap, leading to heart attack and
sudden cardiac death. It provides a cellular mechanism for the
promotion of atherosclerosis by ApoCI and the triglyceride-rich
lipoproteins. It extends the role of N-SMase in atherosclersois and
apoptosis. It also provides for the first time, a regulatory
molecule in blood that effects the expression of two cell surface
molecules essential in HDL metabolism and reverse cholesterol
transport, the SR-BI and ABCA-1 proteins.
[0040] The ApoCI modulators identified according to the methods of
the invention can be used to inhibit ApoCI expression and/or
activity, including inhibition of N-SMase activity and inhibition
of apoptosis, and are, therefore, useful in treating or diagnosing
atherosclerosis and apotosis-associated disorders, e.g., plaque
rupture and myocardial infarction. For example, inhibition of the
activity of an ApoCI molecule can cause decreased apoptosis in the
vascular smooth muscle cells of a subject Thus, the ApoCI
inhibitors used in the methods of the invention can be used to
treat disorders characterized by excessive apoptosis.
[0041] As used herein, an "apoptosis-associated disorder" includes
a disease, disorder, or condition which is associated with abnormal
or aberrant apoptosis.
[0042] As used interchangeably herein, "ApoCI activity,"
"biological activity of ApoCI" or "functional activity of ApoCI,"
includes an activity exerted by an ApoCI protein, polypeptide or
nucleic acid molecule on an ApoCI responsive cell or tissue (e.g.,
a vascular smooth muscle cell) or on an ApoCI protein substrate
(e.g., N-SMase), as determined in vivo, or in vitro, according to
standard techniques. ApoCI activity can be a direct activity, such
as an association with an ApoCI-target molecule. As used herein, a
"substrate" or "target molecule" or "binding partner" is a molecule
with which an ApoCI protein binds or interacts in nature, such that
ApoCI-mediated function, e.g., modulation of apoptosis, is
achieved. An ApoCI target molecule can be a non-polypeptide
molecule, or a protein or polypeptide (e.g., an ApoCI receptor or
an ApoCI effector). Alternatively, an ApoCI activity is an indirect
activity, such as a cellular signaling activity mediated by
interaction of the ApoCI protein with an ApoCI target molecule. The
biological activities of ApoCI are described herein. For example,
the ApoCI proteins can have one or more of the following
activities: 1) they modulate N-SMase activity; 2) they modulate
apoptosis; 3) they modulate expression of ABCA1 and/or SR-B1; 4)
they modulate egress of cholesterol from macrophages; 5) they
modulate plaque rupture; and 6) they modulate myocardial
infarction. In the context of ApoCI-enriched HDL, referred to
herein as "dysfunctional HDL", ApoCI stimulates N-Smase activity,
promotes apoptosis, downregulates expression of ABCA1 and SR-B1,
downregulate egress of cholesterol from macrophages, increase the
risk of plaque rupture, and increase the risk of myocardial
infarction.
[0043] Various aspects of the invention are described in further
detail in the following subsections:
I. Screening Assays:
[0044] As described herein, ApoCI induces apoptosis of arterial
smooth muscle cells through Neutral Sphingomyelinase (N-SMase)
stimulation. Accordingly, the invention provides methods (also
referred to herein as "screening assays") for identifying
inhibitors, i.e., candidate or test compounds or agents (e.g.,
peptides, peptidomimetics, small molecules, ribozymes, or ApoCI
antisense molecules) which bind to ApoCI proteins, have an
inhibitory effect on ApoCI expression or ApoCI activity, or an
inhibitory effect on the expression or activity of an ApoCI target
molecule. Compounds identified using the assays described herein
may be useful for treating atherosclerosis associated disorders.
Preferably, an ApoCI inhibitor inhibits ApoCI stimulation of
N-SMase, decreases apoptosis, and/or prevents rupture of unstable
plaques and/or myocardial infarctions. In another embodiment, the
screening assays described herein may be used to identify compounds
which have a stimulatory effect on ApoCI activity (and thus, which
increase apoptosis), which may be useful in the treatment of
proliferative disorders (e.g., cancer, polycystic kidney
disease).
[0045] Candidate/test compounds include, for example, 1) peptides
such as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam, K. S. et al.
(1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature
354:84-86) and combinatorial chemistry-derived molecular libraries
made of D- and/or L-configuration amino acids; 2) phosphopeptides
(e.g., members of random and partially degenerate, directed
phosphopeptide libraries, see, e.g., Songyang, Z. et al. (1993)
Cell 72:767-778); 3) antibodies (e.g., polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric, and single chain antibodies as
well as Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0046] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145).
[0047] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al (1994) Angew. Chem. Int.
Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem.
37:1233.
[0048] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA
89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390;
Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.
Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310;
Ladner supra.).
[0049] In one aspect, an assay is a cell-based assay in which a
cell is contacted with an ApoCI protein or biologically active
portion thereof and with a test compound, and the ability of the
test compound to modulate ApoCI activity is determined In a
preferred embodiment, the biologically active portion of the ApoCI
protein includes a domain or motif which can modulate N-SMase
activity. Determining the ability of the test compound to modulate
ApoCI activity can be accomplished by monitoring, for example,
N-SMase activity, or by measuring apoptosis in the cell. The cell,
for example, can be of mammalian origin, e.g., a vascular smooth
muscle cell such as an aortic smooth muscle cell, an endothelial
cell, a macrophage, an epithelial cell, a fibroblast, or a T
lympocyte
[0050] In a preferred embodiment N-SMase activity is measured using
methods described herein in the Examples section, or using a
standard activity gel assay such as the assay disclosed in Example
1, part 6 of U.S. Pat. No. 5,919,687, and includes measuring
activity of the N-SMase peptide using [.sup.14C]-sphingomyelin.
[0051] In another embodiment, N-SMase activity is measured by
measuring the expression levels of two key cell surface molecules
involved in HDL metabolism, ABCA-1 and SR-BI.
[0052] The ability of the test compound to modulate ApoCI binding
to a target molecule can also be determined Determining the ability
of the test compound to modulate ApoCI binding to a target molecule
can be accomplished, for example, by coupling the ApoCI target
molecule with a radioisotope, fluorescent, or enzymatic label such
that binding of the ApoCI target molecule to ApoCI can be
determined by detecting the labeled ApoCI target molecule in a
complex. Alternatively, ApoCI could be coupled with a radioisotope
or enzymatic label to monitor the ability of a test compound to
modulate ApoCI binding to an ApoCI target molecule in a complex.
Determining the ability of the test compound to bind ApoCI can be
accomplished, for example, by coupling the compound with a
radioisotope or enzymatic label such that binding of the compound
to ApoCI can be determined by detecting the labeled compound in a
complex. For example, test compounds and/or ApoCI target molecules
can be labeled with .sup.125I, .sup.35S, .sup.14C, or .sup.3H,
either directly or indirectly, and the radioisotope detected by
direct counting of radioemission or by scintillation counting.
Alternatively, compounds can be enzymatically labeled with, for
example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0053] It is also within the scope of this invention to determine
the ability of a compound to interact with ApoCI without the
labeling of any of the interactants. For example, a
microphysiometer can be used to detect the interaction of a
compound with ApoCI and/or with a ApoCI target molecule without the
labeling of any of the interatants (McConnell, H. M. et al. (1992)
Science 257:1906-1912). As used herein, a "microphysiometer" (e.g.,
Cytosensor) is an analytical instrument that measures the rate at
which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate
can be used as an indicator of the interaction between a compound
and ApoCI.
[0054] Because ApoCI modulates N-SMase activity and apoptosis,
compounds which modulate N-SMase activity and/or apoptosis can be
identified by the ability to modulate ApoCI expression. To
determine whether a test compound modulates ApoCI expression, a
cell which expresses ApoCI is contacted with a test compound, and
the ability of the test compound to modulate ApoCI expression can
be determined by measuring ApoCI mRNA by, e.g., Northern blotting,
quantitative PCR (e.g., RT-PCR), or in vitro transcriptional
assays. To perform an in vitro transcriptional assay, the full
length promoter and enhancer of ApoCI can be linked to a reporter
gene such as chloramphenicol acetyltransferase (CAT), luciferase,
or a fluorescent protein (e.g., GFP and variants thereof) and
introduced into host cells. The same host cells can then be
transfected with or contacted with the test compound. The effect of
the test compound can be measured by reporter gene activity and
comparing it to reporter gene activity in cells which do not
contain the test compound. An increase or decrease in reporter gene
activity indicates a modulation of ApoCI expression and is,
therefore, an indicator of the ability of the test compound to
modulate apoptosis.
[0055] In yet another embodiment, an assay of the present invention
is a cell-free assay in which an ApoCI protein or biologically
active portion thereof is contacted with a test compound and the
ability of the test compound to bind to or to modulate (e.g.,
inhibit) the activity of the ApoCI protein or biologically active
portion thereof is determined. Preferred biologically active
portions of the ApoCI proteins to be used in assays of the present
invention include fragments which participate in interactions with
non-ApoCI molecules. Binding of the test compound to the ApoCI
protein can be determined either directly or indirectly as
described above. Determining the ability of the ApoCI protein to
bind to a test compound can also be accomplished using a technology
such as real-time Biomolecular Interaction Analysis (BIA)
(Sjolander, S, and Urbaniczky, C. (1991) Anal Chem. 63:2338-2345;
Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). As used
herein, "BIA" is a technology for studying biospecific interactions
in real time, without labeling any of the interactants (e.g.,
BIAcore). Changes in the optical phenomenon of surface plasmon
resonance (SPR) can be used as an indication of real-time reactions
between biological molecules.
[0056] In yet another embodiment, the cell-free assay involves
contacting an ApoCI protein or biologically active portion thereof
with a known compound which binds the ApoCI protein to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with
the ApoCI protein, wherein determining the ability of the test
compound to interact with the ApoCI protein comprises determining
the ability of the ApoCI protein to preferentially bind to or
modulate the activity of an ApoCI target molecule.
[0057] The cell-free assays of the present invention are amenable
to use of both soluble and/or membrane-bound forms of isolated
proteins. In the case of cell-free assays in which a membrane-bound
form of an isolated protein is used it may be desirable to utilize
a solubilizing agent such that the membrane-bound form of the
isolated protein is maintained in solution. Examples of such
solubilizing agents include non-ionic detergents such as
n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,
octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton.RTM.
X-100, Triton.RTM. X-114, Thesit.RTM., Isotridecypoly(ethylene
glycol ether).sub.n,
3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0058] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either
ApoCI or an ApoCI target molecule to facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay. Binding of a test
compound to an ApoCI protein, or interaction of an ApoCI protein
with an ApoCI target molecule in the presence and absence of a test
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/ApoCI fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or ApoCI protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtitre plate wells are washed to remove any
unbound components, the matrix is immobilized in the case of beads,
and complex formation is determined either directly or indirectly,
for example, as described above. Alternatively, the complexes can
be dissociated from the matrix, and the level of ApoCI binding or
activity determined using standard techniques. In a preferred
embodiment, ApoCI protein or ApoCI target molecule may be
immobilized (e.g., for high-throughput screening) in microtiter
plates coated with polyvinyldiflouride (PVDF).
[0059] Other techniques for immobilizing proteins or cell membrane
preparations on matrices can also be used in the screening assays
of the invention. For example, either an ApoCI protein or an ApoCI
target molecule can be immobilized utilizing conjugation of biotin
and streptavidin. Biotinylated ApoCI protein or target molecules
can be prepared from biotin-NHS(N-hydroxy-succinimide) using
techniques known in the art (e.g., biotinylation kit, Pierce
Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies which are reactive with ApoCI protein or
target molecules but which do not interfere with binding of the
ApoCI protein to its target molecule can be derivatized to the
wells of the plate, and unbound target or ApoCI protein is trapped
in the wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the ApoCI protein or target
molecule, as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the ApoCI protein or target
molecule.
[0060] In yet another aspect of the invention, the ApoCI protein or
fragments thereof can be used as "bait proteins" in a two-hybrid
assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317;
Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol.
Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques
14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent
WO 94/10300) to identify other proteins which bind to or interact
with ApoCI ("ApoCI-binding proteins" or "ApoCI-bp) and are involved
in ApoCI activity. Such ApoCI-binding proteins may be ApoCI
inhibitors or activators, and are preferably inhibitors.
[0061] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for an ApoCI
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming an ApoCI-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the ApoCI protein.
[0062] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a
cell-free assay, and the ability of the agent to modulate the
activity of an ApoCI protein can be confirmed in vivo, e.g., in an
animal such as an animal model for atherosclerosis.
[0063] Moreover, an ApoCI modulator identified as described herein
(e.g., an antisense ApoCI nucleic acid molecule, an ApoCI-specific
antibody, or a small molecule) can be used in an animal model to
determine the efficacy, toxicity, or side effects of treatment with
such a modulator. Alternatively, an ApoCI modulator identified as
described herein can be used in an animal model to determine the
mechanism of action of such a modulator.
