Author + information
- Received April 10, 2011
- Revision received June 9, 2011
- Revision received July 11, 2011
- Published online October 11, 2011.
- Sotirios Tsimikas, MD⁎,⁎ (, )
- Atsushi Miyanohara, PhD⁎,
- Karsten Hartvigsen, PhD⁎,
- Esther Merki, PhD⁎,
- Peter X. Shaw, PhD⁎,
- Meng-Yun Chou, PhD⁎,
- Jennifer Pattison, BA⁎,
- Michael Torzewski, MD§,
- Janina Sollors∥,
- Theodore Friedmann, MD†,
- N. Chin Lai, PhD‡,
- H. Kirk Hammond, MD‡,
- Godfrey S. Getz, MD, PhD¶,
- Catherine A. Reardon, PhD¶,
- Andrew C. Li, MD⁎,
- Carole L. Banka, PhD⁎ and
- Joseph L. Witztum, MD⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Sotirios Tsimikas or Dr. Joseph L. Witztum, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0682
Objectives We sought to assess the in vivo importance of scavenger receptor (SR)–mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis.
Background The unregulated uptake of OxLDL by macrophage SR contributes to foam cell formation, but the importance of this pathway in vivo is uncertain.
Methods Cholesterol-fed low-density lipoprotein receptor knockout (LDLR−/−) mice were treated with intraperitoneal infusion of human IK17-Fab (2.5 mg/kg) 3 times per week for 14 weeks. Because anti-human antibodies developed in these mice, LDLR−/−/low-density lipoprotein receptor Rag 1 double-knockout mice (lacking the ability to make immunoglobulins due to loss of T- and B-cell function) were treated with an adenoviral vector encoding adenovirus expressed (Adv)–IK17-scFv or control adenovirus-enhanced green fluorescent protein vector intravenously every 2 weeks for 16 weeks.
Results In LDLR−/− mice, infusion of IK17-Fab was able to sustain IK17 plasma levels for the first 8 weeks, but these diminished afterward due to increasing murine anti-IK17 antibody titers. Despite this, after 14 weeks, a 29% decrease in en face atherosclerosis was noted compared with phosphate-buffered saline–treated mice. In LDLR−/−/low-density lipoprotein receptor Rag 1 double-knockout mice, sustained levels of plasma IK17-scFv was achieved by Adv-IK17-scFv–mediated hepatic expression, which led to a 46% reduction (p < 0.001) in en face atherosclerosis compared with adenovirus-enhanced green fluorescent protein vector. Importantly, peritoneal macrophages isolated from Adv-IK17-scFv treated mice had decreased lipid accumulation compared with adenovirus-enhanced green fluorescent protein–treated mice.
Conclusions These data support an important role for SR-mediated uptake of OxLDL in the pathogenesis of atherosclerosis and demonstrate that oxidation-specific antibodies reduce the progression of atherosclerosis, suggesting their potential in treating cardiovascular disease in humans.
The pathogenesis of atherosclerosis is complex and involves the impact of many well-documented traditional risk factors. Among those, hypercholesterolemia plays a dominant role in the initiation of the fatty streak, the earliest morphologic change in the artery. After penetration and binding to the matrix of the intima, it is generally thought that modification(s) of low-density lipoprotein (LDL) lead to its recognition and unregulated uptake by macrophage scavenger receptors (SRs), resulting in cholesteryl ester accumulation. Oxidized LDL (OxLDL) is generally thought to be one of these important modifications, and a variety of macrophage SRs redundantly bind OxLDL, including SR-A (I, II, III), CD36, SR-B1 MARCO, LOX-1, and others (1–3). Although there is controversy about their quantitative role in foam cell formation, considerable evidence supports important roles for these receptors in atherogenesis (4–6). We have shown that certain antibodies recognizing oxidation-specific epitopes can block the ability of OxLDL to be taken up by macrophages. For example, the natural immunoglobulin (Ig)M antibody E06/T15, which binds to the phosphocholine (PC) group of oxidized but not native phospholipids (7), blocks the binding and uptake of OxLDL mediated by CD36 and SR-B1 on macrophages (8–10). Indeed, marked in vivo elevation of E06/T15 IgM titers in cholesterol-fed low-density lipoprotein receptor knockout (LDLR−/−) mice, achieved by immunization with Streptococcus pneumoniae, which contains the same PC epitope, ameliorated the progression of atherosclerosis (11). Similarly, infusion of IgM T15 decreased lesion formation in a vein graft model (12) and immunization with PC-keyhole limpet hemocyanin, which also increased PC-specific antibodies that bound to OxLDL, also retarded lesion progression (13). These and other data (reviewed in Hartvigsen et al. (14) suggest the hypothesis that enhanced titers of antibodies that block OxLDL binding to macrophage SR should decrease foam cell formation and decrease atherosclerosis.
