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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:52-60

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:52.)
© 2000 American Heart Association, Inc.

Vascular Biology

Retrovirus-Mediated, Stable Scavenger-Receptor Gene Transfer Leads to Functional Endocytotic Receptor Expression, Foam Cell Formation, and Increased Susceptibility to Apoptosis in Rabbit Aortic Smooth Muscle Cells

Pauliina Lehtolainen; Motohiro Takeya; Seppo Ylä-Herttuala

From the A.I. Virtanen Institute (P.L., S.Y.-H.) and the Department of Medicine (S.Y.-H.), University of Kuopio, Kuopio, Finland, and the Kumamoto University School of Medicine (M.T.), Kumamoto, Japan.

Correspondence to Seppo Ylä-Herttuala, MD, PhD, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, Neulaniementie 2, FIN-70211 Kuopio, Finland. E-mail Seppo.Ylaherttuala{at}uku.fi


*    Abstract
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Abstract—The type II, class A macrophage scavenger receptor (SR-A) plays an important role in the pathogenesis of atherosclerosis and foam cell formation. However, its role in nonmacrophage cell lines remains unknown. To test the hypothesis that SR-A activity leads to proatherogenic changes in nonmacrophage cell lines, we generated Moloney murine leukemia virus– and vesicular stomatitis virus G protein–pseudotyped retroviruses containing SR-A type II cDNA, which were used for stable transfection of SR-A activity into mouse fibroblasts and rabbit aortic smooth muscle cells (SMCs). ß-Galactosidase–transfected cell lines were used as controls. Transfected cell lines expressed functional SR-A mRNA and protein. Expression of SR-A activity was stable for at least 9 months. By electron microscopy, transfected receptors were located in coated pits and in intracellular structures resembling endocytotic vesicles. Expression of SR-A on the cell surface was verified by flow cytometry and by uptake and degradation of 125I-labeled acetylated low density lipoprotein (LDL). Increases of 5- to 25-fold and of 6- to 8-fold in the rate of acetylated LDL degradation were observed in transfected fibroblasts and SMCs, respectively, compared with ß-galactosidase–transfected control cell lines. Incubation of the transfected SMCs and fibroblasts with acetylated or oxidized LDL led to foam cell formation. Incubation with oxidized LDL also led to increased apoptosis and cell death. An altered morphology with increased cell size and granularity was observed in the most active SR-A SMC clones. It is concluded that stable overexpression of SR-A leads to foam cell formation and other proatherogenic changes in nonmacrophage cell lines. Stable SMC and fibroblast cell lines can be used as models for foam cell formation. The results also suggest that increased SR activity may play an important role in SMC-related pathology in atherosclerotic arteries.


Key Words: atherogenesis • gene transfer • retrovirus • oxidized LDL


*    Introduction
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 *Introduction
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The class A macrophage scavenger receptor (SR-A) belongs to a family of receptors that mediate the uptake of several negatively charged macromolecules, such as oxidized (ox) LDL.1 2 3 4 SR-A also participates in cell adhesion and defense against microorganisms.5 In atherogenesis, SR-A–mediated uptake of oxLDL plays an important role in the formation of lipid-filled macrophages.3 6 7 Expression of SR-A in macrophages in human, rabbit, and mouse atherosclerotic lesions has been demonstrated,6 7 8 9 10 and SR-A–knockout mice develop less atherosclerosis than do their littermates.11 SR-A expression is also induced >100-fold in macrophages in developing atherosclerotic lesions.12

Although the importance of SR-A activity in macrophages has been clearly established, the role of SR-A in nonmacrophage cell lines remains unclear. Although cultured rabbit fibroblasts and smooth muscle cells (SMCs) express SR-A activity on stimulation with phorbol esters,13 no or very limited SR-A expression has been detected in SMCs in human, Watanabe heritable hyperlipidemic rabbit, or cholesterol-fed New Zealand White rabbit atherosclerotic lesions.6 8 9 Also, SR-A expression in atherosclerotic lesions of apoE3-Leiden transgenic mice has been detected in macrophages but not in SMCs.10 However, in cholesterol-fed New Zealand White rabbits, in which the arterial wall was damaged by balloon denudation, SR-A immunoreactivity was detected in a small number of proliferating, intimal SMCs.14 15 Whether expression of the SR-A in SMCs and fibroblasts in vivo leads to foam cell formation and other proatherogenic changes remains unknown.

Retrovirus-mediated gene transfer can be used for stable transfection of eukaryotic cell lines in which gene expression is driven by a retroviral long-terminal repeat (LTR) promoter.16 Cell lines transfected by retroviral techniques have proved valuable for the analysis of transgene functions because they can lead to long-lasting, high-level expression of the transgene that is not regulated by natural promoters. Retroviruses also open up possibilities for in vivo gene transfer with prolonged transgene expression in target tissues.16

The aim of the study was to test the hypothesis that SR-A activity leads to proatherogenic changes in nonmacrophage cell lines. It was found that SR-A expression leads to foam cell formation, enhanced cell death, and apoptosis in stably transfected SMCs and fibroblasts.


*    Methods
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Virus Vectors and Cell Cultures
Retroviral vector (Figure 1Down) was constructed from a parental vector pLRNL (where L=LTR, R=Rous sarcoma virus promoter, N=neomycin, and L=LTR)17 by insertion of a bovine SR-A type II cDNA.1 2 in sense (pLSRNL) and antisense (pLSrRNL) orientation under control of the Moloney murine leukemia virus (MMLV) LTR. The internal neomycin-resistance gene is controlled by an RSV promoter. Plasmid sequences were verified by automated sequencing (A.L.F DNA sequencer, Pharmacia). Two C-deletion mutations occurred in the noncoding region of pLSRNL. Retrovirus vector containing Escherichia coli ß-galactosidase (LacZ) cDNA18 (pLZRNL) was used as a control.19



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  		Figure 1. Retroviral plasmids. A, Fragment (nucleotides 1 to 2003) of the bovine SR-A cDNA1 2 was cloned into a vector containing the MMLV LTR cassette with a neomycin-resistance gene (neo) under the control of the internal RSV promoter.17 B, A full-length E coli ß-galactosidase (ß-gal) cDNA18 was cloned into the same retroviral vector and was used as a control. SR-A and ß-galactosidase are expressed from the constructs by the 5'-LTR promoter. Polyadenylation signals are provided by the 3'-LTR. The rest of the plasmid (not shown) is derived from pBR322.