II. Predictive Medicine:
[0064] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining ApoCI protein levels in the
context of a biological sample (e.g., blood, serum, or plasma) to
thereby determine whether an individual is at risk for
atherosclerotic plaque rupture and/or myocardial infarction. Such
assays can be used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of plaque
rupture and/or myocardial infarction.
[0065] Another aspect of the invention pertains to monitoring the
influence of ApoCI inhibitors (e.g., anti-ApoCI antibodies,
ribozymes, or small molecules) on the expression or activity of
ApoCI in clinical trials.
[0066] The present invention further pertains to methods for
identifying subjects having or at risk of developing
atherosclerosis, plaque rupture, and/or myocardial infarction
associated with aberrant ApoCI expression or activity.
[0067] As used herein, the term "aberrant" includes an ApoCI
expression or activity which deviates from the wild type ApoCI
expression or activity. Aberrant expression or activity generally
includes increased expression or activity, as well as expression or
activity which does not follow the wild type developmental pattern
of expression.
[0068] In one embodiment, a subject is already known to have
atherosclerosis and/or myocardial infarction. In another
embodiment, a subject has known risk factors for developing
atherosclerosis and/or myocardial infarction (e.g., age,
overweight, diabetes, smoking, and/or high-fat diet). In still
another preferred embodiment, a subject is a low-birthweight and/or
low gestational-age infant. In another embodiment, a subject has
been diagnosed as having a ruptured or rupturing plaque using
magnetic resonance imaging (MRI)>
[0069] To determine whether a subject is at risk for developing
plaque rupture and/or myocardial infarction, a biological sample
may be obtained from a subject and the biological sample may be
contacted with a compound or an agent capable of detecting an ApoCI
protein in the biological sample.
[0070] A preferred agent for detecting ApoCI protein in a sample is
an antibody capable of binding to ApoCI protein, preferably an
antibody with a detectable label. Antibodies can be polyclonal, or
more preferably, monoclonal. An intact antibody, or a fragment
thereof (e.g., Fab or F(ab')2) can be used. The term "labeled",
with regard to the probe or antibody, is intended to encompass
direct labeling of the probe or antibody by coupling (i.e.,
physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of direct substances that can be coupled to an antibody or a
nucleic acid probe include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive materials. Examples of indirect labeling
include detection of a primary antibody using a fluorescently
labeled secondary antibody and end-labeling of a DNA probe with
biotin such that it can be detected with fluorescently labeled
streptavidin.
[0071] The term "biological sample" is intended to include tissues,
cells, and biological fluids isolated from a subject, as well as
tissues, cells, and fluids present within a subject. Preferably, a
biological sample is plasma, serum, or blooed. The detection method
of the invention can be used to detect ApoCI protein in a
biological sample in vitro as well as in vivo. In vitro techniques
for detection of ApoCI protein include, but are not limited to,
enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. In vivo techniques for
detection of ApoCI protein include introducing into a subject a
labeled anti-ApoCI antibody. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
[0072] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting ApoCI
protein, such that the presence of ApoCI protein is detected in the
biological sample, and comparing the presence of ApoCI protein in
the control sample with the presence of ApoCI protein in the test
sample.
[0073] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an ApoCI
inhibitor (e.g., an antagonist, peptidomimetic, protein, peptide,
nucleic acid, or small molecule) to effectively treat
atherosclerosis.
[0074] The present invention further provides methods for
determining the effectiveness of an ApoCI inhibitor (e.g., an ApoCI
inhibitor identified herein) in treating atherosclerosis in a
subject. For example, the effectiveness of an ApoCI inhibitor in
decreasing ApoCI protein levels, gene expression or activity, can
be monitored in clinical trials of subjects exhibiting increased
ApoCI gene expression, protein levels, or activity. In such
clinical trials, the expression or activity of an ApoCI protein,
and preferably, other genes that have been implicated in, for
example, atherosclerosis, plaque rupture, and/or myocardial
infarction, can be used as a "read out" or marker of the phenotype
of a particular cell.
[0075] For example, and not by way of limitation, genes, including
N-SMase, that are modulated in cells by treatment with an agent
which inhibits ApoCI activity (e.g., identified in a screening
assay as described herein) can be identified. Thus, to study the
effect of agents which modulate ApoCI activity on subjects
suffering from a atherosclerosis in, for example, a clinical trial,
cells can be isolated and analyzed for the levels of activity or
expression of N-SMase and other genes (e.g., caspases) implicated
in atherosclerosis (e.g., apoptosis associated genes). The levels
of gene expression (e.g., a gene expression pattern) can be
quantified by Northern blot analysis or RT-PCR, as described
herein, or alternatively by measuring the amount of protein
produced, by one of the methods described herein, or by measuring
the levels of activity of N-SMase or other genes. In this way, the
gene expression or activity pattern can serve as a marker,
indicative of the physiological response of the cells to the agent
which inhibits ApoCI activity. This response state may be
determined before, and at various points during treatment of the
individual with the agent which inhibits ApoCI activity.
[0076] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent which inhibits ApoCI activity (e.g., an antagonist,
peptidomimetic, protein, peptide, nucleic acid, or small molecule
identified by the screening assays described herein) including the
steps of (i) obtaining a pre-administration sample from a subject
prior to administration of the agent; (ii) detecting the level of
expression of an ApoCI protein in the pre-administration sample, or
the level of N-SMase activity in the pre-administration sample;
(iii) obtaining one or more post-administration samples from the
subject; (iv) detecting the level of expression or activity of the
ApoCI protein in the post-administration samples, or the level of
activity of N-SMase in the post-administration sample; (v)
comparing the level of expression or activity of the ApoCI protein,
or the level of N-SMase in the pre-administration sample with the
ApoCI protein, or the level of N-SMase in the post administration
sample or samples; and (vi) altering the administration of the
agent to the subject accordingly. For example, increased
administration of the agent may be desirable to decrease the
expression or activity of ApoCI to lower levels than detected,
i.e., to increase the effectiveness of the agent. Alternatively,
decreased administration of the agent may be desirable, e.g., to
decrease side effects. According to such an embodiment, ApoCI
expression or activity may be used as an indicator of the
effectiveness of an agent, even in the absence of an observable
phenotypic response.
III. Methods of Treatment of Subjects Suffering from or at Risk for
Atherosclerosis
[0077] The present invention provides for both prophylactic and
therapeutic methods of treating a subject, e.g., a human, at risk
of (or susceptible to) developing plaque rupure and/or myocardial
infarction. As used herein, "treatment" of a subject includes the
application or administration of a therapeutic agent to a subject,
or application or administration of a therapeutic agent to a cell
or tissue from a subject, who has a diseases or disorders has a
symptom of a disease or disorder, or is at risk of (or susceptible
to) a disease or disorder, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the disease or disorder, the symptom of the disease or disorder, or
the risk of (or susceptibility to) the disease or disorder. As used
herein, a "therapeutic agent" includes, but is not limited to,
small molecules, peptides, polypeptides, antibodies, ribozymes, and
antisense oligonucleotides.
[0078] A. Prophylactic Methods
[0079] In one aspect, the invention provides a method for
preventing in a subject, plaque rupture and/or myocardial
infarction by administering to the subject an agent which inhibits
ApoCI expression or ApoCI activity, e.g., modulation of N-SMase in
cells, e.g., vascular smooth muscle cells. Subjects at risk for
plaque rupture and/or myocardial infarction can be identified by,
for example, any or a combination of the diagnostic or prognostic
assays described herein. Administration of a prophylactic agent can
occur prior to the manifestation of symptoms characteristic of
aberrant ApoCI expression or activity, such that plaque rupture
and/or myocardial infarction (e.g., acute unstable angina pectoris)
is prevented or, alternatively, delayed in its progression.
Depending on the type of ApoCI aberrancy, for example, an ApoCI
antagonist agent can be used for treating the subject. The
appropriate agent can be determined based on screening assays
described herein.
[0080] B. Therapeutic Methods
[0081] Another aspect of the invention pertains to methods for
treating a subject suffering from atherosclerosis. These methods
involve administering to a subject an agent which inhibits ApoCI
expression or activity (e.g., an agent identified by a screening
assay described herein), or a combination of such agents.
[0082] Inhibition of ApoCI activity is desirable in situations in
which ApoCI is abnormally upregulated and/or in which decreased
ApoCI activity is likely to have a beneficial effect, e.g., a
decrease in N-SMase activity.
[0083] In another embodiment, the invention pertains to methods for
treating a subject suffering from a proliferative disorder, e.g., a
disorder in which induction of apoptosis by ApoCI activity may be
beneficial, for example, cancer or polycystic kidney disease.
Subjects suffering from such a disorder may benefit from
administration of an ApoCI agonist.
[0084] The agents which inhibit ApoCI activity can be administered
to a subject using pharmaceutical compositions suitable for such
administration. Such compositions typically comprise the agent
(e.g., nucleic acid molecule, protein, or antibody) and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0085] A pharmaceutical composition used in the therapeutic methods
of the invention is formulated to be compatible with its intended
route of administration. Examples of routes of administration
include parenteral, e.g., intravenous, intradermal, subcutaneous,
oral (e.g., inhalation), transdermal (topical), transmucosal, and
rectal administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include
the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0086] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, and sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0087] Sterile injectable solutions can be prepared by
incorporating the agent that inhibits ApoCI activity in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0088] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, strawberry,
cherry, mango, lemon, lime, raspberry, or orange flavoring.
[0089] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0090] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0091] The agents that inhibit ApoCI activity can also be prepared
in the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[0092] In one embodiment, the agents that inhibit ApoCI activity
are prepared with carriers that will protect the compound against
rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0093] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the agent that inhibits ApoCI activity
and the particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding such an agent for
the treatment of subjects.
[0094] In another embodiment, ApoCI antagonist or agonist compounds
may be administered by adsorbing the compounds to a biopolymer,
e.g., a biopolymer coated-stent, which may be delivered directly to
a site in the body, e.g., to a blood vessel containing a plaque
(e.g., a rupturing or ruptured plaque).
[0095] Toxicity and therapeutic efficacy of such agents can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
can be expressed as the ratio LD50/ED50. Agents which exhibit large
therapeutic indices are preferred. While agents that exhibit toxic
side effects may be used, care should be taken to design a delivery
system that targets such agents to the site of affected tissue in
order to minimize potential damage to uninfected cells and,
thereby, reduce side effects.
[0096] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such ApoCI inhibitors lies preferably within
a range of circulating concentrations that include the ED50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any agent used in the therapeutic
methods of the invention, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0097] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments.
[0098] In a preferred example, a subject is treated with antibody,
protein, or polypeptide in the range of between about 0.1 to 20
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. It will also be appreciated that the effective dosage of
antibody, protein, or polypeptide used for treatment may increase
or decrease over the course of a particular treatment. Changes in
dosage may result and become apparent from the results of
diagnostic assays as described herein.
[0099] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (I.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds. It is understood that appropriate doses of small
molecule agents depends upon a number of factors within the ken of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention. Exemplary doses
include milligram or microgram amounts of the small molecule per
kilogram of subject or sample weight (e.g., about 1 microgram per
kilogram to about 500 milligrams per kilogram, about 100 micrograms
per kilogram to about 5 milligrams per kilogram, or about 1
microgram per kilogram to about 50 micrograms per kilogram). It is
furthermore understood that appropriate doses of a small molecule
depend upon the potency of the small molecule with respect to the
expression or activity to be modulated. Such appropriate doses may
be determined using the assays described herein. When one or more
of these small molecules is to be administered to an animal (e.g.,
a human) in order to modulate expression or activity of a
polypeptide or nucleic acid of the invention, a physician,
veterinarian, or researcher may, for example, prescribe a
relatively low dose at first, subsequently increasing the dose
until an appropriate response is obtained. In addition, it is
understood that the specific dose level for any particular animal
subject will depend upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, gender, and diet of the subject, the time of
administration, the route of administration, the rate of excretion,
any drug combination, and the degree of expression or activity to
be modulated.
[0100] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0101] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
alpha-interferon, beta-interferon, nerve growth factor, platelet
derived growth factor, tissue plasminogen activator; or biological
response modifiers such as, for example, lymphokines, interleukin-1
("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophage colony stimulating factor ("GM-CSF"),
granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
[0102] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies for Immunotargeting of Drugs in Cancer Therapy", in
Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
for Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological and Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, and Future Prospective of the Therapeutic Use
of Radiolabeled Antibody in Cancer Therapy", in Monoclonal
Antibodies for Cancer Detection and Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al. (1982) "The
Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58. Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
[0103] The nucleic acid molecules used in the methods of the
invention can be inserted into vectors and used as gene therapy
vectors. Gene therapy vectors can be delivered to a subject by, for
example, intravenous injection, local administration (see U.S. Pat.
