(Circulation. 2000;101:1091.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Adenovirus-Mediated Gene Transfer of a Secreted Form of Human Macrophage Scavenger Receptor Inhibits Modified Low-Density Lipoprotein Degradation and Foam-Cell Formation in Macrophages
From the A.I. Virtanen Institute (J.L., P.L., S.Y.-H.) and Department of Medicine, University of Kuopio (S.Y.-H.), Kuopio, Finland; and the Sir William Dunn School of Pathology, University of Oxford, Oxford, UK (P.J.G., D.R.G., S.G.).
Correspondence to Seppo Ylä-Herttuala, MD, PhD, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland. E-mail seppo.ylaherttuala{at}uku.fi
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Abstract |
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Background—Macrophage scavenger receptors (MSRs) play an important role in the pathogenesis of atherosclerosis. Therefore, local modulation of MSR activity could have a beneficial effect on atherogenesis.
Methods and Results—We cloned a secreted "decoy" MSR (sMSR) that contains an extracellular portion of the human MSR type AI and constructed an adenoviral vector that directs high-level expression of sMSR in macrophages under the control of the human CD68 promoter. Expression of the sMSR protein inhibited the degradation of 125I-labeled acetylated LDL and oxidized LDL by murine macrophages up to 90%. sMSRs also reduced acetylated LDL degradation in MSR knockout mouse peritoneal macrophages by 60% to 80%, which suggests that the decoy construct can compete for the uptake mediated via other related scavenger receptors. In addition, sMSRs inhibited foam-cell formation in murine macrophages in the presence of cytochalasin D. The mechanism of inhibition is through ligand binding to the sMSRs, which prevents the ligand binding to MSRs on cell membranes.
Conclusions—The demonstration that recombinant adenovirus–mediated gene transfer of decoy sMSRs can block foam-cell formation suggests a possible new strategy for gene therapy of atherosclerosis and for the treatment of lipid accumulation after arterial manipulations.
Key Words: viruses • genes • receptors • lipoproteins • cells
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Macrophage scavenger receptors (MSRs) type AI/II are trimeric membrane glycoproteins that are involved in the deposition of lipids in the arterial wall during atherogenesis.1 2 3 4 5 They also mediate cation-independent adhesion of macrophages in vitro.6 MSRs belong to a family of receptor proteins that have cysteine-rich extracellular domains and exhibit a broad ligand-binding specificity.1 2 3 4 5 They can efficiently bind and internalize several polyanions and modified proteins, such as acetylated (ac) LDL and oxidized (ox) LDL, maleylated BSA, several polysaccharides, endotoxin, some phospholipids, and polyribonucleotides.1 2 3 4 5 Common to all MSR ligands is that they are negatively charged macromolecules.4 5 Members of the receptor family may also participate in host-defense functions.5 6 7
MSR type AI/II receptors have been implicated in the deposition of lipids in the arterial wall during atherogenesis.8 9 10 Because MSR is not downregulated by excess cholesterol accumulation in the cell, it can mediate continuous accumulation of lipids in the lesion macrophages.3 4 5 MSR expression is also strongly induced during lesion development.11 Studies with MSR AI/II knockout mice and their cells show that MSR accounts for 80% of the degradation of acLDL and 30% to 50% of the degradation of oxLDL,10 which is one of the MSR ligands in the vessel wall.12 Lesion formation is also significantly reduced in MSR AI/II knockout mice.10 Therefore, modulation of the MSR activity could have an important effect on atherogenesis.