{psi}2 and PA317 mouse fibroblast cell lines,20 21 rat fibroblast cell line F209,17 human embryonal kidney 293GP cell line,22 and rabbit aortic SMC line RAASMC (established in our laboratory from a newborn New Zealand White rabbit aortic explant19 ) were used in the study. Cells were verified as SMCs by {alpha}-actin immunostaining. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) at 37°C in a 5% CO2 atmosphere. The growth medium was supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine (Gibco). Neomycin-resistant cells were maintained in the same medium containing neomycin analogue G418 (400 µg/mL, Sigma). Peritoneal macrophages were harvested from the peritoneal cavity of 8-week-old male mice.23 Macrophages were cultured in RPMI containing 10% FBS and used for the experiments within 36 hours after isolation.

Production of Retroviruses and Stable Cell Lines
MMLV Retroviruses
For MMLV retroviruses, the ecotropic packaging cell line {psi}220 was transfected with retroviral plasmids by using a standard calcium phosphate precipitation method. Viruses were harvested from {psi}2 cells after 48 hours and used for infection of amphotropic PA317 packaging cells21 as follows. On day 0, PA317 cells were plated at 5x105 cells/10-cm dish. On day 1, monolayers were incubated for 16 hours with filtered (0.45 µm) {psi}2 supernatants in the presence of 8 µg/mL Polybrene (Sigma). On day 2, the medium was replaced with G418-containing medium. After 10 to 12 days of selection, G418-resistant clones were isolated and expanded.19

Pseudotyped Retroviruses
Pseudotyped retroviral vector pLSRNL containing the G glycoprotein of vesicular stomatitis virus (VSV-G) was produced in human kidney 293GP cells cotransfected with MMLV gag and pol genes.24 293GP cells were transfected by using a standard calcium phosphate coprecipitation with 50 µg of pLSRNL DNA. Stable clones expressing SR-A were selected by using 600 µg/mL geneticin. A stable producer clone was selected for the production of pseudotyped pLSRNL virus. To generate pseudotyped virus, the producer clone was transfected with pHCMV-G expression plasmid as described.24 Fresh medium containing 10% FBS was changed after 8 to 12 hours of transfection. Pseudotyped viruses generated from the transfected cells were collected 24, 48, and 72 hours after the change of medium. Collected supernatants were filtered (0.45 µm) and subjected to ultracentrifugation in a Beckman SW-28 rotor at 50 000g (25 000 rpm) at 4°C for 120 minutes. The pellets were resuspended overnight at 4°C in 200 µL of 0.1x Hanks’/1% sucrose in PBS and combined before titration and storage.25 The concentrated viruses containing either SR-A or LacZ were used to transduce RAASMCs. A multiplicity of infection of 10 to 100 viruses was used to transduce RAASMCs in the presence of 8 µg/mL Polybrene for 16 hours. After 48 hours the cells were trypsinized, and selection was started with 600 µg/mL geneticin. After 10 to 12 days of selection, G418-resistant SMC clones were isolated and expanded.19

Determination of Virus Titers
MMLV- and VSV-G–pseudotyped viruses were titrated on F209 cells in the presence of Polybrene (8 µg/mL).19 Titers of the best MMLV producer clones for pLSRNL, pLSrRNL, and pLZRNL were 7x104, 2x103, and 8x104 colony-forming units (cfu)/mL, respectively. Titers of the unconcentrated, pseudotyped viruses pLSRNL and pLZRNL were 104 to 106 cfu/mL. After concentration the titers were 106 to 107 cfu/mL.

Northern and Southern Blot Analyses
Poly(A)+ mRNA was isolated from the clones by an SDS/proteinase K method and used for Northern blot analysis.26 Hybridizations were carried out overnight at 42°C with 106 counts per minute per milliliter of random-primed 32P-labeled bovine SR-A probe, neomycin probe, or ß-actin probe.26 Genomic DNA was isolated from stably transfected clones and from untransduced control cells by proteinase K treatment for Southern blot analysis.27 Genomic DNA (30 µg) was digested with SduI, BspTI, and Mva1269I restriction enzymes (Fermantas, Finnzymes), and the fragments were separated in 0.8% agarose gel, blotted onto a nylon membrane, and hybridized with the SR-A probe. Autoradiography was used for signal detection.

Immunostaining
Cells were plated on glass chamber slides and were grown in 10% lipid-deficient serum/DMEM. Cells were fixed with 4% paraformaldehyde for 10 minutes at 20°C, permeabilized in 0.2% Triton X-100/PBS for 10 minutes, and washed with PBS. Thereafter, the cells were blocked in 3% BSA/PBS for 2 hours and incubated with SRKO 4 antibody15 for 1 hour and subsequent incubation with biotinylated horse anti-mouse IgG (Vector Laboratories) for an additional 30 minutes. An avidin-biotin–horseradish peroxidase system (Vector Laboratories) was used for immunostaining. The nuclei were stained with hematoxylin. Cells incubated without the primary antibody were used as controls.

Immunoelectron Microscopy
To observe the localization of SR-A and SR-A–mediated endocytosis by immunoelectron microscopy, cell samples of untransduced and transduced PA317 fibroblasts were incubated for 48 hours in the presence of acetylated (ac) LDL (5 µg/mL) and fixed for 10 minutes with 0.05% glutaraldehyde in 0.165 mol/L cacodylate buffer, pH 7.4. Immunostaining was carried out for 1 hour with an anti-bovine SR-A IgG-D2.28 The samples were then incubated for 1 hour with an anti-mouse immunoglobulin [F(ab')2] conjugated with peroxidase (Amersham). Between each staining step, washing with PBS containing 0.005% saponin (Sigma) was performed as previously described29 to facilitate permeabilization of the cells. Peroxidase activity was visualized with 3,3'-diaminobenzidine as a substrate. After diaminobenzidine staining, the cells were postfixed with 1% OsO4 for 60 minutes, dehydrated through a graded series of ethanols, embedded in Epon 812, and sectioned serially. Ultrathin sections were observed with an H-7500 electron microscope (Hitachi) without counterstaining.