Immunization with antigens to increase titers of oxidation-specific antibodies initiates a cascade of immunologic responses that could affect lesion formation aside from the direct impact of the humoral antibody responses. Furthermore, antibodies of different isotypes have different effector functions, such as the ability to opsonize antigens and fix complement and also to bind to different Fc receptors, which in turn differ in their biological responses. Therefore, even though an oxidation-specific antibody has the capacity to block OxLDL uptake in vitro, it does not rule out the possibility that its ability to inhibit lesion formation in vivo is due to other immunologic properties.
We previously reported the cloning of the first human antibody to OxLDL from a Fab antibody phage display library (15). The Fab antibody IK17 was shown to bind to both OxLDL and malondialdehyde-modified LDL (MDA-LDL) but not to native LDL or to unrelated antigens, including tetanus toxoid, chicken ovalbumin, type VI collagen, and calf thymus single-stranded DNA. The dissociation constant (Kd) for IK17 was 3.7 × 10−8 mol/l calculated according to Klotz plots. MDA-LDL and copper oxidized low-density lipoprotein (Cu-OxLDL) were effective competitors, whereas native LDL, native high-density lipoprotein, MDA-modified bovine serum albumin, 4-hydroxynonenal-modified LDL (another prominent epitope of OxLDL), MDA-polylysine, and MDA-murine IgG did not compete. On Western blots after sodium dodecylsulfate–polyacrylamide gel electrophoresis under reduced conditions, IK17 bound extensively to the protein moiety (apoB) of Cu-OxLDL and MDA-LDL, but not to native LDL or native high-density lipoprotein. IK17 inhibited the uptake of OxLDL by macrophages and also bound to apoptotic cells and inhibited their phagocytosis by macrophages. Intravenously injected IK17 also was targeted to and effectively imaged atherosclerotic lesions in vivo (15,16).
Because neither IK17-Fab nor IK-17 single-chain Fv fragment (IK17-scFv) has immunologic properties of intact antibodies other than their ability to inhibit uptake of OxLDL and apoptotic by macrophages, we hypothesized that if mice treated with these IK17 antibody fragments had reduced atherosclerosis, this would support an important role for SR-mediated uptake of OxLDL in the pathogenesis of atherosclerosis.
A detailed description of selected methods is provided in the Online Appendix.
Preparation of IK17-Fab and IK17-scFv
Preparation of IK17-Fab
Recombinant IK17-Fab was produced in BL21 (DE3) Escherichia coli cells (Invitrogen, Carlsbad, California) and purified using a goat anti-human Fab affinity column (Pierce, Rockford, Illinois) to >99% purity as shown by Western blotting (15). Residual endotoxin was removed (to >99%) using TritonX 114 (Sigma-Aldrich, St. Louis, Missouri) as previously described (17). The purified IK17-Fab fully retained its immunoreactivity to MDA-LDL and copper OxLDL (Cu-OxLDL), using chemiluminescent immunoassays described in the following, similar to the parent antibody.