We constructed a chimeric fusion protein that consists of the bovine growth hormone signal sequence and the human MSR AI extracellular domain. This secreted MSR (sMSR) was cloned into an adenoviral vector, and sMSR recombinant adenoviruses were produced under the control of the promoter of macrophage gene CD68. Adenoviruses were used to transduce murine macrophages, and the conditioned media were shown to reduce degradation of acLDL and oxLDL by 70% to 90% and to inhibit macrophage foam-cell formation.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Materials
pRc/CMV plasmid was from Invitrogen and pNASSµ plasmid from Clontech. All cell culture reagents were from Gibco BRL. Oligo-dT cellulose, 5-bromo-4-chloro-3-indolyl-phosphate, and 4-nitro blue tetrazolium chloride were from Boehringer-Mannheim. 125I and nylon membrane were from Amersham, nitrocellulose membrane from Bio-Rad, and [32P]CTP from New England Nuclear. Poly(G) conjugated resin, cytochalacin D, and anti-Flag monoclonal antibody M5 were from Sigma. RAW 264 mouse macrophages (TIB-71), ECV304 cells (CRL-1998), and NIH 3T3 fibroblasts (CRL-6361) were obtained from ATCC. Chamber slides were from LabTek Brand, Nunc International.
Cloning of the sMSR and Adenovirus Plasmids
sMSR was constructed in 3 steps (Figure 1
). First, a 109-bp fragment was created
by polymerase chain reaction (PCR) coding for 28 amino acids of the
bovine growth hormone signal sequence with HindIII and
XbaI sites and an Eco47III site encoding the last
2 amino acids of the signal peptide. This fragment was cloned into
pRc/CMV plasmid. Second, oligonucleotides encoding a
"Flag" epitope (DYKDDDDK), Eco47III, and XbaI
sites and a NotI site following the Flag epitope were
synthesized. This insert and the vector from step 1 were ligated.
Third, a 5' oligonucleotide containing a
NotI site and a 3' oligonucleotide specific
for the C-terminus of the type AI MSR with XbaI restriction
site after the stop codon were used in a PCR reaction to generate the
extracellular domain of the receptor.
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An E1/E3-deleted adenovirus was generated by subcloning into a
pAdenogal plasmid13 a 2940-bp BstXI fragment
(-2938 to +2) of the human CD68 promoter.14 SV40
first intron, Escherichia coli ß-galactosidase cDNA, and
the SV40 poly(A) were cut from plasmid NASSß and subcloned in the 3'
end of the CD68 promoter. ß-Galactosidase cDNA was excised and sMSR
ligated to the vector to create plasmid psMSRA-1 (Figure 1
).
Correct structure of the construct was verified by sequencing.
Generation of Adenoviruses
Adenovirus plasmid psMSRA-1 was linearized and cotransfected
with sub360 adenovirus genomic DNA in 293 cells by
CaPO4 precipitation.15 Viruses from
1 expanded plaque were used for a large-scale preparation of the
recombinant adenoviruses.16 Purified virus preparation was
analyzed for the absence of toxicity, wild-type viruses,
microbiological contaminants, and lipopolysaccharide as
described.16
Southern and Northern Blots
Adenovirus genomic DNA was digested with HindIII and
analyzed in a Southern blot with a random-primed DNA probe
specific for both MSR and sMSR.15 RAW 264
macrophages and rabbit aortic smooth muscle cells (SMCs) were
transduced with sMSR adenoviruses at 1000 MOI. Uninfected control
plates were treated similarly to the transduced plates. mRNA was
isolated with oligo-dT cellulose resin and analyzed in a
Northern blot with a random-primed probe specific for
sMSR.15 The membrane was stripped and reprobed with a
human ß-actin probe.
Western Blot
RAW 264, ECV 304, and NIH 3T3 cells and SMCs were transduced
with sMSR adenoviruses; the medium was changed to Optimem containing
0.5% FBS, and the cells were grown for 48 hours. Medium was collected
and lyophilized, electrophoresed under reducing and nonreducing 10%
SDS-PAGE, and transferred to a nitrocellulose membrane.15
sMSR was detected with a mouse anti-Flag monoclonal antibody (M5)
according to the manufacturer’s instructions.
Poly(G) Resin Incubation Test
Control medium and medium containing sMSR were incubated with
poly(G) resin overnight as described.17 Medium was
electrophoresed under nonreducing 8% SDS-PAGE and transferred to a
nitrocellulose membrane,15 and sMSR was detected as
described above.