No. 5,328,470) or by stereotactic injection (see, e.g., Chen et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
[0104] In a preferred embodiment, an ApoCI inhibitor may be
adminstered to a subject in conjuction with any known treatment
and/or agent useful for treating atherosclerosis and/or myocardial
infarction.
IV. Recombinant Expression Vectors and Host Cells Used in the
Methods of the Invention
[0105] The methods of the invention (e.g., the screening assays
described herein) include the use of vectors, preferably expression
vectors, containing nucleic acid molecules encoding an ApoCI
protein (or a portion thereof), as well as ApoCI target molecules
(e.g., N-SMase), or portions thereof. As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One type of
vector is a "plasmid", which refers to a circular double stranded
DNA loop into which additional DNA segments can be ligated. Another
type of vector is a viral vector, wherein additional DNA segments
can be ligated into the viral genome. Certain vectors are capable
of autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0106] The recombinant expression vectors to be used in the methods
of the invention comprise a nucleic acid of the invention in a form
suitable for expression of the nucleic acid in a host cell, which
means that the recombinant expression vectors include one or more
regulatory sequences, selected on the basis of the host cells to be
used for expression, which is operatively linked to the nucleic
acid sequence to be expressed. Within a recombinant expression
vector, "operably linked" is intended to mean that the nucleotide
sequence of interest is linked to the regulatory sequence(s) in a
manner which allows for expression of the nucleotide sequence
(e.g., in an in vitro transcription/translation system or in a host
cell when the vector is introduced into the host cell). The term
"regulatory sequence" is intended to include promoters, enhancers
and other expression control elements (e.g., polyadenylation
signals). Such regulatory sequences are described, for example, in
Goeddel (1990) Methods Enzymol. 185:3-7. Regulatory sequences
include those which direct constitutive expression of a nucleotide
sequence in many types of host cells and those which direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, and the like. The expression vectors of the invention can
be introduced into host cells to thereby produce proteins or
peptides, including fusion proteins or peptides, encoded by nucleic
acids as described herein (e.g., ApoCI proteins, ApoCI target
molecules, mutant forms of ApoCI proteins and/or ApoCI target
molecules, fusion proteins, and the like).
[0107] The recombinant expression vectors to be used in the methods
of the invention can be designed for expression of ApoCI proteins
in prokaryotic or eukaryotic cells. For example, ApoCI proteins can
be expressed in bacterial cells such as E. coli, insect cells
(using baculovirus expression vectors), yeast cells, or mammalian
cells. Suitable host cells are discussed further in Goeddel (1990)
supra. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase. Expression of proteins in
prokaryotes is most often carried out in E. coli with vectors
containing constitutive or inducible promoters directing the
expression of either fusion or non-fusion proteins. Fusion vectors
add a number of amino acids to a protein encoded therein, usually
to the amino terminus of the recombinant protein. Such fusion
vectors typically serve three purposes: 1) to increase expression
of recombinant protein; 2) to increase the solubility of the
recombinant protein; and 3) to aid in the purification of the
recombinant protein by acting as a ligand in affinity purification.
Often, in fusion expression vectors, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and
Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs,
Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
[0108] Purified fusion proteins can be utilized in ApoCI activity
assays (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for ApoCI
proteins. In a preferred embodiment, an ApoCI fusion protein
expressed in a retroviral expression vector of the present
invention can be utilized to infect bone marrow cells which are
subsequently transplanted into irradiated recipients. The pathology
of the subject recipient is then examined after sufficient time has
passed (e.g., six weeks).
[0109] In another embodiment, a nucleic acid of the invention is
expressed in mammalian cells using a mammalian expression vector.
Examples of mammalian expression vectors include pCDM8 (Seed, B.
(1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.
6:187-195). When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40. For other
suitable expression systems for both prokaryotic and eukaryotic
cells see chapters 16 and 17 of Sambrook, J. et al., Molecular
Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0110] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
[0111] The methods of the invention may further use a recombinant
expression vector comprising a DNA molecule of the invention cloned
into the expression vector in an antisense orientation. That is,
the DNA molecule is operatively linked to a regulatory sequence in
a manner which allows for expression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to ApoCI mRNA.
Regulatory sequences operatively linked to a nucleic acid cloned in
the antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific,
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid, or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes, see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0112] Another aspect of the invention pertains to the use of host
cells into which an ApoCI or ApoCI target molecule (such as
N-SMase) nucleic acid molecule of the invention is introduced,
e.g., an ApoCI or ApoCI target molecule nucleic acid molecule
within a recombinant expression vector or an ApoCI or ApoCI target
molecule nucleic acid molecule containing sequences which allow it
to homologously recombine into a specific site of the host cell's
genome. The terms "host cell" and "recombinant host cell" are used
interchangeably herein. It is understood that such terms refer not
only to the particular subject cell but to the progeny or potential
progeny of such a cell. Because certain modifications may occur in
succeeding generations due to either mutation or environmental
influences, such progeny may not, in fact, be identical to the
parent cell, but are still included within the scope of the term as
used herein.
[0113] A host cell can be any prokaryotic or eukaryotic cell. For
example, an ApoCI protein or ApoCI target molecule can be expressed
in bacterial cells such as E. coli, insect cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS
cells). Other suitable host cells are known to those skilled in the
art.
[0114] Vector DNA can be introduced-into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual. 2.sup.nd. ed., Cold Spring Harbor Laboratory,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989), and other laboratory manuals.
[0115] A host cell used in the methods of the invention, such as a
prokaryotic or eukaryotic host cell in culture, can be used to
produce (i.e., express) an ApoCI protein or ApoCI target molecule.
Accordingly, the invention further provides methods for producing
an ApoCI protein or ApoCI target molecule using the host cells of
the invention. In one embodiment, the method comprises culturing
the host cell of the invention (into which a recombinant expression
vector encoding an ApoCI protein or ApoCI target molecule has been
introduced) in a suitable medium such that an ApoCI protein or
ApoCI target molecule is produced. In another embodiment, the
method further comprises isolating an ApoCI protein or ApoCI target
molecule from the medium or the host cell.
V. Isolated Nucleic Acid Molecules Used in the Methods of the
Invention
[0116] In a preferred embodiment, the ApoCI polypeptide sequence is
set forth as TPDVSSALDKLKEFGNTLEDKARELIBRINKQSELSAKMREWFSETFQKVKEL
KIDS (SEQ ID NO:1). The ApoCI sequence of SEQ ID NO:1 is a mature
polypeptide sequence, i.e., a polypeptide sequence in which the
signal sequence has been cleaved off. The cDNA sequence of the
isolated human ApoCI gene and the predicted amino acid sequence of
the human ApoCI polypeptide are also set forth in GenBank Accession
Nos. NM.sub.--001645 and NP.sub.--001636, respectively. The
predicted ApoCI polypeptide disclosed in GenBank Accession No.
NP.sub.--001636 contains the signal sequence (the signal sequence
comprises the first 26 amino acid residues of precursor protein).
The contents of all of the above-referenced GenBank records are
herein incorporated by reference.
[0117] The methods of the invention also use isolated nucleic acid
molecules that encode ApoCI target molecules, i.e., proteins which
ApoCI interacts with and/or modulates the activity and/or
expression of. In a preferred embodiment, an ApoCI target molecule
is a protein such as ABCA-1 or SR-BI. Nucleic acid and polypeptide
sequences for any of these types of transporters are well-known in
the art.
[0118] The methods of the invention include the use of isolated
nucleic acid molecules that encode ApoCI proteins and ApoCI target
molecules or biologically active portions thereof, as well as
nucleic acid fragments sufficient for use as hybridization probes
to identify ApoCI-encoding and ApoCI target molecule-encoding
nucleic acid molecules (e.g., ApoCI and ApoCI target molecule mRNA)
and fragments for use as PCR primers for the amplification or
mutation of ApoCI and ApoCI target molecule nucleic acid molecules.
As used herein, the term "nucleic acid molecule" is intended to
include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules
(e.g., mRNA) and analogs of the DNA or RNA generated using
nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0119] A nucleic acid molecule used in the methods of the present
invention, e.g., an ApoCI or ApoCI target molecule nucleic acid
molecule, or a portion thereof, can be isolated using standard
molecular biology techniques and the sequence information provided
herein. Using all or portion of the nucleic acid sequence of an
ApoCI or ApoCI target molecule nucleic acid molecule as a
hybridization probe, an ApoCI or ApoCI target molecule nucleic acid
molecules can be isolated using standard hybridization and cloning
techniques (e.g., as described in Sambrook, J. et al., Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989).
[0120] Moreover, a nucleic acid molecule encompassing all or a
portion of an ApoCI or ApoCI target molecule nucleic acid molecule
can be isolated by the polymerase chain reaction (PCR) using
synthetic oligonucleotide primers designed based upon the sequence
of an ApoCI or ApoCI target molecule nucleic acid molecule (e.g.,
the nucleic acid sequence of SEQ ID NO:1).
[0121] A nucleic acid used in the methods of the invention can be
amplified using cDNA, mRNA or, alternatively, genomic DNA as a
template and appropriate oligonucleotide primers according to
standard PCR amplification techniques. Furthermore,
oligonucleotides corresponding to ApoCI or ApoCI target molecule
nucleotide sequences can be prepared by standard synthetic
techniques, e.g., using an automated DNA synthesizer.
[0122] Moreover, the nucleic acid molecules used in the methods of
the invention can comprise only a portion of the nucleic acid
sequence of an ApoCI or ApoCI target molecule nucleic acid
molecule, for example, a fragment which can be used as a probe or
primer or a fragment encoding a portion of a an ApoCI protein or
ApoCI target molecule, e.g., a biologically active portion of an
ApoCI protein or ApoCI target molecule. The probe/primer typically
comprises substantially purified oligonucleotide.
[0123] In preferred embodiments, the probe further comprises a
label group attached thereto, e.g., the label group can be a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissue which misexpress an ApoCI
protein or ApoCI target molecule, such as by measuring a level of
an ApoCI-encoding nucleic acid in a sample of cells from a subject
e.g., detecting ApoCI or ApoCI target molecule mRNA levels or
determining whether a genomic ApoCI or ApoCI target molecule gene
has been mutated or deleted.
[0124] The methods of the invention further include the use of
allelic variants of human ApoCI, e.g., functional and
non-functional allelic variants. Functional allelic variants are
naturally occurring amino acid sequence variants of the human ApoCI
protein that maintain an ApoCI activity. Functional allelic
variants will typically contain only conservative substitution of
one or more amino acids of SEQ ID NO:1, or substitution, deletion
or insertion of non-critical residues in non-critical regions of
the protein. Non-functional allelic variants are naturally
occurring amino acid sequence variants of the human ApoCI protein
that do not have an ApoCI activity. Non-functional allelic variants
will typically contain a non-conservative substitution, deletion,
or insertion or premature truncation of the amino acid sequence of
SEQ ID NO:1, or a substitution, insertion or deletion in critical
residues or critical regions of the protein.
[0125] The methods of the present invention may further use
orthologues of the ApoCI protein. Orthologues of the ApoCI protein
are proteins that are isolated from other organisms and possess the
same ApoCI activity.
[0126] The methods of the present invention further include the use
of nucleic acid molecules comprising in which a mutation has been
introduced. The mutation may lead to amino acid substitutions at
"non-essential" amino acid residues or at "essential" amino acid
residues. A "non-essential" amino acid residue is a residue that
can be altered from the wild-type sequence of ApoCI (e.g., the
sequence of SEQ ID NO:1) without altering the biological activity,
whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are
conserved among the ApoCI proteins from different organisms are not
likely to be amenable to alteration.
[0127] Mutations can be introduced into SEQ ID NO:1 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., asparagine, glutamine,
serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
glycine, alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in an ApoCI protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of an ApoCI coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for ApoCI biological activity to identify
mutants that retain activity. Following mutagenesis of SEQ ID NO:1,
the protein can be expressed recombinantly and the activity of the
protein can be determined using an assay described herein.
[0128] In other embodiments, the oligonucleotide used in the
methods of the invention may include other appended groups such as
peptides (e.g., for targeting host cell receptors in vivo), or
agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;
Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No. WO 88/09810) or the blood-brain bather (see, e.g.,
PCT Publication No. WO 89/10134). In addition, oligonucleotides can
be modified with hybridization-triggered cleavage agents (See,
e.g., Krol et al. (1988) Biotechniques 6:958-976) or intercalating
agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end,
the oligonucleotide may be conjugated to another molecule (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
VI. Isolated ApoCI Proteins and ApoCI Target Molecules Used in the
Methods of the Invention
[0129] The methods of the invention include the use of isolated
ApoCI proteins and ApoCI target molecules, and biologically active
portions thereof, as well as polypeptide fragments suitable for use
as immunogens to raise anti-ApoCI and anti-ApoCI target molecule
antibodies. In one embodiment, native ApoCI and ApoCI target
molecule proteins can be isolated from biological sources by an
appropriate purification scheme using standard protein purification
techniques. In another embodiment, ApoCI proteins and ApoCI target
molecules are produced by recombinant DNA techniques. Alternative
to recombinant expression, an ApoCI protein or polypeptide or ApoCI
target molecule can be synthesized chemically using standard
peptide synthesis techniques.