Generation of IK17-scFv and its Adenoviral Vector
A full description of these procedures can be found in the Online Appendix. In brief, to convert IK17-Fab into a single-chain Fv fragment, 2 rounds of polymerase chain reaction (PCR) were used to introduce a 7-amino acid linker connecting the VL and VH regions and restriction sites for cloning. The coding region of IK17-scFv was amplified by PCR and then subcloned into HindIII and NotI sites of the eukaryotic expression vector pSecTag2A (Invitrogen), which contains a mouse kappa signal sequence for expression and secretion, and a c-myc tag, allowing detection with an anti-myc antibody. The expression and secretion cassette of the IK17-scFv gene was isolated from the pSecTag-IK17-scFv plasmid by a PCR reaction and then inserted into the EcoRV (blunt) site of the adenovirus shuttle plasmid pDeltaE1Z, which expresses the transgene from the cytomegalovirus promoter. The resulting adenovirus shuttle plasmid pDeltaE1Z-IK17 was cotransfected with E1-deleted adenovirus backbone genome JM17 DNA into HEK293 cells. Two to 3 weeks after cotransfection, plaques were isolated and amplified to examine the IK17-scFv expression. Adenoviral vectors were then purified through 2 rounds of caesium chloride centrifugation, and titers were measured in 2 ways: plaque formation on HEK293 cells (plaque-forming units per milliliter) and OD260 reading (particles per milliliter). The enhanced green fluorescent protein gene was inserted into the same adenoviral vector to generate adenovirus expressed enhanced green fluorescent protein (Adv-EGFP), which was used as a control.
Murine models and atherosclerosis studies
Animal protocols were approved by the University of California San Diego and the Veterans Affairs Institutional Animal Care and Use Committee. All mice used were on the C57BL/6 background and included wild-type C57BL/6 (Jackson Laboratories, Bar Harbor, Maine), LDLR−/−, and LDL receptor Rag 1 double-knockout (LDLR−/−/Rag1−/−) mice (18). The mice were bred and maintained under specific pathogen-free conditions unless otherwise noted.
Study 1: Infusion of IK17-Fab in LDLR−/− Mice
Twenty-two male LDLR−/− mice (6 weeks old) were placed on a 1.25% cholesterol/21% milk fat diet for 2 weeks to rapidly increase total cholesterol levels and to initiate atherosclerosis and then switched to a normal mouse chow diet enriched with 0.5% cholesterol for another 2 weeks as previously described (19). This diet was then continued for the remainder of the study, and the mice (n = 11 in each group) were randomized to receive either IK17-Fab (2.5 mg/kg in sterile phosphate-buffered saline [PBS]) or similar volume PBS, given intraperitoneally 3 times per week for 14 weeks. Blood samples were obtained before the 1.25% cholesterol/21% milk fat diet, before the 0.5% cholesterol diet, and then at 4-week intervals until the end of the study (total of 6 time points). The animals were then killed by CO2 inhalation, at which time the extent of atherosclerosis was determined.
Study 2: Adenoviral Expression of IK17-scFv in C57BL/6 and in LDLR−/−/Rag1−/− Mice
In initial studies to validate the ability of adenovirus expressed (Adv)-IK17-scFv to express biologically active IK17 into plasma, we injected C57BL/6 mice with varying doses of virus and then measured IK17-scFv titers in plasma as described in the following text. Biologically active IK17-scFv was secreted into plasma, but sustained expression was not possible because of rapidly extinguished hepatic expression of IK17-scFv and because of murine humoral responses to the human IK17. To overcome this, we expressed the Adv-IK17-scFv in LDLR−/−/Rag1−/− mice. For gene transfer, 10-week-old male mice (20 to 25 g) were anesthetized with 2% isoflurane inhalation, and adenoviral vectors were diluted into 100 μl of PBS and injected through the retro-orbital plexus with a 27-gauge needle.
The male LDLR−/−/Rag1−/− mice were divided into 2 groups of 15 animals each. Group A was injected with 1011 viral particles of Adv-IK17-scFv and group B was injected with the same amount of Adv-EGFP. One week after the first injection, the mice were started on a high cholesterol (HC) diet (Harlan-Teklad 8604 standard rodent diet containing 1.25% cholesterol) for 16 weeks. These mice were subsequently treated by repeat injections every 2 weeks. Blood (∼100 μl) was collected 2 weeks before the study and at 2, 4, 8, and 16 weeks after initiation of the HC diet. After 16 weeks of the HC diet, the mice were killed by CO2 inhalation, and the entire aorta and heart were removed for atherosclerosis measurements. Two of the mice injected with Adv-IK17-scFv died before the end of the study, and their data are not included.