Analysis of Lipoprotein Metabolism
Fluorescence Microscopy and Flow Cytometry Analysis to Measure DiI-acLDL Uptake
LDL (d=1.019 to 1.063 g/mL) from fresh, human plasma was isolated by ultracentrifugation as described.30 LDL was labeled with fluorescent DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular Probes) as described31 and then acetylated. For competition studies, LDL was acetylated with acetic anhydride32 and oxidized by overnight incubation with copper ions (20 µmol/L).23 For the uptake assay of fluorescent lipoproteins, cells were incubated at 37°C on chamber slides with growth medium containing 10 µg/mL DiI-acLDL for 4 to 8 hours in the presence or absence of acLDL, oxLDL, or polyinosinic acid (Sigma). Cells were either analyzed by flow cytometry or fixed with 0.05% glutaraldehyde or 4% paraformaldehyde (10 minutes, 20°C) for fluorescence microscopy.33

For flow cytometry analysis, untransduced cells and stably transduced clones were grown in 10% FBS/DMEM and trypsinized 2 days before the assay. In some experiments, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 24 hours. Cultured fibroblasts or SMCs (1 to 2x106 cells) were washed with PBS and incubated with 10 µg/mL DiI-acLDL in 10% lipid-deficient serum/DMEM at 37°C for 5 hours. For competition assays, a 25-fold excess of acLDL, oxLDL, and polyinosinic acid was used. Immediately after the incubations, the cells were placed on ice and washed twice with ice-cold PBS. Cells were harvested by trypsinization and suspended at a concentration of 106 cells/mL in 2% FBS/PBS. Cells (5x103 to 104) were analyzed by fluorescence-activated cell sorter (FACS, Becton Dickinson, research software version B 2/88) and an argon-ion laser (514-nm excitation) and a 580-nm filter. The data collected from the cells were analyzed with Consort 30 software.

Degradation Assay
For the degradation assay, LDL was iodinated with carrier-free Na125I,23 acetylated with acetic anhydride,32 and oxidized by overnight incubation with copper ions (20 µmol/L).23 Modified, iodinated LDL was used for the degradation analyses at 5 to 10 µg/mL. A 25-fold excess of unlabeled acLDL or oxLDL was used in the competition assays. After the cells were washed, the amounts of 125I-labeled, acid-soluble material in the medium (degradation) and in the cells (cell-associated activity) were determined.23 Values obtained from cell-free wells were subtracted before calculating the results. Protein concentrations were determined by the method of Lowry et al.34

Lipid Staining
Cells (5x103 to 104) were preincubated on chamber slides for 2 days in 10% lipid-deficient serum/DMEM. On the third day, the medium was removed and fresh medium with 100 to 360 µg/mL acLDL was added. For competition analysis, a 5-fold excess of polyinosinic acid was used. After a 1- to 3-day incubation, the cells were washed with 1x PBS, fixed with 1.25% glutaraldehyde, and stained with oil red O for 15 minutes, followed by hematoxylin counterstaining. Cells were then washed in tap water, and lipid staining was evaluated by light microscopy.

Apoptosis and Cell Death Assay
Annexin V (annexin V–FITC apoptosis detection kit, Genzyme) was used for identification of the cells undergoing an early phase of apoptosis. Annexin V binds to exposed phosphatidylserine in the outer leaflet of the plasma membrane due to disruption of phospholipid asymmetry characteristic of early apoptotic events. Because translocation of phosphatidylserine to the external cell surface also occurs during necrosis, annexin V–FITC was used in conjunction with the vital dye propidium iodide (PI) to distinguish apoptotic cells (annexin+/PI-) from necrotic cells (annexin-/PI+) or those cells that had already died due to apoptosis (annexin+/PI+). Uptake of the fluorescent dyes was measured from 104 cells by flow cytometry. The protocol was standardized by using etoposide (8 µmol/L) and okadaic acid (5 nmol/L) treatments, which are known to cause apoptosis in cultured cells.35 36


*    Results
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 *Results
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Northern and Southern Blot Analyses
After gene transfer, 33 pLSRNL retrovirus producer clones were selected for further analysis. All of the isolated clones were positive for neomycin mRNA (Figure 2Down). Seventy-five percent of the clones were also positive for SR-A mRNA. Northern blot analysis of poly(A)+ mRNA showed one 5.5-kb band in the transduced pLSRNL cells with the bovine SR-A probe and 5.5- and 2-kb bands with the neomycin probe, corresponding to transcripts driven by the LTR (polycistronic mRNA) and the internal RSV promoter, respectively. Clones 11 and 23 expressed only the 2-kb internal neomycin transcript.



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  		Figure 2. Northern blot analysis of poly(A)+ RNA prepared from untransduced PA317 cells and stably transduced SR-A and LacZ clones. mRNA (1 µg) was electrophoresed on 1% agarose/2.2 mol/L formaldehyde gel, followed by hybridization with SR-A, neomycin, and actin probes. St indicates standard total RNA isolated from rabbit lung; C, untransduced PA317 cells, which showed no hybridization with SR-A or neomycin probes. Lanes 3 to 10, PA317 SR-A clones 5, 10, 21, 14, 17®, 11®, 27, and 23, respectively (® after clone number represents SR-A in reverse orientation). The clones showed varying amounts of SR-A and neomycin mRNA. Z, the PA317/LacZ-expressing control cells, showed no SR-A mRNA, but polycistronic LacZ+neo- retrovirus cassette (6.5 kb) and monocistronic neo mRNA (2 kb) were detectable. 28S and 18S are used as RNA size markers. They were determined from the standard total RNA samples run on lanes 1 and 12.

To document the integration of the SR-A cDNA into the genome of the transduced cells, Southern blot analysis of several clones was performed (data not shown). After digestion of the clones with BspTI, a 5-kb band was obtained in all but 2 clones, thus verifying integration of the complete retroviral cassette. Three smaller bands were present in the 2 aberrant clones. The reason for the additional bands in these clones remains unknown, but it may be due to rearrangements in the retroviral cassette during the integration step (data not shown). Thus, retroviral gene transfer does not always lead to a properly oriented functional expression cassette in the transduced cells.