Dot-Blot Assay
The amount of sMSR protein in the transfection medium was
measured with a dot-blot assay15 from several dilutions of
the transfection medium, which were spotted onto a nitrocellulose
membrane with standards ranging from 0.01 to 200 ng/100 µL. Proteins
were detected with anti-Flag M5 antibody and quantified with an image
analyzer and MCID-M4 program (Imaging Research Inc). Generation
of the purified sMSR-AI protein standard will be described in detail
elsewhere (Gough P.J. et al, manuscript in preparation).
Isolation and Modification of LDL
LDL was isolated from fasting plasma of healthy normolipidemic
donors by sequential ultracentrifugation12
and radioiodinated18 before
acetylation19 or oxidation.12
Specific activity of the labeled LDLs was 90 to 250 cpm/ng protein.
Degradation Assay
RAW 264 macrophages were transduced with sMSR
adenoviruses, and Optimem containing 10% lipoprotein-deficient serum
(LPDS) was added. Conditioned medium was collected every 12 hours and
used for lipoprotein degradation and competition assays.12
Untransduced control plates were treated similarly and used as
controls. In earlier experiments, it was determined that conditioned
medium from lacZ control adenovirus16 –transfected cells
did not have any effect on the degradation of acLDL in
macrophages. RAW 264 cells and peritoneal macrophages
from MSR-A knockout mice10 and MSR-A/LDL receptor
double-knockout mice20 were incubated with 10 µg/mL of
125I-labeled acLDL or
125I-labeled copper oxLDL12 and
conditioned medium for 9 hours. After incubation, the media were
analyzed for LDL degradation products.12
Foam-Cell Formation
RAW 264 cells were plated on chamber slides and incubated for 24
hours with Optimem/10% LPDS containing 100 µg/mL acLDL and 4
µg/mL of phagocytosis inhibitor cytochalasin D with or
without sMSR (2.5 µg/mL). After the incubation, the cells were washed
with PBS, fixed with 4% paraformaldehyde, and stained
with oil red O in 60% isopropanol, 0.4% dextrin and hematoxylin.
Organ Distribution of sMSR–125I-acLDL Complex
Medium without or with sMSR protein (2.5 µg) was combined with
25 µg of 125I-acLDL for 30 minutes and injected
into the tail vein of anesthetized MSR-A/LDL receptor
double-knockout mice (n=4)20 in a total volume of 200 µL
(2.1x107 cpm). Mice were euthanized 30 minutes
later, and organs were analyzed for radioactivity as
described.21
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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To study the effects of "decoy" scavenger receptor on the metabolism of modified LDL in macrophages, we cloned a soluble sMSR that contains the extracellular domains of human MSR AI and produced replication-deficient adenoviruses in which transgene expression is controlled by the promoter of the macrophage gene CD68 (Figure 1
). Because adenoviruses
undergo RNA splicing during their replication cycle, Southern
analysis was performed to confirm the presence of the
full-length transgene (Figure 2a). An
expected 7.9-kb HindIII fragment containing the 4.6-kb
expression cassette was visualized by hybridization with MSR probe,
whereas wild-type virus did not contain the fragment (Figure 2b).
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To confirm that the DNA was transcribed to mRNA RAW 264 cells and SMCs
were transduced with sMSR adenovirus, mRNA was isolated and
analyzed on Northern blot. A 1.5-kb sMSR fragment was detected
in the transduced RAW 264 cells and SMCs. Controls show that the probe
is specific for the sMSR (Figure 3a). The
same membrane was stripped and hybridized with ß-actin probe to
confirm that similar amounts of mRNA were loaded on each lane (Figure 3b).
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Western blot analysis was performed with RAW 264
macrophages, SMCs, NIH 3T3 fibroblasts, and ECV 304 cells
transduced with sMSR adenoviruses, and lyophilized medium was
analyzed in SDS-PAGE (Figure 4).