[0130] As used herein, a "biologically active portion" of an ApoCI
protein includes a fragment of an ApoCI protein having an ApoCI
activity. Biologically active portions of an ApoCI protein include
peptides comprising amino acid sequences sufficiently identical to
or derived from the amino acid sequence of the ApoCI protein, e.g.,
the amino acid sequence shown in SEQ ID NO:1, which include fewer
amino acids than the full length ApoCI proteins, and exhibit at
least one activity of an ApoCI protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the ApoCI protein. A biologically active portion of an
ApoCI protein can be a polypeptide which is, for example, 5, 8, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more
amino acids in length. Biologically active portions of an ApoCI
protein or ApoCI target molecule can be used as targets for
developing agents which modulate an ApoCI activity.
[0131] In a preferred embodiment, the ApoCI protein used in the
methods of the invention has an amino acid sequence shown in SEQ ID
NO:1. In other embodiments, the ApoCI protein is substantially
identical to SEQ ID NO:1, and retains the functional activity of
the protein of SEQ ID NO:1, yet differs in amino acid sequence due
to natural allelic variation or mutagenesis, as described in detail
in subsection V above. Accordingly, in another embodiment, the
ApoCI protein used in the methods of the invention is a protein
which comprises an amino acid sequence at least about 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more
identical to SEQ ID NO:1.
[0132] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-identical
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to an
amino acid sequence of having 100 amino acid residues, at least 30,
preferably at least 40, more preferably at least 50, even more
preferably at least 60, and even more preferably at least 70, 80,
90 or more amino acid residues are aligned). The amino acid
residues or nucleotides at corresponding amino acid positions or
nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0133] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package (available online through the Genetics Computer
Group), using either a Blosum 62 matrix or a PAM250 matrix, and a
gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,
2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent
identity between two nucleotide sequences is determined using the
GAP program in the GCG software package (available online through
the Genetics Computer Group), using a NWSgapdna.CMP matrix and a
gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3,
4, 5, or 6. In another embodiment, the percent identity between two
amino acid or nucleotide sequences is determined using the
algorithm of Meyers, E. and Miller, W. (Comput. Appl. Biosci.
4:11-17 (1988)) which has been incorporated into the ALIGN program
(version 2.0 or 2.0U), using a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4.
[0134] The methods of the invention may also use ApoCI and ApoCI
target molecule chimeric or fusion proteins. As used herein, an
ApoCI "chimeric protein" or "fusion protein" comprises an ApoCI
polypeptide operatively linked to a non-ApoCI polypeptide. A "ApoCI
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to an ApoCI molecule, whereas a "non-ApoCI
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
the ApoCI protein, e.g., a protein which is different from the
ApoCI protein and which is derived from the same or a different
organism. Within an ApoCI fusion protein the ApoCI polypeptide can
correspond to all or a portion of an ApoCI protein. In a preferred
embodiment, an ApoCI fusion protein comprises at least one
biologically active portion of an ApoCI protein. In another
preferred embodiment, an ApoCI fusion protein comprises at least
two biologically active portions of an ApoCI protein. Within the
fusion protein, the term "operatively linked" is intended to
indicate that the ApoCI polypeptide and the non-ApoCI polypeptide
are fused in-frame to each other. The non-ApoCI polypeptide can be
fused to the N-terminus or C-terminus of the ApoCI polypeptide.
[0135] For example, in one embodiment, the fusion protein is a
GST-ApoCI fusion protein in which the ApoCI sequences are fused to
the C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant ApoCI.
[0136] In another embodiment, this fusion protein is a protein
containing a heterologous signal sequence at its N-terminus In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of a protein can be increased through use of a
heterologous signal sequence.
[0137] Moreover, the ApoCI-fusion proteins used in the methods of
the invention can be used as immunogens to produce anti-ApoCI
antibodies in a subject, to purify ApoCI ligands and in screening
assays to identify molecules which inhibit the interaction of ApoCI
with an ApoCI target molecule.
[0138] Preferably, a chimeric or fusion protein used in the methods
of the invention is produced by standard recombinant DNA
techniques. For example, DNA fragments coding for the different
polypeptide sequences are ligated together in-frame in accordance
with conventional techniques, for example by employing blunt-ended
or stagger-ended termini for ligation, restriction enzyme digestion
to provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al., John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). An ApoCI or ApoCI target molecule-encoding
nucleic acid can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the ApoCI protein or ApoCI
target molecule.
[0139] The present invention also pertains to the use of variants
of the ApoCI proteins or ApoCI target molecules which function as
ApoCI antagonists. Variants of the ApoCI proteins or ApoCI target
molecules can be generated by mutagenesis, e.g., discrete point
mutation or truncation of an ApoCI protein or ApoCI target
molecule. An agonist of the ApoCI proteins or ApoCI target
molecules can retain substantially the same, or a subset, of the
biological activities of the naturally occurring form of the
protein. An antagonist of a protein can inhibit one or more of the
activities of the naturally occurring form of the protein by, for
example, competitively modulating an ApoCI-mediated activity of an
ApoCI protein. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. In one embodiment,
treatment of a subject with a variant having a subset of the
biological activities of the naturally occurring form of the
protein has fewer side effects in a subject relative to treatment
with the naturally occurring forms of the ApoCI protein or ApoCI
target molecules.
[0140] In one embodiment, variants of an ApoCI protein which
function as ApoCI antagonists can be identified by screening
combinatorial libraries of mutants, e.g., truncation mutants, of an
ApoCI protein for ApoCI protein antagonist activity. In one
embodiment, a variegated library of ApoCI variants is generated by
combinatorial mutagenesis at the nucleic acid level and is encoded
by a variegated gene library. A variegated library of ApoCI
variants can be produced by, for example, enzymatically ligating a
mixture of synthetic oligonucleotides into gene sequences such that
a degenerate set of potential ApoCI sequences is expressible as
individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display) containing the set of
ApoCI sequences therein. There are a variety of methods which can
be used to produce libraries of potential ApoCI variants from a
degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an
appropriate expression vector. Use of a degenerate set of genes
allows for the provision, in one mixture, of all of the sequences
encoding the desired set of potential ApoCI sequences. Methods for
synthesizing degenerate oligonucleotides are known in the art (see,
e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984)
Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056;
Ike et al. (1983) Nucleic Acids Res. 11:477).
[0141] In addition, libraries of fragments of an ApoCI protein
coding sequence can be used to generate a variegated population of
ApoCI fragments for screening and subsequent selection of variants
of an ApoCI protein. In one embodiment, a library of coding
sequence fragments can be generated by treating a double stranded
PCR fragment of an ApoCI coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes
N-terminal, C-terminal and internal fragments of various sizes of
the ApoCI protein.
[0142] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of ApoCI proteins. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify ApoCI variants (Arkin and Youvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al.
(1993) Prot. Eng. 6(3):327-331).
[0143] The methods of the present invention further include the use
of anti-ApoCI antibodies and anti-ApoCI target molecule antibodies.
An isolated ApoCI protein or target molecule, or a portion or
fragment thereof, can be used as an immunogen to generate
antibodies that bind ApoCI using standard techniques for polyclonal
and monoclonal antibody preparation. A full-length protein can be
used or, alternatively, antigenic peptide fragments of the protein
can be used as immunogens. The antigenic peptide of ApoCI comprises
at least 8 amino acid residues of the amino acid sequence shown in
SEQ ID NO:1 and encompasses an epitope of ApoCI such that an
antibody raised against the peptide forms a specific immune complex
with the ApoCI protein. Preferably, the antigenic peptide comprises
at least 10 amino acid residues, more preferably at least 15 amino
acid residues, even more preferably at least 20 amino acid
residues, and most preferably at least 30 amino acid residues.
[0144] Preferred epitopes encompassed by the antigenic peptide are
regions of ApoCI that are located on the surface of the protein,
e.g., hydrophilic regions, as well as regions with high
antigenicity.
[0145] An ApoCI immunogen is typically used to prepare antibodies
by immunizing a suitable subject (e.g., rabbit, goat, mouse, or
other mammal) with the immunogen.
[0146] An appropriate immunogenic preparation can contain, for
example, recombinantly expressed ApoCI protein or a chemically
synthesized ApoCI polypeptide. The preparation can further include
an adjuvant, such as Freund's complete or incomplete adjuvant, or
similar immunostimulatory agent Immunization of a suitable subject
with an immunogenic ApoCI preparation induces a polyclonal
anti-ApoCI antibody response.
[0147] The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
which specifically binds (immunoreacts with) an antigen, such as an
ApoCI. Examples of immunologically active portions of
immunoglobulin molecules include F(ab) and F(ab').sub.2 fragments
which can be generated by treating the antibody with an enzyme such
as pepsin. The invention provides polyclonal and monoclonal
antibodies that bind ApoCI molecules. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope of ApoCI. A monoclonal antibody composition thus
typically displays a single binding affinity for a particular ApoCI
protein with which it immunoreacts.
[0148] Polyclonal anti-ApoCI antibodies can be prepared as
described above by immunizing a suitable subject with an ApoCI
immunogen. The anti-ApoCI antibody titer in the immunized subject
can be monitored over time by standard techniques, such as with an
enzyme linked immunosorbent assay (ELISA) using immobilized ApoCI.
If desired, the antibody molecules directed against ApoCI can be
isolated from the mammal (e.g., from the blood) and further
purified by well known techniques, such as protein A chromatography
to obtain the IgG fraction. At an appropriate time after
immunization, e.g., when the anti-ApoCI antibody titers are
highest, antibody-producing cells can be obtained from the subject
and used to prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by Kohler and
Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981)
J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.
255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA
76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more recent human B cell hybridoma technique (Kozbor et al. (1983)
Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
monoclonal antibody hybridomas is well known (see generally
Kenneth, R. H. in Monoclonal Antibodies: A New Dimension in
Biological Analyses, Plenum Publishing Corp., New York, N.Y.
(1980); Lerner, E. A. (1981) Yale J. Biol. Med. 54:387-402; Gefter,
M. L. et al. (1977) Somat. Cell Genet. 3:231-36). Briefly, an
immortal cell line (typically a myeloma) is fused to lymphocytes
(typically splenocytes) from a mammal immunized with an ApoCI
immunogen as described above, and the culture supernatants of the
resulting hybridoma cells are screened to identify a hybridoma
producing a monoclonal antibody that binds ApoCI.
[0149] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-ApoCI monoclonal antibody (see, e.g.,
Galfre, G. et al. (1977) Nature 266:55052; Gefter et al. (1977)
supra; Lerner (1981) supra; and Kenneth (1980) supra). Moreover,
the ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from ATCC. Typically, HAT-sensitive mouse myeloma cells
are fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind ApoCI, e.g., using a standard
ELISA assay.
[0150] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-ApoCI antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with ApoCI to
thereby isolate immunoglobulin library members that bind ApoCI.
Kits for generating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene
SurfZAP.TM. Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in
generating and screening antibody display library can be found in,
for example, Ladner et al., U.S. Pat. No. 5,223,409; Kang et al.,
PCT International Publication No. WO 92/18619; Dower et al., PCT
International Publication No. WO 91/17271; Winter et al., PCT
International Publication No. WO 92/20791; Markland et al., PCT
International Publication No. WO 92/15679; Breitling et al., PCT
International Publication No. WO 93/01288; McCafferty et al, PCT
International Publication No. WO 92/01047; Garrard et al., PCT
International Publication No. WO 92/09690; Ladner et al., PCT
International Publication No. WO 90/02809; Fuchs et al. (1991)
Biotechnology (NY) 9:1369-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992)
J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature
352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA
89:3576-3580; Garrard et al. (1991) Biotechnology (NY) 9:1373-1377;
Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et
al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty
et al. (1990) Nature 348:552-554.
[0151] Additionally, recombinant anti-ApoCI antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the methods of
the invention. Such chimeric and humanized monoclonal antibodies
can be produced by recombinant DNA techniques known in the art, for
example using methods described in Robinson et al., International
Application No. PCT/US86/02269; Akira et al., European Patent
Application No. 184,187; Taniguchi, M., European Patent Application
No. 171,496; Morrison et al., European Patent Application No.
173,494; Neuberger et al., PCT International Publication No. WO
86/01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,
European Patent Application No. 125,023; Better et al. (1988)
Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA
84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et
al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.
(1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559;
Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986)
BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.
(1986) Nature 321:552-525; Verhoeyen et al (1988) Science 239:1534;
and Beidler et al. (1988) J. Immunol. 141:4053-4060.