Immunocytochemistry and Immunoelectron Microscopy
The presence of SR-A in the transduced cells was studied by immunocytochemistry. Protein was located in the cytoplasm and was concentrated in areas near the nucleus (Figure 3Down). The presence of SR-A in the transduced mouse fibroblasts was studied by immunoelectron microscopy. A positive reaction for SR-A was detected in the transduced cells (Figures 4ADown and 4BDown). At higher magnification, the reaction for SR-A was observed on the plasma membrane condensed at coated-pit–like structures and in some intracellular structures similar to endocytotic vesicles (Figure 4CDown). These findings suggest, but do not prove, participation of the transduced SR-A in a functional endocytotic pathway. No positive reaction was observed in the untransduced control cells (Figure 4DDown).



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  		Figure 3. Presence of SR-A protein in transduced RAASMCs as studied by immunostaining with SRKO 4 antiserum, 1:200 dilution. A, SR-A–transduced RAASMCs (clone 14). B, Higher magnification of cells in A. C, LacZ-transduced control RAASMCs. D, Nonimmune control without primary antibody. Diaminobenzidine staining. Magnification of A, C, and D, x50; B, x100.



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  		Figure 4. Immunoelectron microscopic localization of SR-A protein in stably transduced fibroblasts cultured in the presence of acLDL (5 µg/mL). A, Stably transduced mouse fibroblasts (clone 5), x2500. B, Serial section of the same cell shown in A, x5600. C, Enlargement of the rectangle in B, x18 000. D, Untransduced control cells, x18 000. Reaction products for SR-A are localized on the plasma membrane on coated pits (A, B, and C, arrowheads) and in some intracellular structures resembling endocytotic vesicles (B and C, arrows). Untransduced control cells showed no reaction for SR-A on the plasma membrane coated pits (D, arrowhead) nor in the intracellular compartment.

Fluorescence Microscopy and Flow Cytometry Analysis of DiI-acLDL Uptake
As analyzed by fluorescence microscopy, transduced RAASMC clones efficiently internalized DiI-acLDL, whereas untransduced cells did not express any detectable SR-A activity (Figure 5Down). In the transduced cells, DiI-acLDL was detected in spherical vesicles that were evenly distributed throughout the cytoplasm (Figure 5Down).



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  		Figure 5. DiI-acLDL binding and uptake in untransduced SMCs (A and C) and SR-A–transduced SMCs (clone 14; B and D). A and B, Fluorescence images of the cells; C and D, phase-contrast images of the same cells. Cells were incubated with 10 µg/mL DiI-acLDL for 5 hours at 37°C, fixed, and analyzed by fluorescence microscopy. Magnification x100.

Transduced fibroblasts and SMCs analyzed by flow cytometry showed functional SR-A receptors on the cell surface. No uptake of DiI-acLDL was detected in the untransduced control cells (Figure 6ADown) unless they were stimulated. SMC clone 14 showed 60% to 80% higher DiI-acLDL uptake activity than the untransduced control SMCs, and the uptake was totally inhibited by a 40-fold excess of unlabeled acLDL or polyinosinic acid. Cytochalasin D was used to inhibit phagocytosis, but it had no effect on the uptake of DiI-acLDL by the transduced SMCs (data not shown). Flow cytometry analysis also revealed altered morphology of the SR-A–transduced SMCs compared with untransduced, control SMCs. The transduced cells were bigger and more granular than the control cells (Figure 6BDown). PMA (50 ng/mL) pretreatment for 24 hours increased the binding of DiI-acLDL in LacZ-transduced control cells, but only a small increase was seen in SR-A–transduced cells after PMA treatment (Figure 6CDown).



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  		Figure 6. Flow cytometry analysis of binding of DiI-acLDL by transduced SMCs. A, Measurement of 104 cells revealed DiI-acLDL binding in 88% of SR-A–transduced RAASMCs (black area) compared with untransduced control cells (hatched line, white area). B, The forward/side light scatter of untransduced SMCs (control, upper panel) and SR-A–transduced SMCs (clone 14, lower panel), where green dots represents negative fluorescence and red dots positive fluorescence, showing the binding of DiI-acLDL. Analysis showed increased cell size and granularity in SR-A–transduced SMCs, which were able to bind DiI-acLDL (red dots). Solid line indicates gated area; ie, cells used for the analysis. Same cells as in Figure 6AUp. C, Graph showing effect of PMA (50 ng/mL, 24 hours) on the binding of DiI-acLDL in LacZ-transduced control SMCs (control, upper panel) and in SR-A–transduced SMCs (clone 14, lower panel) from 104 living cells. Flow cytometry analysis of PMA-stimulated cells are shown as a black area and from unstimulated cells as dotted lines.

Degradation Assays
To demonstrate that the transduced SR-A was functional, degradation rates of 125I-labeled acLDL were studied. Degradation activity was measured in 8 fibroblast clones and 2 SMC clones (Figure 7Down). Six fibroblast clones had SR-A in the right orientation (clones 5, 10, 14, 16, 21, and 23) and 2 clones (11 and 17) in the reverse orientation (derived from pLSrNL). Degradation of 125I-acLDL in the best-transduced clones (5, 16, 21, and 14) having SR-A in the right orientation was 9- to 22-fold higher than in the control cells. The degradation activity of the untransduced control cells, SR-A clones 17, 11, and 23, and in LacZ-transduced cells was <10 ng/mg cell protein, whereas corresponding values for the best-transduced clones 5, 16, 21, and 14 were 216±59, 126±96, 114±44, and 86±11 ng/mg cell protein, respectively (Figure 7ADown). Displacement assays with a 25-fold excess of unlabeled acLDL showed a 42% decrease in the degradation of 125I-acLDL in the most active clone 5. For comparison, the degradation of 125I-acLDL in mouse peritoneal macrophages was {approx}2.5 to 8 µg/mg cell protein (data not shown). Two transduced SMC clones (clones 12 and 14) showed 6- to 8-fold increased degradation of 125I-acLDL (Figure 7BDown). Degradation was effectively competed by unlabeled acLDL, oxLDL, and polyinosinic acid. In long-term cultures, the degradation rates of the transfected clones were somewhat lower but remained stable for at least 9 months (data not shown). Average degradation activities of the transduced clones were 487 ng/mg cell protein for clone 14 and 293 ng/mg cell protein for clone 12. A 25-fold excess of the unlabeled acLDL or polyinosinic acid decreased the degradation to 20% and 10% of the control values, respectively, whereas oxLDL decreased the degradation to 40% of the controls (Figure 7BDown). The degradation of 125I-oxLDL was 2-fold higher in SMC clone 14 compared with untransduced SMCs (data not shown), indicating an increased uptake of oxLDL via SR-A.