Easily detectable amounts of sMSR protein were found in RAW 264 and ECV
304 cell supernatants. Fibroblasts and SMCs expressed only a barely
detectable amount of sMSR protein (Figure 4). Most of the sMSR
protein was present as a monomer and a dimer, but some trimer was
also detected. Quantitative analysis based on a dot-blot assay
from the conditioned medium indicated that RAW 264 macrophages
and ECV 304 cells produced 0.1 to 2.5 ng/µL sMSR protein in the
medium during a 12-hour incubation. Without concentration, the
production of sMSR protein in SMCs and NIH 3T3 fibroblasts was
below the reliable detection limit of the dot-blot assay (1 ng). Under
reducing conditions, almost all the sMSR protein from RAW 264 cells was
detected as a monomer (Figure 4).
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We hypothesized that the sMSR binds to modified LDLs in the growth
medium and thus prevents the ligand binding to native scavenger
receptors. To test this, we incubated the sMSR medium with poly(G)
conjugated resin. Original sMSR medium, poly(G) resin–incubated
medium, and proteins released from the poly(G) resin were
analyzed with a Western blot along with the similarly treated
control medium. The poly(G) resin was able to bind almost all (>90%)
of the sMSR from the medium (Figure 5).
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Functional analysis of the sMSR protein was performed with a
lipoprotein degradation assay (Figure 6).
125I-acLDL (10 µg/mL) was added to the
conditioned medium and incubated with cells for 9 hours. In the
presence of sMSR, the rate of degradation of
125I-acLDL decreased by 70% to 90% (Figure 6a). The decrease was comparable to a 15-fold excess of
unlabeled acLDL or 30-fold excess of oxLDL (data not shown). A similar
decrease in the degradation was also seen with
125I-labeled oxLDL: sMSR was able to decrease the
degradation of oxLDL by 60% to 80% (Figure 6b). The decrease
was comparable to a 30-fold excess of unlabeled oxLDL or acLDL (data
not shown). sMSR was also able to reduce the degradation of
125I-acLDL in peritoneal macrophages
isolated from either MSR-A knockout mice (Figure 6c) or
MSR-A/LDL receptor double-knockout mice (data not shown) by 60% to
80%. On the basis of the quantitative dot-blot assay (see above), it
was calculated that the conditioned medium used for the competition
studies contained 2.5 µg/mL sMSR protein versus 10 µg/mL labeled
acLDL. If molecular weights of 2.5x106 Da for
acLDL and 135 kDa were assumed for dimeric sMSR, the conditioned medium
contained 
5 sMSR molecules for every LDL particle.
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We next tested the possibility that part of the reduction in the
degradation of modified LDLs could have been due to the interaction of
the sMSR with the native scavenger receptor on cell membranes.
sMSR-containing medium was incubated with RAW 264 cells for 9 hours and
then replaced with control medium containing
125I-acLDL. However, preincubation did not
decrease 125I-acLDL degradation, whereas
simultaneous addition of the sMSR and
125I-acLDL decreased the degradation by 70%
(Figure 7).
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In experiments with 125I-labeled LDLs, the sMSR
was able to decrease the degradation of oxLDL and acLDL when the
modified LDL was present in small quantities (ie, 10 µg/mL). To
test whether the sMSR could inhibit the uptake of larger quantities of
modified lipoproteins, we tested the effect of sMSR on foam-cell
formation in RAW 264 macrophages. Cells were incubated with 100
µg/mL of acLDL in the presence of 4 µg/mL of cytochalasin D and in
the absence or presence of sMSR medium. Addition of medium containing
sMSR caused a marked decrease in RAW 264 macrophage lipid
accumulation and foam-cell formation (Figure 8).
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Organ distribution of sMSR–125I-acLDL complex
was evaluated in vivo by injecting the complex into the tail vein of
MSR-A/LDL receptor double-knockout mice. The results suggest that the
accumulation of the complex was enhanced in liver compared with mice
injected with 125I-acLDL alone (Figure 9).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We have engineered a recombinant adenovirus that expresses a secreted form of human MSR under the control of the human macrophage CD68 promoter. It was found that sMSR effectively inhibited lipid accumulation and foam-cell formation induced by modified LDL in RAW 264 macrophages.