[0152] An anti-ApoCI antibody can be used to detect ApoCI protein
(e.g., in a biological sample, cellular lysate or cell supernatant)
in order to evaluate the abundance and pattern of expression of the
ApoCI protein. Anti-ApoCI antibodies can be used diagnostically to
monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling (i.e.,
physically linking) the antibody to a detectable substance.
Examples of detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, .beta.-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin, and examples
of suitable radioactive material include 125I, 131I, 35S or 3H.
[0153] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the sequence listing and
the figures, are incorporated herein by reference.
EXAMPLES
Materials and Methods
[0154] The following materials and methods were used in Examples
1-7.
Isolation of Human ApoCI-Enriched HDL and ApoCI Poor HDL
[0155] Two plasma pools (A and B) were prepared from umbilical cord
blood. Pool A and pool B consisted of plasma from normal infants
with increased or decreased amounts of large HDL particles,
respectively. The concentration of ApoCI in pool A was 7.2 mg/dL,
and was 3.9 mg/dL in pool B. The ApoB-containing lipoproteins
(VLDL, IDL, LDL, and Lp (a)) were then precipitated with heparin
manganese (Warnick, G. R., and Albers J J. (1978) J. Lipid Res. 19,
65-76). No ApoB was detected in the heparin manganese supernatant
(HMS). All of the ApoCI remained with the HDL in the HMS. The
supernatants were then dialyzed against the running buffer (0.5 M
Tris HCl, pH 7.4 containing 0.15 M NaCl, and 1.5 mg/ml EDTA), and
aliquots applied to an antiapoA-II immunosorber. The preparation of
the anti-apoA-II immunosorber and the fractionation of the Lp-A-I
and Lp-AI:A-II particles have been previously described (Bekaert,
E. D., Alaupovic, P., Knight-Gibson, C., Blackett, P.,
Ayrault-Jarrier, M. (1991). Pediatr. Res. 29, 315-321).
[0156] Briefly, the HMS was applied to the antiA-II immunosorber
and incubated overnight. The unretained plasma proteins and the
Lp-A-I particles were eluted with the running buffer. The retained
Lp-A-I:A-II were eluted with running buffer. The sodium thiocyanate
was removed as described (Bekaert, E. D., Alaupovic, P.,
Knight-Gibson, C., Blackett, P., Ayrault-Jarrier, M. (1991).
Pediatr. Res. 29, 315-321), and the concentrated retained fraction
consisting of Lp-A-I:A-II particles was applied in the running
buffer to an anti-ApoCI-immunosorber, which was prepared with
affinity purified ApoCI antibodies according to the procedure used
for the preparation of the antiA-II immunosorber. The ApoA-II
retained-ApoCI unretained fraction was eluted with the running
buffer and the apoA-II retained, ApoCI retained fraction was eluted
with 4.5 M sodium thiocyanate, the thiocyanate removed, and the
eluate dialyzed against the starting buffer. The apoA-II retained,
ApoCI retained particles from pool A, and the apoA-II retained,
ApoCI unretained particles from pool B constituted the
ApoCI-enriched HDL and the ApoCI-poor HDL, respectively (Table 1).
Aliquots of both fractions were taken for determining the lipid and
apolipoprotein composition (see also below) (Table 1).
TABLE-US-00001 TABLE I Lipid and Apolipoprotein Composition of
ApoCI-enriched HDL, ApoCI-poor HDL and ApoCI-enriched HMS. ApoCI-
enriched ApoCI-enriched ApoCI-poor Heparin-Mn ++ HDL HDL
Supernatant Lipids (%) Cholesteryl Esters 9.8 16.2 16.3 Free
Cholesterol 1.8 2.2 3.9 Triglycerides 2.6 1.7 0.9 Phospholipids
33.4 30.4 37.5 Total Apolipoproteins 52.2 49.5 41A.sup. 100.0 100.0
100.0 Apolipoproteins (%) Apo AI 47.3 74.3 55.5 Apo AII 10.2 19.1
19.6 Apo CI 6.4 ND 5.9 Apo CII 4.2 ND 2.2 Apo CIII 14.0 0.3 3.5 Apo
D 8.2 3.2 3.9 Apo E 9.5 3.0 7.5 100.0 100.0 100.0 ApoCI-enriched
HDL and ApoCI-poor HDL were isolated from the heparin manganese
supernatants of human plasma from umbilical cord blood by
immunoaffinity chromatography as described in Methods.
ApoCI-enriched HDL is the apoA-II-retained, ApoCI-retained fraction
of LpA-I:A-II particles. ApoC-poor HDL is the apoA-II-retained,
ApoCI-unretained fraction of LpAI:A-II particles.
[0157] These two fractions were then stored frozen at -80.degree.
C. until used for cell experiments. The fractions were thawed at
that time, dialyzed against the culture medium (see below) and
aliquots added to the cell culture dishes.
ApoCI-Enriched Heparin Manganese Supernatant
[0158] In several cell experiments, ApoCI-enriched HMS was used.
This fraction was prepared from a separate plasma pool from normal
infants with increased large HDL. After the apoB-containing
lipoproteins were precipitated with heparin manganese (Warnick, G.
R., and Albers J J. (1978) J. Lipid Res. 19, 65-76), the
supernatant was dialyzed against 0.5 M Tris HCl, pH 7.4 containing
0.15 M NaCl, and 1.5 mg/ml EDTA. Aliquots were taken for
determining the lipid and apolipoprotein composition (see also
below) (Table 1). No apoB was detected in the supernatant. The
supernatant was frozen at -80.degree. C. until used for cell
experiments. The supernatant was thawed at that time, dialyzed
against the culture medium (see below) and aliquots added to the
cell culture dishes.
Lipid and Apolipoprotein Composition
[0159] Neutral lipids (cholesteryl esters, free cholesterol and
triglycerides) were determined by gas chromatography in
ApoCI-enriched HDL, ApoCI-poor HDL and HMS by a modified procedure
(Lee, D. M. (1999) Atherosclerosis 146, 221-235) of the method of
Kuksis et al (Kuksis, A., Myher, J. J., Marai, L., Geher, K. (1975)
J. Chromatogr. Sci. 13, 423-430). Phospholipids were measured by
the method of Gerlach and Deuticke (Gerlach, E., Deuticke, B.
(1963) Biochem. Z. 337, 477-479). The quantitative determination of
apolipoproteins was performed by electroimmunoassay for
apolipoprotein A-I and apolipoprotein A-II (Curry, M. D.,
Alaupovic, P., Suenram, C. A. (1976) Clin. Chem. 22, 315-322),
apolipoprotein B (Curry, M. D., Gustafson, A., Alaupovic, P.,
McConathy, W. J. (1978) Clin. Chem. 24, 280-286), ApoCI and
apolipoprotein C-II (Curry, M. D., McConathy, W. J., Fesmire, J.
D., Alaupovic, P. (1981) Clin. Chem. 27, 543-548), ApoCIII (Curry,
M. D., McConathy, W. J., Fesmire, J. D., Alaupovic, P. (1980)
Biochim Biophys. Acta. 617, 503-513), apolipoprotein D (Curry, M.
D., McConathy, W. J., Alaupovic, P. (1977) Biochim Biophys. Acta.
491, 232-241), and apolipoprotein E (Curry, M. D., McConathy, W.
J., Alaupovic, P., Ledford, J. H., Popovic, M. (1976) Biochim
Biophys. Acta. 439, 413-425).
Cultured Cells
[0160] Human AoSMC that were negative for mycoplasma, hepatitis B
and C and HIV-1 were purchased from Clonetics (San Diego, Calif.)
and cultured in Dulbecco's minimal essential medium (Invitrogen,
Carlsbad, Calif.) with 10% fetal bovine serum (Hyclone, Salt Lake
City, Utah), 100-units/ml penicillin, 100-.mu.g/ml streptomycin to
confluence. The cells were incubated in serum-free medium for 30
min prior to initiating the experiments. Cells were then washed and
harvested in PBS, and centrifuged at 1500 rpm for 5 min at
4.degree. C. Protease inhibitors were then added and the pellets
stored frozen at -30.degree. C. until use.
Purified human ApoCI and ApoCIII
[0161] Human ApoCI and ApoCIII were purchased from Academy
Bio-Medical Company (Houston, Tex.) and were >99% homogenous by
SDS gel-electrophoresis and were lipopolysaccharide-free.
Assessment of Apoptosis
[0162] Fluorescence Microscope-Quantitative Assay of Apoptotic
Death
[0163] 1.times.10.sup.3 cells were grown on sterilized glass cover
slips in 6-well trays and treated with ApoCI (2.5 .mu.g/ml medium),
ApoCIII (2.5 .mu.g/ml medium), tumor necrosis factor (TNF)-.alpha.
(30 ng/ml), ApoCI-enriched HDL (1 .mu.g apoA-I/ml medium) and
ApoCI-poor HDL (5 .mu.g apoA-I/ml medium). After 24 h incubation,
the medium was removed, cells fixed with ethanol:acetic acid 70:30
(v/v) and stored at 4.degree. C. Next, the fixing solution was
removed and cells stored frozen at -30.degree. C. Cover slips were
mounted on the microscope slides and stained with DAPI reagent
(4,6-diamino-2-phenylindol, dihydrochloride). The stained nuclei
were visualized by fluorescence microscopy (Zeiss Axiovert 25).
Apoptotic cells were determined after counting -500 cells in a
double blind fashion.
DNA Laddering Assay
[0164] Cells were treated with either TNF-.alpha. (30 ng/ml), ApoCI
or ApoCIII (2.5 .mu.g/ml) for 6 h. The cells were then washed twice
with minimal essential medium and harvested with buffer (10 mM
HEPES, pH 7.4. 5 mM EDTA, 0.25 mM EGTA, 50 mM NaF, 5 mM
.beta.-mercaptoethanol, 0.35 M sucrose, 0.1% Nonidet and protease
inhibitors, 1 mM phenylmethylsulfonyl fluoride, 2 .mu.g/ml
leupeptin and 5 .mu.g/ml pepstatin), and pelleted. Genomic DNA was
prepared employing standard protocol. Genomic DNA was subjected to
electrophoresis, stained with ethidium bromide and the gel was
photographed. The gel was calibrated using DNA fragments of known
molecular weight.
Neutral Sphingomyelinase Assay
[0165] After stimulation with ApoCI, ApoCI-enriched HDL and
ApoCI-poor HDL, the cells were washed with 5 ml of PBS and
harvested. The pellets were stored frozen at -70.degree. C. and
resuspended in 0.5 ml of buffer (100 mM Tris-HCl pH 7.4, 0.1%
Triton X-100, 1 mM EDTA and protease inhibitors). The cell
suspension was sonicated 3 times (3-s bursts) using a probe
sonicator and centrifuged at 500.times.g at 4.degree. C. for 5 mM
The supernatant was used as the enzyme source. 100 .mu.g of protein
was assayed for neutral sphingomyelinase activity in a buffer
consisting of 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 0.1 mg of
bovine serum albumin, 5 mM MgCl.sub.2 and 50 .mu.mol of
[.sup.14C]sphingomyelin (specific activity 50 mCi/mmol, from
American Radiolabled Chemicals (St. Louis, Mo.). The samples were
incubated at 37.degree. C. for 2 h and terminated with the addition
of 1 ml of 10% trichloroacetic acid. The precipitate was pelleted
(1000.times.g at 4.degree. C. for 30 mM) and 1 ml of the
supernatant was extracted with 1 ml of anhydrous diethyl ether.
About 0.5 ml of the aqueous phase was removed for liquid
scintillation counting (Ghosh, N., and Chatterjee, S. (1998) J.
Biol. Chem. 264, 12554-12561).
Measurement of the Ceramide Level
[0166] Cells were pelleted and extracted with
chloroform-methanol-acetic acid 100:100:2 (v/v/v). Lipids in
organic phase were dried in N.sub.2 atmosphere and re-extracted,
and the organic phase was dried. Detergent solution (30 .mu.l of
7.5% n-octyl-.beta.-glucopyranoside with 5 mM cardiolipin in 1 mM
diethylenimine-pentaacetic acid) was added, and the samples were
sonicated. Total inorganic phosphate level was measured employing
the method of Bartlett et al. (Bartlett, G R. (1959). J. Biol.
Chem. 234, 449-458). Ceramide was measured using the
sn-1,2-diacylglycerolkinase-assay reagent system and labeling for
30 mM with 1 mCi of [.sup.32P] ATP in 10 .mu.l of 5 mM ATP
(Signorelli, P., and Hannun, Y. A. (2002) Meth. Enzymol. 345,
275-294). Diacylglycerol kinase was from Streptomyces species
(Calbiochem, San Diego, Calif.). [.sup.32P] ATP (specific activity
3000 Ci/mmol) was from New England Nuclear Company (Boston, Mass.).
Ceramide-1-phosphate and diacylglycerol-1-phosphate were resolved
by thin-layer chromatography on silica gel plates (Whatman,
Clinton, N.J.), using a solvent composed of
chloroform-acetone-methanol-acetic acid-water 10:4:3:2:1(v/v) and
detected by autoradiography using an Instant Imager (Packard
Canberra Company). Ceramide was expressed as pmol ceramide/nmol
phosphate.