Macrophage Binding and Competition Assay
Binding of biotinylated Cu-OxLDL and MDA-LDL ligands to J774 murine macrophages plated in microtiter wells and the ability of IK17-Fab or IK17-scFv in culture supernatants or plasma to inhibit binding were assessed by a chemiluminescent binding assay as previously described (11,20) with modifications. A detailed protocol can be found in the Online Appendix.
Peritoneal Macrophage Isolation and Lipid Content Assessment
Foam cell formation in elicited peritoneal macrophages of experimental mice was determined as previously described (21). To derive mechanistic information for study 2, in a separate experiment, LDLR−/−/Rag1−/− mice were treated with regular mouse chow, an HC diet, an HC diet and Adv-IK17-scFv, or an HC diet and Adv-EGFP every 2 weeks for 8 weeks. Peritoneal macrophages were isolated from mice 4 days after intraperitoneal injection of thioglycolate. Cells 1.0 × 105 were resuspended into 0.5 ml of Dulbecco's modified Eagle medium containing 20% fetal calf serum and added to each well of a 24-well culture plate laid with a round cover slip. After the macrophages were attached to the cover slip (about 3 h), the cells were washed with PBS and fixed with formaldehyde/sucrose solution. The cells were then stained with heated Oil red O/propylene glycol solution and mounted. The lipid-loaded macrophages were counted using a microscope with a visual grid and expressed as the percentage foam cells per total cells counted.
Chemiluminescent Immunoassays, Immunohistochemistry, and Atherosclerosis Quantification
Methodologies for these assays are described in the Online Appendix.
Differences between groups in atherosclerosis measures and immunohistochemistry parameters were analyzed by Student t test. Analysis of quantitative parameters of oxidation biomarkers within groups of mice over time (4 time points) (Fig. 1B) and of the percentage of Oil Red O staining (4 groups, Fig. 6B statistics) was performed with repeated measures analysis of variance with post hoc Bonferroni correction. p values <0.05 are considered significant. Data are given as mean ± SEM unless otherwise noted.
Study 1: IK17-Fab infusion inhibits atherosclerosis progression in LDLR−/− mice
There were no significant differences in weight gain, plasma cholesterol, or triglyceride levels between groups over time (Table 1). Significant increases in autoantibodies to MDA-LDL, apoB-immune complexes, and IgM T15/E06 levels occurred in both groups in response to the diet, but no significant differences were present between groups (Fig. 1 in the Online Appendix).
IK17-Fab Plasma Concentrations and IK17-Fab Immune Complexes
As expected, substantial elevations in plasma levels of IK17-Fab concentrations were documented at 4 to 8 weeks during the ongoing IK17-Fab infusions. However, IK17-Fab plasma levels decreased significantly by the end of the study (Fig. 1A), corresponding to an increase in titers of murine IgG (31,140 ± 8,198 vs. 335 ± 105 relative light units, p < 0.001) and IgM (17,140 ± 3,397 vs. 2,009 ± 262 relative light units, p < 0.001) anti-IK17 antibodies in the IK17-Fab–injected group (data at 1:500 plasma dilution). This was also associated with increased plasma levels of murine IgM and IgG IK17-Fab/immune complexes (Fig. 1B).
Impact of IK17-Fab on Atherosclerosis Progression
Despite the immune response to IK17-Fab, there was a 29% decrease in en face atherosclerosis in the IK17-treated LDLR−/− mice compared with mice treated with PBS (4.9 ± 0.25% vs. 6.9 ± 0.47%, p < 0.001) (Figs. 1C and 1D). In fact, the mice with the lowest anti-IK17 immune response also had the least atherosclerosis, and for the group as a whole, the plasma titers of IgG anti-IK17 antibodies correlated positively with the extent of en face lesion formation in the IK17-Fab–treated mice (Spearman correlation, r = 0.87; p < 0.001) Analysis of cross sections at the level of the aortic valve did not reveal differences between the 2 groups (1.93 ± 0.20 mm2 vs. 2.12 ± 0.13 mm2 total lesion area, p = 0.42). Immunohistochemistry of the aortic root for macrophages, smooth muscle cells, collagen, and MDA epitopes revealed a strong trend (p = 0.08) for reduced MDA staining but no differences in other measures (Online Fig. 2).