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  		Figure 7. Degradation of 125I-acLDL in transduced fibroblasts and SMCs. A, Values from untransduced fibroblasts (C), 8 SR-A clones, and LacZ (Z) -transduced fibroblasts. B, Values from LacZ-transduced SMCs (C) and 2 SR-A–transduced SMC clones. Cells were incubated for 9 hours with 5 µg/mL (A) and 10 µg/mL (B) 125I-acLDL in the absence or presence of unlabeled competitors. The results are mean values ±SD of 3 experiments.

Lipid Staining
Clear differences were seen in lipid loading after oil red O staining in the transduced and untransduced fibroblasts and SMCs (Figure 8Down). Incubation with 100 µg/mL acLDL led to foam cell formation in SR-A–transduced fibroblasts, whereas 360 µg/mL acLDL was needed to cause foam cell formation in the SR-A–transduced SMCs. Some differences were observed in the distribution pattern of lipid vesicles in fibroblasts and SMCs after incubation with 100 µg/mL acLDL. In fibroblasts, the lipid vesicles were mostly seen in perinuclear areas, whereas in SMCs the lipid vesicles were mostly located in the periphery of the cell (Figure 8Down).



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  		Figure 8. Foam cell formation in transduced fibroblasts (A through F) and SMCs (G through L). Cells were incubated with 100 µg acLDL (C through F, I through K) or with 360 µg/mL acLDL (L) for 3 days and stained with oil red O. A, Untreated control fibroblasts; B, untreated, transduced fibroblast clone 5; C, incubated control fibroblasts; D, incubated, transduced fibroblast clone 5 showing massive lipid loading; E, same as D in the presence of 500 µg/mL polyinosinic acid, which inhibits foam cell formation; F, higher magnification of a foam cell in D; G, untreated control SMCs; H, untreated SMC clone 14; I, incubated control SMCs; J, incubated SMC clone 14 showing lipid loading; K, distribution pattern of lipid vesicles in incubated SMC clone 14 cells; L, clone 14 SMCs incubated with 360 µg/mL acLDL showing foam cell formation. Magnification x50 (A through E, G through J, L), x100 (F, K).

Apoptosis and Cell Death
The effects of oxLDL on apoptosis and cell death were measured in SR-A–transduced SMCs and control cells by flow cytometry. OxLDL induced apoptosis and cell death in SMCs during a 48- to 72-hour incubation at concentrations of 200 to 500 µg/mL. A similar apoptotic response was seen in fibroblasts with less amount of oxLDL (50 to 200 µg/mL). After a 72-hour incubation in the presence of 200 µg/mL oxLDL, 18% of the SR-A–transduced SMCs were in early apoptosis (annexin+/PI-) compared with 7% in the control cells (Figure 9ADown). The proportion of late apoptotic (annexin+/PI+) cells was also moderately increased. Untreated control and transduced SMCs were primarily annexin-/PI-, indicating that they were viable and not undergoing enhanced apoptosis or cell death. Incubation with acLDL or native LDL did not induce any significant apoptosis (data not shown). A 48-hour incubation with a higher concentration of oxLDL (500 µg/mL) increased annexin V positivity in the SR-A–transduced SMCs by 3.6-fold compared with the control cells (Figure 9BDown). In the transduced SMCs, a 4.3-fold increase in PI+ but annexin- cells (ie, necrotic cell death) and a 2.9-fold increase in the control cells were seen when compared with untreated control cells, indicating that the higher concentration of oxLDL also induced cell death without apoptosis.



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  		Figure 9. Flow cytometry analysis of apoptosis and cell death as measured by annexin V–FITC/PI staining in transduced SMC clone 14 after incubation with oxLDL. A, Results are shown as relative fluorescence units (% of untreated, control cells), where annexin+/PI-, annexin+/PI+, and annexin-/PI+ are each 100%. After a 72-hour incubation in the presence of 200 µg/mL oxLDL, a 2.3-fold increase was seen in the proportion of early apoptotic cells (annexin+/PI-) in the SR-A–transduced SMC clone compared with a 1.5-fold increase in control cells. The proportion of late apoptotic cells (annexin+/PI+) was increased 2.6-fold in the transduced clone and 2.3-fold in the control cells. B, Incubation with oxLDL (48 hours, 500 µg/mL) caused a 3.6-fold (lower panel) increase in annexin V positivity (FITC fluorescence intensity, left panel) in SR-A–transduced SMCs compared with control SMCs (upper panel). A 4.3-fold increase in PI positivity (PI fluorescence intensity, right panel) in transduced SMCs compared with a 2.9-fold increase in control SMCs indicated an increased susceptibility to apoptosis and cell death in the transduced cells.


*    Discussion
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 *Discussion
down arrowReferences
 
Retrovirus techniques were used to generate cell lines that express SR-A activity.37 Advantages of retroviral gene transfer include stability of the cell lines over long periods of time; a several-fold increase in the transgene activity that can be achieved under the viral LTR promoter; and inclusion of a selection marker in the expression cassette, which permits isolation and maintenance of homogeneous clones of the transduced cells. Thus, these stable cell lines offer several advantages over conventional transient transfections wherein significant variation exists in transfection efficiency and duration of the transgene activity. Possible limitations of the retroviral techniques include unpredictable alterations in target cell functions due to random integration of the transgene and the inability to regulate transgene function by endogenous regulators.37

The role of SR-A in the development of macrophage-derived foam cells is well established. In human, rabbit, and mouse arteries, SR-A receptors are expressed in lesion macrophages.6 7 8 9 10 Because oxLDL is present in atherosclerotic lesions,23 38 it is conceivable that SR-A could lead to lipid accumulation and foam cell formation. The role of SR-A in atherogenesis has been confirmed in SR-A gene–disrupted mice, which develop 60% less atherosclerosis when bred with apoE-knockout mice compared with control mice.11 Also, marked variability in the induction of SR-A activity has been reported in 2 strains of rabbit that differ in their susceptibility to atherosclerosis.39