Measurements of Cytochrome c Release and Caspase
Activation--Mitochondrial and Cytosolic Protein Isolation
[0167] After incubation with ApoCI (2.5 .mu.g/ml medium), ApoCIII
(2.5 .mu.g/ml medium), apoC-1-enriched HDL (1 .mu.g apoA-I/ml
medium), or ApoCI-poor HDL (5 apoA-I/ml medium) for 24 h and 48 h,
the cells were subjected to extraction with a buffer (30 mM HEPES,
pH 7.5, 10 mM KCl, 1.5 mM MgCl.sub.2, 5 mM DTI, 2 mM
phenylmethylsulfonyl fluoride (PMSF), 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 2 .mu.g/ml leupeptin, 2 .mu.g/ml aprotinin,
0.5 .mu.g/ml benzamidine, and 250 mM sucrose) on ice for 30 mM
Next, the samples were centrifuged at 10,000.times.g at 4.degree.
C. The supernatant was utilized as a source of cytochrome c in
Western immunoblot assay. The pellets were lysed in RIPA buffer
(phosphate buffer saline, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, and cocktail of inhibitors described
above). The samples were passed through a 21-gauge needle to shear
the DNA. Next, 10 .mu.l of PMSF (10 mg/ml) was added and incubation
continued for 60 mM on ice. The samples were centrifuged at
10,000-x g for 10 mM at 4.degree. C. The supernatants were used as
a source of protein in Western immunoblot assay for determination
of caspase activation. Proteins (100 .mu.g) were separated using
4-15% SDS-polyacrylamide gel under denaturing conditions and
electro transferred onto nitrocellulose. Membranes were then
incubated with primary antibody (anti cytochrome c and anti
caspase-3 rabbit polyclonal antibody (1:300) (Santa Cruz
Biotechnology, Santa Cruz, Calif.) and horseradish labeled
anti-rabbit antibody (1:3000).
Immunofluorescence of Cytochrome c in Human Cultured Aortic Smooth
Muscle Cells
[0168] AOSMC (5.times.10.sup.3) were grown on cover slips to
confluence and pretreated with serum free medium for 30 min as
described above. The cells were washed three times with phosphate
buffered saline (PBS) containing 1 mM MgCl.sub.2 and 0.1 mM
CaCl.sub.2 (solution A). The cells were fixed with 3%
paraformaldehyde in solution A for 10 mM, and permeabilized with
0.5% Triton X-100 in solution A for 5 min at room temperature. The
cover slips were then washed 3 times for 5 min with solution A.
Primary antibody (anti-cytochrome c) was used at a dilution of 2
.mu.g/ml in PBS and applied for 1 h with gentle shaking. The cells
were washed with PBS and a fluorescein isothiocyanate-conjugated
anti-rabbit Ig was applied for 1 h according to the manufacturer.
The cover slips were washed, mounted on microscope slides, viewed
and photographed on a Zeiss Axiovert 25 fluorescent microscope.
Analysis of p53/p56 Lyn Kinase Expression
[0169] Confluent cultures of AoSMC were incubated with ApoCI (2.5
.mu.g/ml medium), ApoCIII (2.5 .mu.g/ml medium), ApoCI-enriched HDL
(1 .mu.g apoA-I/ml medium), or apoA-1-poor HDL (5 .mu.g apoA-I/ml
medium). Cells were harvested in PBS at various time intervals and
stored frozen in the presence of the protease inhibitor cocktail
above. Western analysis for p53/p56 Lyn kinase was performed using
primary anti-Lyn p53/p56 antibody (1:300) (Santa Cruz
Biotechnology) and chemiluminescence (Amersham Pharmacia Biotech,
Piscataway, N.J.).
Measurement of the Levels of Caveolin-I, Scavenger Receptor BI, and
ABC Transporter ABCAI
[0170] Confluent cultures of AoSMC were preincubated with
serum-free medium for 30 mM and then treated with ApoCI (2.5
.mu.g/ml medium), HMS (58.8 .mu.g apoA-I/ml medium and 6.2 .mu.g
ApoCI/ml medium), and C2-Ceramide (30 .mu.M). At indicated time
periods cells were harvested in PBS and pellets lysed in 10 mM
HEPES buffer (pH 7.4) 5 mM EDTA, 0.25 mM EGTA, 50 mM NaF, 0.35 M
sucrose, 0.1% Nonidet P-40 and protease inhibitors 2 .mu.g/ml
aprotinin, 10 .mu.g/ml leupeptin, 5 .mu.g/ml pepstatin, and 1 mM
PMSF. Protein concentration was determined using B CA-kit (Pierce,
Rockford, Ill.). 100 .mu.g of protein was subjected to
electrophoresis in denaturing conditions on 4-15% polyacrylamide
gel and electro transferred onto nitrocellulose (BioRad, Rochester,
N.Y.). After the transfer, membranes were blocked for 1 h at room
temperature in TBS-T/5% milk. Western assay was performed using
rabbit caveolin-I (1:1000), SR-BI (1:5000) or goat ABCA1 (1:1000)
primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) and
horseradish peroxidase-conjugated anti-rabbit or anti-goat antibody
(Amersham Pharmacia Biotech, Piscataway, N.J.). Blots were
developed using ECLP.sup.his Western blotting detection reagents
(Amersham Pharmacia Biotech) and were scanned by laser
densitometry. The intensity of the signals were quantified with the
ImageGuant program (Molecular Dynamic, Sunnyvale, Calif.).
Example 1
ApoCI and ApoCI-Enriched HDL Particles Stimulate Apoptosis in
ASMC
[0171] Cells incubated with ApoCI contained many apoptotic cells,
as judged by white staining of the nucleus after treatment with
4',6-diamidino-2-phenylindole dihydrochloride (DAPI) reagent (FIG.
1A). In contrast, cells incubated with ApoCIII for 24 hours had
only a few apoptotic cells (FIG. 1A). After fluorescence
microscopic quantitative analysis of DAPI staining of the nucleus
in attached cells after treatment (FIG. 1B), 2.24% of normal
(control) cells, 4.78% of ApoCIII-treated cells, and 26.19% of
ApoCI-treated cells were apoptotic. In addition, .about.50% of ASMC
incubated with ApoCI-enriched HDL particles were apoptotic; whereas
only .about.2% of the cells incubated with ApoCI-poor HDL were
apoptotic (FIG. 1B; Table 1).
[0172] Apoptotic cells undergo endonucleosomal cleavage, resulting
in the fragmentation of DNA into 180 to 200 base-pair fragments
that resolve as a ladder on agarose gel electrophoresis. Cells
incubated with tumor necrosis factor .alpha. (TNF-.alpha.)
(positive control), or with ApoCI, exhibited DNA laddering. In
contrast, control cells, and those incubated with ApoCIII, did not
exhibit significant DNA laddering (FIG. 1C).
Example 2
Neutral Sphingomyelinase Inhibitor GW4869 Abrogates ApoCI-Induced
Apoptosis
[0173] As shown in FIG. 2A and the corresponding densitometric scan
(FIG. 2B), N-SMase inhibitor GW4869 (20 .mu.mol/L) completely
abrogated the ApoCI-induced apoptosis (.about.12% compared with
control, .about.2%), an effect that was also observed using a lower
dose (10 .mu.mol/L) of GW4869. However, when C2-ceramide was added
to the ASMC with ApoCI and GW4869, the inhibitory effect of GW4869
on N-SMase was bypassed, and apoptosis was restored (FIGS. 2A and
B) (.about.12.5% of apoptotic cells compared with control of 2.2%
apoptotic cells).
Example 3
ApoCI and ApoCI-Enriched HDL Particles Stimulate the Activity of
N-SMase in Cultured ASMC
[0174] Within 5 minutes of incubation of cells with TNF-.alpha.
(positive control) and ApoCI, there was .about.2-fold increase in
the activity of N-SMase that reached a maximum at 10 minutes and
then decreased to baseline by 30 to 60 minutes (FIG. 3A). Such a
pattern was not seen in the control cells or in those incubated
with ApoCIII. The stimulation of N-SMase activity with
ApoCI-enriched HDL particles was even more pronounced than that
seen with TNF-.alpha. and ApoCI (FIG. 3B). At 5 minutes, there was
a 2.6-fold stimulation of activity of N-SMase that reached a
maximum of 2.7-fold stimulation at 10 minutes. In contrast to
TNF-.alpha. and ApoCI, the stimulation of N-SMase activity with
ApoCI-enriched HDL was still manifested (2.2-fold) at 30 minutes
and then approached baseline at 60 minutes (FIG. 3B). ApoCI-poor
HDL also stimulated N-SMase activity in a similar pattern, but to a
considerably lesser extent than ApoCI-enriched HDL (FIG. 3B). ApoCI
exerted a concentration-dependent increase in the activity of
N-SMase in ASMC (FIG. 3C), with a maximum increase in N-SMase
activity (1.5-fold) at a concentration of ApoCI of 2.5 .mu.g/mL
medium). In additional experiments to examine the specificity of
this inhibitor, we found that GW4869 did not alter the acid
sphingomyelinase activity (control: 400 nmol/mg protein; GW4869:
340 nmol/mg protein) in cultured human ASMC.
Example 4
Antibody Against N-SMase Abrogates ApoCI-Enriched HDL-Induced
N-SMase Activity in Cultured ASMC
[0175] Previous studies in human renal proximal tubular cells
(Chatterjee, S. (1993) Adv. Lipid Res. 26:25-48) and neuronal cells
(Spence, M. W. (1993) Adv. Lipid Res. 26:3-23) have shown that
N-SMase is localized on the outer leaflet of the plasma membrane.
Using N-SMase antibody and FITC-conjugated secondary antibody, we
made similar observations in human ASMC. We therefore preincubated
ASMC with an antibody (IgG) against N-Smase (1:500 dilution) and
found that anti-N-SMase, but not rabbit IgG (control), inhibited
the stimulation of N-SMase activity by ApoCI-enriched HDL by 77%.
This result suggested that ApoCI mediated the increased activity of
N-SMase in human ASMC by the ApoCI-enriched HDL particle at the
cell surface.
Example 5
ApoCI and ApoCI-Enriched HDL Particles Stimulate the Generation of
Ceramide in Cultured Human ASMC
[0176] ApoCI exerted a time-dependent increase in ceramide levels,
which reached a maximum effect (1.7-fold) by 5 minutes and then
decreased to a value similar to that in control cells by 10 minutes
(FIG. 4A). Incubation of ASMC with ApoCI-enriched HDL particles
with time reached a maximum increase (.about.1.7-fold) in the level
of ceramide by 30 minutes, compared with control cells, whereas
ApoCI-poor HDL only stimulated the ceramide level slightly (FIG.
4B). GW4869 (20 .mu.mol/L) significantly decreased the cellular
level of ceramide induced by ApoCI after 5 minutes and 6 hours of
incubation. GW4869 completely inhibited ApoCI-induced ceramide
production after 12 hours of incubation but reversed it to a high
level 24 hours later (FIG. 4C). Using an anti-ceramide antibody,
cells treated with ApoCI reacted strongly, as judged by
immunofluorescence microscopy, compared with control cells (FIG.
4D). Most fluorescence was localized in the perinuclear area, an
effect of ApoCI on the ceramide level that was eliminated by GW
4869, an inhibitor of N-SMase.
Example 6
ApoCI and ApoCI-Enriched HDL Particles Stimulate the Release of
Cytochrome c From Mitochondria in ASMC
[0177] The release of cytochrome c from mitochondria into cytosol
is a key pro-apoptotic event. ApoCI stimulated the release of
cytochrome c 4.1-fold. ApoCIII and ApoCI-enriched HDL particles
also showed some stimulation of cytochrome c (2.3- and 1.2-fold,
respectively) (FIGS. 5A and B). Using immunofluorescence, a marked
increase in the release of cytochrome c from mitochondria into the
cytoplasm with ApoCI (even greater than that with TNF-.alpha.) was
noted, an effect that was inhibited by GW4869 (FIG. 5C).
Example 7
ApoCI and ApoCI-Enriched HDL Particles Stimulate the Expression of
Caspase-3 in Human ASMC
[0178] ApoCI stimulated the caspase-3 level 4-fold compared with
control medium. ApoCI-enriched HDL stimulated caspase-3 expression
in ASMC 1.7-fold, whereas ApoCI-poor HDL did not. ApoCIII also
stimulated caspase-3 expression, but to a lesser extent than
ApoCI-enriched HDL.