Several transmembrane receptors for growth factors and cytokines have a soluble counterpart that may participate in the regulation of growth hormone or cytokine effects by preventing the ligand from interacting with the transmembrane receptor.22 23 24 25 Secreted proteins have also been used in therapeutic studies to block the activity of the native transmembrane receptor.22 23 24 Thus, secreted decoy receptors offer potential new tools to influence pathological processes in vivo. Major problems with this approach have been the low affinity of engineered soluble receptors for physiological ligands and inability to achieve high enough concentrations of soluble decoy receptors in target tissues. Also, it may be difficult to obtain long enough expression of the transgene in the target tissue and avoid potential immunological problems related to repeated adenoviral gene transfer. However, secreted receptors could still prove useful in diseases in which even a relatively small amount of protein delivered locally could have a therapeutic effect and in which the most important therapeutic target is to clear the ligand from the treated area.
Adenoviral gene transfer of the sMSR was chosen because it was recently shown that catheter-mediated delivery of adenoviral vectors can lead to 5% transfection efficiency in human atherosclerotic arteries, which may be enough to achieve clinical effects with secreted transgene products.16 Soluble sMSR cloned in this study was placed under the control of the CD68 promoter, which directs a high expression level in macrophages.14 This was considered desirable because most of the scavenger receptor activity in human atherosclerotic lesions is expressed by macrophages.8 9 26 Transcriptional targeting of adenoviral gene transfer to certain cell types is also an additional safety feature in the use of recombinant viruses. The 2.9-kb CD68 promoter does not have a complete macrophage locus control region,14 and it was found that the promoter was also active in ECV 304 cells, whereas only a weak expression was detected in fibroblasts and SMCs. Expression in endothelial-like cells was considered beneficial in terms of possible local in vivo applications in the treatment of atherosclerosis and restenosis. Thus, the engineered sMSR adenovirus was able to direct clearly detectable transcriptional targeting to cell types involved in the processing of modified LDL in the arterial wall.
Poly(G) resin was able to bind almost all of the sMSR in the medium, indicating that sMSR presents ligand binding characteristics similar to those of the native MSR. Similar binding characteristics have previously been reported by Resnick et al17 and by Andersson et al.27 However, there are no reports about the ability of sMSR to show biological effects on macrophages or to prevent foam-cell formation. The findings suggest that ligand binding to sMSR in the growth medium is the mechanism of the inhibition of the native scavenger receptor activity. This conclusion is further supported by the fact that sMSR was also effective in reducing the degradation of acLDL in MSR-A knockout mouse macrophages in vitro via other putative scavenger receptors that presumably rely on the recognition of binding domains similar to those of MSR.
Our results suggest that sMSR may be a useful tool for studies regarding atherogenesis and macrophage functions. Because MSRs play an important role in foam-cell formation in vivo,8 9 10 it is important to note that sMSR inhibited foam-cell formation in RAW 264 macrophages even in the presence of high concentrations of modified LDL. Our results also suggest that in vivo, the accumulation of sMSR-modified LDL complex is enhanced in liver. This should be beneficial, because the liver is the major organ responsible for the secretion of excess cholesterol from the body. It is also possible that sMSR may affect monocyte/macrophage adhesion in progressive atherosclerotic lesions. The results warrant in vivo evaluation of the adenovirus-mediated gene delivery of sMSR for the treatment of atherosclerosis and lipid accumulation after intravascular manipulations.
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Acknowledgments |
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This study was supported by grants from the Finnish Foundation for Cardiovascular Research, the Sigrid Jusélius Foundation, the Finnish Academy, and Kuopio University Hospital (EVO grants 5102 and 5130). Dr Gordon is supported by the UK Medical Research Council and the British Heart Foundation. Dr Gough held an Arthritis and Rheumatism Council studentship, and Dr Greaves is supported by Glaxo-Wellcome. The authors thank Dr Tatsuhiko Kodama, MD (University of Tokyo) for providing knockout mice; Aila Erkinheimo, Eila Pelkonen, and Anne Martikainen for skillful technical assistance; and Marja Poikolainen for preparing the manuscript.
Received July 8, 1999; revision received September 21, 1999; accepted October 6, 1999.
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