Inconsistent data exist regarding the role of SR-A in nonmacrophage cell lines. Immunohistochemical studies suggest that SR-A is expressed in some SMCs in the balloon-injured, hypercholesterolemic rabbit neointima,14 40 whereas in other studies, SR-A mRNA or immunoreactive protein has not been identified in human, rabbit, or mouse lesion SMCs.6 8 9 10 However, in vitro rabbit and human SMCs express SR-A after PMA stimulation. Endothelial cells are known to take up acLDL, and SR-A immunoreactivity has been detected in the endothelium with the use of anti-peptide antibodies,41 whereas no such reactivity has been found in other studies.6 8 9 10 40 It is possible that conflicting results are at least partly due to differences in the specificity of reagents used for the studies.

We have demonstrated that functional SR-A activity can be stably expressed in SMC and fibroblast cell lines. Transfection leads to foam cell formation with both acLDL and oxLDL. Even though human atherosclerotic lesions contain a significant portion of SMC-derived foam cells, it has been difficult to generate SMC foam cells in vitro.32 42 Accordingly, the consequences of foam cell formation to SMC biology have been difficult to study. Stably transfected cell lines can be used as models of foam cell formation and lipid accumulation in vitro. The capacity of transfected SMCs to take up acLDL or oxLDL was much lower than that of macrophages, which suggests that SMC-derived foam cells might not develop as rapidly as macrophage-derived foam cells. However, human lesions develop slowly during the first 3 decades of life,43 44 and it is conceivable that even a low SR-A activity could lead to a gradual oxLDL accumulation in arterial SMCs over several years. Increased oxLDL accumulation without foam cell formation may also alter SMC functions through various effects of oxidized lipids on transcription factors and gene expression.45 46 47 48 Other lipoprotein receptors than SR-A and uptake mechanisms may also contribute to lipid uptake in lesion SMCs.4

It was found that after incubation with oxLDL, the stably transduced SMC lines were more susceptible to apoptosis and cell death, which together with foam cell formation could contribute to atheroma formation and lesion development. These findings are in line with recent reports that oxLDL can cause apoptosis in cultured cells and that apoptotic cell death occurs in human and experimental atherosclerotic plaques.45 46 47 49 50 Susceptibility to apoptosis in response to oxLDL incubation was increased in the SR-A–transduced cells, from 1.5- to 2.6-fold, in comparison with the untransduced cells, which is in line with a 2-fold increase in the degradation of 125I-oxLDL seen in the transduced cells. OxLDL caused more apoptosis compared with acLDL, despite the fact that an 8-fold increase in the rate of 125I-acLDL degradation was seen in the transduced SMCs. However, it is impossible to fully distinguish the degree of apoptosis caused by the SR-A–mediated uptake of oxLDL from that of the non–SR-A–mediated uptake. The results also suggest that the role of SR-A in cell death is indirect and due to an increased uptake of oxLDL. It is important to note that the increased rate of apoptosis was not associated with an increased rate of replication (P.L. et al, unpublished observations, 1998).51

Even though apoptosis has been shown to occur in plaques and not in fatty streaks, SMCs within human fatty streaks express Bax, a proapoptotic protein of the Bcl-2 family52 that increases the susceptibility of cells to apoptosis.53 The expression of Bax can activate a common pathway of apoptosis by triggering a mitochondrial cytochrome c release.54 It has also been shown that apoptosis in vascular SMCs can be induced through caspase-3 activation and Bcl-2 downregulation by lipid peroxides, which are present in oxLDL,55 and that oxidative stress response elements are present in the SR-A promoter of human SMCs.56

In conclusion, our results show that SR-A expression in SMCs and fibroblasts leads to proatherogenic changes by increasing lipid accumulation and predisposing cells to apoptosis. In addition to the significant role of SR-A in macrophages, these receptors may participate in atherogenesis by predisposing nonmacrophage cells to proatherogenic changes. It is possible that modulation of SR-A activity may be useful for the prevention of proatherogenic changes in vivo.


*    Acknowledgments
 
This study was supported by grants from the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation, and the Finnish Academy (to S.Y.H.). We thank Dr Tatsuhiko Kodama and Dr Monty Krieger for bovine SR-A type II cDNA, Dr Theodore Friedmann and Dr Atsushi Miyanohara for the parental pLRNL retroviral construct, and Dr Jukka Pelkonen and Dr Chandan Sen for help in the FACS assays. We also thank Marja Poikolainen for preparing the manuscript.

Received August 27, 1998; accepted April 21, 1999.


*    References
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 *References
 