Material and Methods
[0179] The following materials and methods were used in Examples
8-9
Study Protocol
[0180] All animal studies were performed under approved protocol by
the Animal Care and Use Committee of the Johns Hopkins University,
Baltimore, Md. Animals (purchased from Covance Research Products,
Denver, Pa.) consisted of three sets of different atherosclerotic
stages (group 1: three WHHL rabbits, group 2: six WHHL rabbits fed
with high fat 14% coconut oil and 0.2% cholesterol diet for 3
months, group 3: five WHHL with similar diet but additional
balloon-induced endothelial denudation).
[0181] We acquired MR angiography and vessel wall imaging on a 1.5T
whole body MR scanner. During scanning animals were anaesthetised
with ketamine (25 mg/kg bodyweight IM), xylazine (2.5 mg/kg
bodyweight IM), and acepromazine (0.75 mg/kg bodyweight IM). Mean
anaesthesia time per animal was 2.5 hours. Snake venom (Russell's
viper, 0.15 mg/kg intra-peritoneal) followed by histamine (0.02
mg/kg intravenous) were administered to all animals twice within 48
hours). All supplies were from Sigma Chemical Co, St. Louis,
Mo.
[0182] MR imaging was repeated after 2 days in 10 animals at exact
locations by registration against the baseline study employing
anatomical landmarks.
[0183] After the second MRI, animals were euthanized (pentobarbital
(100 mg/kg i.v.), the aorta excised and sectioned into equal sized
samples for histopathologic and immunohistochemical analysis.
MRI and Image Analysis
[0184] Sub-renal aortic MRI angiography and vessel wall imaging
were performed on a commercial 1.5 Tesla (T) whole body MR system
(Gyroscan ACS-NT, Philips) after developing a novel vector ECG
triggered 3D radial balanced Fast-Field-Echo (FFE) angiography
gradient sequence (TR=7.2 ms, TE=3.6 ms, FOV=360 mm,
matrix=512*512, reconstructed voxel size=0.7*0.7*1.5 mm) Orthogonal
to prior angiography, we acquired 80-100 transverse concomitant
slices of a fat suppressed high resolution 3D black-blood FSE
imaging sequence (TR=3 heart beats, TE=10.5 ms, ETL=18, Inversion
time (TI)=400 ms, FOV=76 mm, Matrix=304*304, slice thickness=2 mm)
to cover entirely the sub-renal aorta to its iliac bifurcation. For
quantitative measurements we analysed signal-to-noise ratios
(SNR=Signal intensity.sub.blood/SD.sub.Air) with a previously
described analysis software (Constantinides, P. and Chakravarti, R.
N. (1961) Arch Pathol. 72:197-208).
Comparison of Histology and MR Images
[0185] In animal groups of plaque rupture, Hematoxylin and eosin
(HE) stained tissue samples were analysed with an Axioskop
microscope and an Axiocam digital camera (Carl Zeiss, Germany) by
one observer. Six measurements were taken per histological slice in
which measurement locations were marked on the image for exact
matching with the MRI slice.
[0186] According to the anatomical specimen coordinates (distance
from right renal artery and iliac bifurcation) equivalent
transverse MRI images were obtained to match MRI thickness
measurements with equivalent histological locations.
Immunohistology
[0187] We stained sections fixed with Tissue-Tec-O.C.T. compound
with Oil-Red-O. The formalin-fixed, paraffin-embedded blocks of
tissues fixed with 10% buffered formalin solution were cut into 4
.mu.m thick slides onto chemmate slides and subjected to
immunohistochemical analysis with antibody against ApoCI (goat
anti-human ApoCI antibody, Academy Bio-Medical Co, Houston, Tex.),
ceramide (IgM-enriched mouse anti-ceramide, Glycobiotech, Kuekels,
Germany), caspase-1 and caspase-3 (rabbit anti-human, Santa Cruz,
Calif.), CD-68 and .beta.-actin (mouse monoclonal, Cell
Marque/Ventara Laboratory, Tucson, Ariz.). The indirect
immuno-peroxidase method using 3-amino-9-ethylcarbazole or vector
Nova Red (Vector Laboratories, California) with Hematoxylin
counterstaining was used.
[0188] Tissue sections were also stained for apoptosis using
4',6-diamidino-2-phenylindole dihydrochloride reagent (DAPI). The
activity of neutral sphingomyelinase in tissue extracts was carried
out employing [.sup.14C] sphingomyelin (American Radiolabelled
Compounds, St Louis, Mo.) as a substrate as described (Kolmakova A.
et al. (2004) Arterioscler Thromb Vasc Biol. 24(2):264-269). The
immunocytochemical slides were analyzed and documented by a
cardiovascular cyto-pathologist.
Statistics
[0189] Data comparisons and correlations were made by ANOVA and
were reported as mean.+-.1 SD whereas a value of P=0.05 was
considered significant.
Example 8
MRI
[0190] Sub-renal MRI angiography and vessel wall images with
in-plane resolution up to 130 .mu.m were consistently obtained in
all animals at different anatomic levels of the aorta (FIG. 6).
[0191] The SNR of the three groups (FIG. 7A) was in the same range
(39-52). Interestingly, cholesterol fed animals from group 2
compared to group 3 had significantly lower SNR (p=0.048), and
compared to group 1, it shows a trend to significantly lower SNR
(p=0.06).
[0192] MRI vessel wall thickness in animal groups of plaque rupture
(mean=0.42.+-.0.3 mm) correlated well with histopathologic
examination (y=0.81x+0.17, R.sup.2=0.64, r=0.8, FIG. 7B).
[0193] We consistently found an inverse correlation between serum
cholesterol levels (mg/dl) and vessel wall MRI signal
(y=-4.9872x+1953.7; R.sup.2=0.58, r=0.76, FIG. 8C).
[0194] Applying 3D high resolution angiography and vessel wall
imaging before (FIGS. 8A and 8B) and after venom application (FIGS.
8D and 8E) to all rabbits, MRI was able to detect in vivo
neo-intimal disruption with concomitant severe thrombosis (>40%
stenosis) in six animals of groups 2 and 3 only (FIGS. 8D-8F).
Example 9
Immunohistology
[0195] Group 2 rabbits exhibited strongly Oil Red 0 (ORO) positive
plaque having large oil droplets (FIG. 9E) and pronounced
immuno-histochemical staining of the aortic intima with the ApoCI
antibody (FIG. 9F, see spots indicated by arrow) as opposed to
group 1 rabbits (FIGS. 9A and 9B). When observed under a higher
magnification, most, if not all the ApoCI from group 2 were found
with the aortic smooth muscle cells and in macrophages. Next, we
investigated the immuno-localization of ceramide, an apoptotic
agent, previously shown to mediate ApoCI, TNF-.alpha. and stress
induced apoptosis. Group 2 rabbit aorta sections contained ceramide
in the intima with massive deposits in the intimal plaque area
(indicated by an arrow) and media but with less intensity (FIG.
9G), whereas group 1 animals only showed discrete ceramide staining
in the media (FIG. 9C).
[0196] Immuno-staining with caspase-1 (FIGS. 9D and 9H) and
caspase-3 antibody (FIGS. 9I and 9M) in group 1 and 2 animals
showed its presence in the cytoplasm in the medial SMC but in sharp
contrast strong and massive caspase-3 immuno-staining was observed
predominantly in the neo-intimal area of dissection/plaque rupture
(FIG. 9M) whereas, group 1 animals only had mild caspase-3 staining
(FIG. 9I). In fact, no significant differences in the caspase-3
immuno-staining in the media of both groups of animals were
observed.
[0197] To determine the cell type present in plaque intima the
tissue sections were stained with CD-68 for macrophages and indeed
these cells appeared to be among the predominant type of cells in
the neo-intimal plaques (FIG. 9N, see arrow). No CD-68 positive
macrophages were observed in control tissue (FIG. 9J) and in media
(FIG. 9N). Also, strong .beta.-actin staining (marker for smooth
muscle cells) was observed in the neo-intima (FIG. 9O) and in the
media in the control group of rabbit tissue. Study with DAPI
reagent revealed the presence of some apoptotic cells in group 2 of
rabbit sections (FIG. 9P, also see inset).
[0198] Western immunoblot assay revealed the presence of both
caspase-1 and caspase-3 in the rabbit tissue sections. In fact, the
basal level of pro-caspase-3 (32 kDa) was similar in group 1 and 2
rabbit aorta sections (FIGS. 10A and 10B). However, the level of
the active form of caspase-3 (17 kDa protein) was markedly
increased in group 2 of rabbit aorta sections (FIG. 10B) compared
to group 1 (FIG. 10A). Biochemical assay revealed that the activity
of neutral sphingomyelinase in group 2 rabbits aorta sections was
2.5.+-.0.5 fold higher than group 1 rabbit tissue sections (control
tissue activity: 5 nmol/mg protein/hr).
[0199] In animals of group 3 with fat diet and endothelial
denudation we observed accumulation of ApoCI, ceramide and
caspase-3 more prominently in the vessel media.
Materials and Methods
[0200] The following materials and methods were used in Examples
10-15.
Patient Population
[0201] The group of 163 infants (31 white males, 39 white females;
47 black males and 46 black females) studied was previously
characterized (Kwiterovich Jr P O. et al. (In press, 2004) Ethn.
Dis.). There were 23 small-for-gestational age (SGA) infants,
defined as a birth weight for gestational age < or =10%
(Kwiterovich Jr P O. et al. (In press, 2004) Ethn. Dis.). The Joint
Committee on Clinical Investigation at Johns Hopkins approved the
study.
Lipid, Lipoprotein and Apolipoprotein Measurement
[0202] Plasma from cord blood was analyzed for levels of
cholesterol, triglycerides, LDL and HDL cholesterol, Lp (a)
lipoprotein, and apolipoproteins A-I, A-II, B, C-I, C-III, and apoE
(Kwiterovich Jr P O. et al. (In press, 2004) Ethn. Dis.; Kolmakova
A. et al. (2004) Arterioscler. Thromb. Vas. Biol. 24:1-9). Fifteen
lipoprotein subclasses, the number of LDL particles, and the
average sizes (nm) of VLDL, LDL and HDL were determined by nuclear
magnetic resonance (NMR) spectroscopy (Kwiterovich Jr P O. et al.
(In press, 2004) Ethn. Dis.; Otvos, J D. (2000) In: Rifai N. et
al., Eds. Handbook of Lipoprotein Testing. Washington, D.C.: AACC
Press, 609-623). Lipoprotein density profiles for VLDL, LDL and HDL
were obtained after sucrose density gradient ultracentrifugation
(DGU) (Macfarlane R D. et al. (1997) Electrophoresis 18:1796-1806;
Farwig Z N. et al. (2003) Anal Chem 75:3823-3830).
Preparation of Lipoprotein Fractions from Sucrose DGU for Capillary
Electrophoresis
[0203] Fractions from the lipoprotein density profile were thawed
and a portion subjected to delipidation (Macfarlane R D. et al.
(1997) Electrophoresis 18:1796-1806; Farwig Z N. et al. (2003) Anal
Chem 75:3823-3830). The samples were analyzed in duplicate by
capillary electrophoresis using the Beckman P/ACE 5510 instrument
at 17 kV for 30 minutes.
Preparation of Lipoprotein Fractions for MALDI-TOF Mass
Spectrometry (MS) and Immobilized pH Gradient (IPG) Isoelectric
Focusing (IEF)
[0204] The lipoprotein fractions were thawed, centrifuged to pellet
particulate matter, and subjected to solid phase extraction
delipidation (Macfarlane R D. et al. (1997) Electrophoresis
18:1796-1806; Farwig Z N. et al. (2003) Anal Chem 75:3823-3830).
The apolipoproteins were eluted, concentrated, and an aliquot taken
for MALDI-TOF MS analysis, using a Voyager Elite XL DE mass
spectrometer. The remaining samples were evaporated to dryness,
reconstituted in 250 .mu.L 8.0 M urea containing 2% CHAPS,
sonicated, degassed, and electrophoresis performed using the IPG
phor unit (Amersham Pharmacia, Sweden) as described (Macfarlane R
D. et al. (1997) Electrophoresis 18:1796-1806; Farwig Z N. et al.
(2003) Anal Chem 75:3823-3830).
Activity of Cholesterol Ester Transfer Protein
[0205] CETP activity was determined by using the CETP Activity Kit
by Roar Biomedical, Inc. (New York, N.Y.), according to the
manufacturer's specifications.
[0206] Statistical Methods
[0207] The relationships between lipids, lipoprotein cholesterols,
apolipoproteins, lipoprotein subclasses and lipoprotein sizes in
the four groups of infants were evaluated, first using ANOVA on
data that were not adjusted for age, and then linear regression to
correct for influence of gestational age. P values were also
estimated using the Kruskal-Wallis test, due to the small size of
two of the groups (n=5 each in group 0 and 3). To evaluate
differences in these lipid-related variables between white and
black and male and female infants, a CHI squared test was
performed. All p values found to be significant at the p<0.05
levels.
Example 10
Lipoprotein Density Profiling after Sucrose DGU
[0208] A prominent feature of the lipoprotein density profiles was
the presence or absence of a distinct peak of d 1.062 and 1.072
g/ml between the major peaks for LDL and HDL (FIGS. 11A, 11B). The
peak density of Lp (a) in adult plasma is close to 1.055 gm/ml, and
thus potentially occurring within the density range between 1.062
and 1.072 gm/ml. Lp (a) levels in cord blood, however, are very low
(Kwiterovich Jr P O. et al. (In press, 2004) Ethn. Dis.), and the
mean Lp (a) levels (mg/dL+/-1 SD) in group 3 (1.2+/-1.3)) and group
0 (0.6+/-0.9) were not significantly different.
Example 11
Characterization of Lipoprotein Peak of d 1.062-1.072 g/ml
[0209] Two infants from group 3 and group 0, respectively, were
selected for detailed analyses of this lipoprotein peak. The Lp (a)
levels in these two infants were low (3 mg/dL in 013 and
undetectable in 021). Three lipoproteins, namely, LDL, the
lipoprotein with a peak of d 1.062-1.072 g/ml, and HDL, were
isolated by sucrose DGU, delipidated and prepared for the following
analyses.
[0210] Capillary Electrophoresis and Isoelectric Focusing
(IEF).
[0211] After capillary electrophoresis, apoA-I (47.7%) was the
major apolipoprotein in the lipoprotein of d 1.062-1.072 g/ml from
the group 3 infant, and ApoCI, ordinarily a minor component of HDL,
was the second most prevalent apolipoprotein (37.6%). Negligible
amounts of apolipoproteins were detected in the same lipoprotein
density segment from the group 0 infant. These results were
confirmed by IEF, showing clearly apoA-I (pI 5.43) and ApoCI (pI
6.70) bands in the prominent lipoprotein peak from the group 3
infant, but not the group 0 infant.
[0212] MALDI-TOF MS Analyses.
[0213] In the prominent lipoprotein peak of d 1.062-1.072 g/ml
(FIG. 12A) from the group 3 infant, the intensity of ApoCI,
relative to the intensity of apoA-I, was notably greater than in
HDL (FIG. 12C). The apolipoproteins in the lipoprotein peak of d
1.062-1.072 from the group 0 infant were barely detectable (FIG.
12B). There was little difference in the spectra for HDL of usual
density between the group 3 (FIG. 12C) and the group 0 infants
(FIG. 12D). The above observations were confirmed in another group
3 infant and a normal control. There was no difference detected in
LDL spectra.
[0214] Gradient Gel Electrophoresis of HDL.
[0215] Plasma from four infants in group 3, one infant in group 2,
and three infants in group 0 were ultracentrifuged at d<1.21
g/ml and GGE performed (Nichols A. et al. (1991) Biochem et Biophys
Acta. 1085:306-14; Genzel-Boroviczeny O. et al. (1988) Pediatr.
Res. 23:543-547). As shown in representative densitometric scans of
the gels (FIG. 13), group 3 infants differed from group 0 infants.
The largest HDL subclass in group 3 infants had a mean diameter of
11.6 nm (range 11.5 to 11.8), compared to 9.4 (range 8.8 to 10.8)
in group 0 infants, and 10.8 in one group 2 infant.
[0216] ApoCI was found in each of different HDL subclasses (FIG.
13), as judged by immunoblots of the GGE gels using an anti-ApoCI
antibody. These results are consistent with those from MALDI-TOS MS
(see above), and indicate that all HDL subclasses contained
ApoCI.
Example 12
Immunochemical Characterization of the Apolipoproteins in Infants
with Prominent Versus Undetectable Amounts of the Lipoprotein of d
1.062-1.072 g/ml
[0217] The distribution of apolipoproteins A-I, A-II, B, C-I, and
C-III, between apoB-containing lipoproteins (VLDL, IDL, LDL and Lp
(a)), and non-apoB-containing lipoproteins (HDL) was determined
without prior ultracentrifugation in infants from group 3 (N=5) and
group 0 (N=5). The apoB-containing lipoproteins in one ml plasma
were precipitated with heparin-manganese chloride, and the
apolipoprotein levels measured in plasma, heparin-manganese
supernatants (non-apoB containing lipoproteins) and resolubilized
precipitates (apoB-containing lipoproteins), using rocket
immunoelectrophoresis (Kolmakova A. et al. (2004) Arterioscler.
Thromb. Vas. Biol. 24:1-9).
[0218] Apolipoprotein B.
[0219] The plasma levels of total apoB were higher in group 3 than
in group 0 infants, but did not reach statistical significance
(Table 2). All of the apoB was in the heparin manganese
precipitates and none was detected in the supernatants.
[0220] Apolipoprotein C-I.
[0221] The mean levels of ApoCI in both whole plasma and the
heparin-manganese supernatants were about twofold higher in group 3
than in group 0 infants (Table 2). In a larger group of infants,
the mean (SD) plasma level of ApoCI (.mu.mol/L) of 11.6 (4.8) in 17
group 3 infants was significantly higher (p=0.036) than that of 7.7
(4.7) in 13 infants from group 0. All of the ApoCI was in the
supernatants and absolutely none was detected in the precipitates,
distinctly different than later in life when a significant portion
of ApoCI is associated with the apoB-containing lipoproteins (Curry
M D. et al. (1981) Clin Chem 27:543-548). These immunochemical
results further indicate that the ApoCI-enriched lipoprotein peak
is an HDL subclass rather than a LDL subclass.
[0222] Apolipoprotein C-III.
[0223] Infants in group 3 had significantly more of their ApoCIII
associated with the non-apoB-containing lipoproteins, while infants
in group 0 had significantly more of their ApoCIII associated with
the apoB-containing lipoproteins (Table 2).
[0224] Apolipoproteins A-I and A-II.
[0225] The apoA-I levels were higher in both the supernatants and
precipitates in the infants in group 3 than in group 0 (Table 2).
The mean apoA-II levels between groups 3 and 0 were very similar
for whole plasma, supernatants and precipitates (Table 2).
[0226] Apolipoprotein E.
[0227] In a separate experiment, the concentration (.mu.mol/L) of
apoE in group 3 infants was higher than in group 0 infants in
pooled whole plasma (3.8 v 1.7) and heparin-manganese supernates
(2.3 v 1.4).
[0228] Definitions of Four Groups of Infants.
[0229] Using lipoprotein density profiles (FIG. 11), four groups of
infants were classified, based on the gray intensity scale in the
area between LDL and HDL: Group 0, no inflection above baseline (no
detectable ApoCI-enriched HDL); Group 1, small inflection (blip)
above baseline (1 to 5 on scale) (possible ApoCI-enriched HDL);
Group 2, a peak above baseline >5 and <50 on scale (probable
ApoCI-enriched HDL; Group 3, a large peak above baseline >50 on
scale (prominent ApoCI-enriched HDL).
Example 13
Lipid, Lipoprotein, Apolipoprotein and Lipoprotein Subclasses and
Lipoprotein Sizes in Group 0, 1, 2, and 3 Infants
[0230] Lipoprotein density profiling was performed in 156 of the
163 infants (95.7%) previously reported (Kwiterovich Jr P O. et al.
(In press, 2004) Ethn. Dis.), to determine the frequency of
appearance and degree of enrichment of the d 1.062-1.072 g/ml peak.
The frequency (%) of the four groups was: 0, 17.3%; 1, 41.0%; 2,
22.4%; and 3, 19.2%.
[0231] The levels of the lipid-related variables were determined in
the four groups of infants (FIG. 14). Because of the influence of
gestational age on the apoB- and apoA-1-containing lipoproteins in
this population (Kwiterovich Jr P O. et al. (In press, 2004) Ethn.
Dis.), the p values were determined using data non-adjusted and
adjusted for gestational age. Before age adjustment, all the
variables except apoB and small VLDL were significantly different.
After age adjustment, the only LDL variables that remained
significantly higher in group 3 were L3, L1 and LDL size (FIG. 14).
In contrast, all the HDL and VLDL related variables remained
significantly different after correction for gestational age (FIG.
14). Large HDL levels were higher while small HDL and the VLDL
related variables were lower in group 3. We also examined
age-corrected means, which were very similar to the measured mean
levels shown in FIG. 14 for the HDL- and VLDL-related variables.
Differences between large LDL and large HDL in groups 0, 1, 2, and
3 (FIG. 14) were independent of triglycerides and VLDL.
[0232] Despite the fact that the gestational ages were very similar
in groups 0, 1 and 2, there were also impressive dose-response
relationships for the levels of all six of the HDL-related
variables from class 0 through class 1, 2 and 3 infants (FIG. 14).
These analyses further support the conclusion that the differences
for the HDL subclasses shown in FIG. 14 were independent of
age.
Example 14
Distribution of Gestational Age in Infants in Groups 0, 1, 2 and
3
[0233] The gestational ages (mean (1SD), in weeks) in groups, 0, 1,
2 and 3 were: 39.7 (1.75); 39.3 (1.28); 38.8 (1.68); and 36.2
(4.16), respectively, and differed significantly (p<0.0001). The
mean gestational age in group 3 infants was not only younger, but
had a distribution that was clearly broader than those in groups 0,
1, and 2 (FIG. 15).
[0234] The birth weights (grams) in group 0, 1, 2 and 3 infants
were (mean (1SD)): 3268.6 (631.9); 3412.2 (548.3); 3240.6 (609.2);
and 2683.7 (783.3), respectively, and differed significantly
(p<0.0001), being particularly low in group 3. After correcting
for gestational age, the birth weights were no longer significant
(p=0.15). There were no significant differences in the numbers of
male and female (p=0.38), white and black (p=0.88), or SGA and AGA
(p=0.34) infants between the four groups.
[0235] We next plotted the levels of large LDL (L3) and largest HDL
(H5) against gestational age for the group 3 and group 0 infants
(FIG. 16). Group 3 infants had higher values of L3 than group 0
infants, but these L3 levels decreased dramatically with increasing
gestational age. In distinct contrast, the higher H5 levels in
group 3 did not fall with gestational age, indicating strongly that
the elevated amount of ApoCI-enriched HDL in group 3 persisted and
was not simply a consequence of younger gestational age (FIG.
16).
Example 15
ApoCI and CETP Activity
[0236] ApoCI levels in adults are associated with decreased CETP
activity and larger HDL particles (Jong M C. et al. (1999)
Arterioscler Thromb Vasc Biol 19:472-484; Gautier T. et al. (2000)
J Biol. Chem. 275:37504-37509). We examined if higher ApoCI levels
in cord blood inhibited CETP activity, accounting for the different
amounts of the large HDL particles. In a subset of 40 infants
(group 0 N=17; group 3 N=13; and groups 1 and 2, N=10), there was
no relationship between CETP activity (uM/ml) (range 0.0620 to
1.398) and ApoCI levels (umol/L) (range 2.3 to 20.7), r=0.087, NS.
In a larger group (N=123) of infants, there was no significant
difference (p=0.149) in CETP activity between the four groups.
TABLE-US-00002 TABLE 2 Apolipoprotein content of plasma,
heparin-manganese supernatant, and heparin-manganese precipitate in
infants with prominent (Group 3) or undetectable (Group 0)
ApoCI-enriched HDL. Group 3 Infants Group 0 Infants Apolipoproteins
(N = 5) (N = 5) P ApoB Plasma 27.4 (12.2)* 21.4 (2.3) 0.53
Heparin-Mn.sup.+2 Supernatant 0 0 1.0 Precipitate 23.1 (12.6) 16.3
(1.5) 0.46 ApoCI Plasma 8.7 (1.9) 4.8 (2.4) 0.08 Heparin-Mn.sup.+2
Supernatant 6.9 (3.1) 3.3 (1.8) 0.03 Precipitate 0 0 1.0 ApoCIII
Plasma 5.4 (1.1) 4.3 (1.1) 0.25 Heparin-Mn.sup.+2 Supernatant 4.7
(0.9) 3.1 (0.6) 0.01 Precipitate 0.6 (0.3) 1.3 (0.6) 0.05 ApoA-I
Plasma 119.7 (38.8) 99.6 (17.1) 0.46 Heparin-Mn.sup.+2 Supernatant
101.6 (30.7) 83.0 (11.6) 0.12 Precipitate 4.9 (0.9) 3.1 (0.8) 0.01
ApoA-II Plasma 21.4 (2.1) 21.1 (1.6) 0.60 Heparin-Mn.sup.+2
Supernatant 19.8 (2.2) 19.8 (0.8) 0.46 Precipitate 0.9 (0.2) 0.9
(0.2) 0.91 *Mean (SD)
[0237] The invention has been described in detail with reference to
preferred embodiments thereof. However, it will be appreciated that
those skilled in the art, upon consideration of this disclosure,
may make modification and improvements within the spirit and scope
of the invention as set forth in the following claims.
* * * * *