  1. Kodama T, Freeman M, Rohrer L, Zabrecky J, Matsudaira P, Krieger M. Type I macrophage scavenger receptor contains {alpha}-helical and collagen-like coiled coils. Nature. 1990;343:531–535.[Medline] [Order article via Infotrieve]
  2. Rohrer L, Freeman M, Kodama T, Penman M, Krieger M. Coiled-coil fibrous domains mediate ligand binding by macrophage scavenger receptor type II. Nature. 1990;343:570–572.[Medline] [Order article via Infotrieve]
  3. Krieger M, Herz J. Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem. 1994;63:601–637.[Medline] [Order article via Infotrieve]
  4. Ylä-Herttuala S. Expression of lipoprotein receptors and related molecules in atherosclerotic lesions. Curr Opin Lipidol. 1996;7:292–297.[Medline] [Order article via Infotrieve]
  5. Fraser I, Hughes D, Gordon S. Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature. 1993;364:343–346.[Medline] [Order article via Infotrieve]
  6. Ylä-Herttuala S, Rosenfeld ME, Parthasarathy S, Sigal E, Särkioja T, Witztum JL, Steinberg D. Gene expression in macrophage-rich human atherosclerotic lesions: 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts. J Clin Invest. 1991;87:1146–1152.
  7. Freeman M, Ekkel Y, Rohrer L, Penman M, Freedman NJ, Chisolm GM, Krieger M. Expression of type I and type II bovine scavenger receptors in Chinese hamster ovary cells: lipid droplet accumulation and nonreciprocal cross competition by acetylated and oxidized low density lipoprotein. Proc Natl Acad Sci U S A. 1991;88:4931–4935.[Abstract/Free Full Text]
  8. Naito M, Suzuki H, Mori T, Matsumoto A, Kodama T, Takahashi K. Coexpression of type I and type II human macrophage scavenger receptors in macrophages of various organs and foam cells in atherosclerotic lesions. Am J Pathol. 1992;141:591–599.[Abstract]
  9. Luoma J, Hiltunen T, Särkioja T, Moestrup SK, Gliemann J, Kodama T, Nikkari T, Ylä-Herttuala S. Expression of {alpha}2-macroglobulin receptor/low density lipoprotein receptor-related protein and scavenger receptor in human atherosclerotic lesions. J Clin Invest. 1994;93:2014–2021.
  10. Leppänen P, Luoma JS, Hofker MH, Havekes LM, Ylä-Herttuala S. Characterization of atherosclerotic lesions in apo E3-Leiden transgenic mice. Atherosclerosis. 1998;136:147–152.[Medline] [Order article via Infotrieve]
  11. Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka M, Jishage K, Ueda O, Sakaguchi H, Higashi T, Suzuki T, Takashima Y, Kawabe Y, Cynshi O, Wada Y, Honda M, Kurihara H, Aburatani H, Doi T, Matsumoto A, Azuma S, Noda T, Toyoda Y, Itakura H, Yazaki Y, Horiuchi S, Takahashi K, Kruijt JK, Van Berkel TJC, Steinbrecher UP, Ishibashi S, Maeda N, Gordon S, Kodama T. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature. 1997;386:292–296.[Medline] [Order article via Infotrieve]
  12. Hiltunen TP, Luoma JS, Nikkari T, Ylä-Herttuala S. Expression of LDL receptor, VLDL receptor, LDL receptor-related protein, and scavenger receptor in rabbit atherosclerotic lesions: marked induction of scavenger receptor and VLDL receptor expression during lesion development. Circulation. 1998;97:1079–1086.[Abstract/Free Full Text]
  13. Pitas RE. Expression of the acetyl low density lipoprotein receptor by rabbit fibroblasts and smooth muscle cells: upregulation by phorbol esters. J Biol Chem. 1990;265:12722–12727.[Abstract/Free Full Text]
  14. Li H, Freeman MW, Libby P. Regulation of smooth muscle cell scavenger receptor expression in vivo by atherogenic diets and in vitro by cytokines. J Clin Invest. 1995;95:122–133.
  15. Gough PJ, Greaves DR, Suzuki H, Hakkinen T, Hiltunen MO, Turunen M, Herttuala SY, Kodama T, Gordon S. Analysis of macrophage scavenger receptor (SR-A) expression in human aortic atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 1999;19:461–471.[Abstract/Free Full Text]
  16. Cornetta K, Morgan RA, Anderson WF. Safety issues related to retroviral-mediated gene transfer in humans. Hum Gene Ther. 1991;2:5–14.[Medline] [Order article via Infotrieve]
  17. Miyanohara A, Sharkey MF, Witztum JL, Steinberg D, Friedmann T. Efficient expression of retroviral vector-transduced human low density lipoprotein (LDL) receptor in LDL receptor-deficient rabbit fibroblasts in vitro. Proc Natl Acad Sci U S A. 1988;85:6538–6542.[Abstract/Free Full Text]
  18. Kalnins A, Otto K, Rüther U, Müller-Hill B. Sequence of the lacZ gene of Escherichia coli. EMBO J. 1983;2:593–597.[Medline] [Order article via Infotrieve]
  19. Ylä-Herttuala S, Luoma J, Viita H, Hiltunen T, Sisto T, Nikkari T. Transfer of 15-lipoxygenase gene into rabbit iliac arteries results in the appearance of oxidation-specific lipid-protein adducts characteristic of oxidized low density lipoprotein. J Clin Invest. 1995;95:2692–2698.
  20. Mann R, Mulligan RC, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983;33:153–159.[Medline] [Order article via Infotrieve]
  21. Miller AD, Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986;6:2895–2902.[Abstract/Free Full Text]
  22. Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A. 1993;90:8033–8037.[Abstract/Free Full Text]
  23. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086–1095.
  24. Emi N, Friedmann T, Yee JK. Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus. J Virol. 1991;65:1202–1207.[Abstract/Free Full Text]
  25. Laitinen M, Pakkanen T, Donetti E, Baetta R, Luoma J, Lehtolainen P, Viita H, Agrawal R, Miyanohara A, Friedmann T, Risau W, Martin JF, Soma M, Ylä-Herttuala S. Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses. Hum Gene Ther. 1997;8:1645–1650.[Medline] [Order article via Infotrieve]
  26. Kingston RE. Preparation of cytoplasmic RNA from tissue culture cells. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current Protocols in Molecular Biology. New York, NY: Greene Publishing Associates and John Wiley and Sons; 1991:4.1.2–4.1.3.
  27. William MS. Preparation of genomic DNA from mammalian tissue. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current Protocols in Molecular Biology. New York, NY: Greene Publishing Associates and John Wiley and Sons; 1991:2.2.1–2.2.3.
  28. Kodama T, Reddy P, Kishimoto C, Krieger M. Purification and characterization of a bovine acetyl low density lipoprotein receptor. Proc Natl Acad Sci U S A. 1988;85:9238–9242.[Abstract/Free Full Text]
  29. Mori T, Takahashi K, Naito M, Kodama T, Hakamata H, Sakai M, Miyazaki A, Horiuchi S, Ando M. Endocytic pathway of scavenger receptors via trans-Golgi system in bovine alveolar macrophages. Lab Invest. 1994;71:409–416.[Medline] [Order article via Infotrieve]
  30. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345–1353.
  31. Pitas RE, Innerarity TL, Weinstein JN, Mahley RW. Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy. Arteriosclerosis. 1981;1:177–185.[Abstract/Free Full Text]
  32. Basu SK, Goldstein JL, Anderson GW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976;73:3178–3182.[Abstract/Free Full Text]
  33. Jaakkola O, Yla-Herttuala S, Sarkioja T, Nikkari T. Macrophage foam cells from human aortic fatty streaks take up ß-VLDL and acetylated LDL in primary culture. Atherosclerosis. 1989;79:173–182.[Medline] [Order article via Infotrieve]
  34. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.[Free Full Text]
  35. Riordan FA, Foroni L, Hoffbrand AV, Mehta AB, Wickremasinghe RG. Okadaic acid-induced apoptosis of HL60 leukemia cells is preceded by destabilization of bcl-2 mRNA and downregulation of bcl-2 protein. FEBS Lett. 1998;435:195–198.[Medline] [Order article via Infotrieve]
  36. Bonelli G, Sacchi MC, Barbiero G, Duranti F, Goglio G, Verdun di Cantogno L, Amenta JS, Piacentini M, Tacchetti C, Baccino FM. Apoptosis of L929 cells by etoposide: a quantitative and kinetic approach. Exp Cell Res. 1996;228:292–305.[Medline] [Order article via Infotrieve]
  37. Cornetta K, Nguyen N, Morgan RA, Muenchau DD, Hartley JW, Blaese RM, Anderson WF. Infection of human cells with murine amphotropic replication-competent retroviruses. Hum Gene Ther. 1993;4:579–588.[Medline] [Order article via Infotrieve]
  38. Ylä-Herttuala S, Rosenfeld ME, Parthasarathy S, Glass CK, Sigal E, Witztum JL, Steinberg D. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Sci U S A. 1990;87:6959–6963.[Abstract/Free Full Text]
  39. Thiery J, Teupser D, Walli AK, Ivandic B, Nebendahl K, Stein O, Stein Y, Seidel D. Study of causes underlying the low atherosclerotic response to dietary hypercholesterolemia in a selected strain of rabbits. Atherosclerosis. 1996;121:63–73.[Medline] [Order article via Infotrieve]
  40. Greaves DR, Gough PJ, Gordon S. Recent progress in defining the role of scavenger receptors in lipid transport, atherosclerosis and host defence. Curr Opin Lipidol. 1998;9:425–432.[Medline] [Order article via Infotrieve]
  41. Daugherty A, Cornicelli JA, Welch K, Sendobry SM, Rateri DL. Scavenger receptors are present on rabbit aortic endothelial cells in vivo. Arterioscler Thromb Vasc Biol. 1997;17:2369–2375.[Abstract/Free Full Text]
  42. Wolfbauer G, Glick JM, Minor LK, Rothblat GH. Development of the smooth muscle foam cell: uptake of macrophage lipid inclusions. Proc Natl Acad Sci U S A. 1986;83:7760–7764.[Abstract/Free Full Text]
  43. McGill HC Jr. Fatty streaks in the coronary arteries and aorta. Lab Invest. 1968;18:560–564.[Medline] [Order article via Infotrieve]
  44. Ylä-Herttuala S, Nikkari T, Hirvonen J, Laaksonen H, Möttönen M, Pesonen E, Raekallio J, Åkerblom HK. Biochemical composition of coronary arteries in Finnish children. Arteriosclerosis. 1986;6:230–236.[Abstract/Free Full Text]
  45. Ares MP, Kallin B, Eriksson P, Nilsson J. Oxidized LDL induces transcription factor activator protein-1 but inhibits activation of nuclear factor-{kappa}B in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1995;15:1584–1590.[Abstract/Free Full Text]
  46. Kinscherf R, Claus R, Wagner M, Gehrke C, Kamencic H, Hou D, Nauen O, Schmiedt W, Kovacs G, Pill J, Metz J, Deigner HP. Apoptosis caused by oxidized LDL is manganese superoxide dismutase and p53 dependent. FASEB J. 1998;12:461–467.[Abstract/Free Full Text]
  47. Bjorkerud B, Bjorkerud S. Contrary effects of lightly and strongly oxidized LDL with potent promotion of growth versus apoptosis on arterial smooth muscle cells, macrophages, and fibroblasts. Arterioscler Thromb Vasc Biol. 1996;16:416–424.[Abstract/Free Full Text]
  48. Nagy L, Tontonoz P, Alvarez JG, Chen H, Evans RM. Oxidized LDL regulates macrophage gene expression through ligand activation of PPAR-{gamma}. Cell. 1998;93:229–240.[Medline] [Order article via Infotrieve]
  49. Geng YJ, Libby P. Evidence for apoptosis in advanced human atheroma: colocalization with interleukin-1ß-converting enzyme. Am J Pathol. 1995;147:251–266.[Abstract]
  50. Kockx MM, De Meyer GR, Muhring J, Bult H, Bultinck J, Herman AG. Distribution of cell replication and apoptosis in atherosclerotic plaques of cholesterol-fed rabbits. Atherosclerosis. 1996;120:115–124.[Medline] [Order article via Infotrieve]
  51. Sakai M, Miyazaki A, Hakamata H, Kodama T, Suzuki H, Kobori S, Shichiri M, Horiuchi S. The scavenger receptor serves as a route for internalization of lysophosphatidylcholine in oxidized low density lipoprotein-induced macrophage proliferation. J Biol Chem. 1996;271:27346–27352.[Abstract/Free Full Text]
  52. Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol. 1994;124:1–6.[Free Full Text]
  53. Kockx MM, De Meyer GR, Muhring J, Jacob W, Bult H, Herman AG. Apoptosis and related proteins in different stages of human atherosclerotic plaques. Circulation. 1998;97:2307–2315.[Abstract/Free Full Text]
  54. Skulachev VP. Cytochrome c in the apoptotic and antioxidant cascades. FEBS Lett. 1998;423:275–280.[Medline] [Order article via Infotrieve]
  55. Nishio E, Watanabe Y. Oxysterols induced apoptosis in cultured smooth muscle cells through CPP32 protease activation and bcl-2 protein downregulation. Biochem Biophys Res Commun. 1996;226:928–934.[Medline] [Order article via Infotrieve]
  56. Mietus-Snyder M, Glass CK, Pitas RE. Transcriptional activation of scavenger receptor expression in human smooth muscle cells requires AP-1/c-Jun and C/EBP-ß: both AP-1 binding and JNK activation are induced by phorbol esters and oxidative stress. Arterioscler Thromb Vasc Biol. 1998;18:1440–1449.[Abstract/Free Full Text]



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