Study 2: adenoviral expression of IK17-scFv inhibits progression of atherosclerosis in LDLR−/−/Rag1−/− mice
To overcome the likely neutralization of the infused IK17-Fab by mouse anti-IK17 antibodies, we generated IK17 as a functional scFv and expressed it via an adenoviral vector in LDLR−/−/Rag1−/− mice, which lack T and B cells.
Generation of Functional IK17-scFv
IK17-scFv was generated and purified to near homogeneity as described in detail in the Online Appendix. Similar to the parent IK17-Fab, the purified IK17-scFv displayed the same binding to OxLDL and MDA-LDL when examined by chemiluminescent enzyme-linked immunosorbent assay (Fig. 2A), and inhibited the binding of both MDA-LDL (Fig. 2B) and OxLDL (Fig. 2C) to J774 macrophages. Examination of the binding of the IK17-scFv gene product from the eukaryotic expression vector pSecTag2A (Fig. 2D) and the adenovirus shuttle vector pDelta-E1Z revealed the same binding properties as purified IK17-Fab. Furthermore, the IK17-scFv recombinant adenovirus (Adv-IK17-scFv) rescued from 293 cells was infectious and cells infected with Adv-IK17-scFv expressed and secreted IK17-scFv into the culture medium (Online Fig. 3) and retained the binding properties of the parent IK17-Fab (Fig. 2D).
The Adenovirus IK17-scFv Transgene was Expressed and Secreted into the Circulation
In an initial study, we injected 2.5 × 1011 viral particles of Adv-IK17-scFv into wild-type C57B6 and LDLR−/−/Rag−/− mice via a tail vein and collected blood samples at 5, 19, and 28 days post-injection. Functional IK17-scFv, detected by binding to MDA-LDL and Cu-OxLDL, was detectable in plasma at day 5, but was lost after 19 days (Fig. 3A). Repeat injection 21 days after the initial injection did not result in the appearance of plasma IK17-scFv binding activity 7 days later. We reasoned that the host immune response extinguished the adenoviral expression of IK17-scFv, both at the level of the liver and by mounting an antibody response to the human transgene. Indeed, we demonstrated murine IgG and IgM titers to the IK17-scFv gene product from the plasma 19 days post-injection of Adv-IK17-scFv in wild-type LDLR−/− mice (data not shown). To overcome this problem, we injected Adv-IK17-scFv into LDLR−/−/Rag1−/− mice. However, even in these mice that lack both B and T cells, IK17-scFv expression in plasma was also limited to <3 weeks, presumably due to methylation of the cytomegalovirus promoter in the liver (21). However, in these mice, repeated injections of Adv-IK17-scFv restored the transgene expression each time for approximately 3 weeks (Fig. 3A).
Sustained Expression of IK17-scFv for 16-Week Intervention Study
By repeatedly injecting Adv-IK17-scFv at 2-week intervals, we maintained a sustained and high titer of IK17-scFv in plasma over a 16-week period. Using real-time PCR to monitor tissue expression of the injected transgene, we demonstrated that it was predominantly expressed in liver as expected (Online Fig. 4). By monitoring IK17-scFv binding to MDA-LDL and OxLDL by enzyme-linked immunosorbent assay, we demonstrated sustained expression of functional IK17-scFv in plasma throughout the 16 weeks of gene transfer (Figs. 3B and 3C). A competitive immunoassay demonstrated that the binding of secreted IK17-scFv to both OxLDL and MDA-LDL was inhibited by both MDA-LDL and OxLDL, but not native LDL, indicating that the plasma IK17-scFv retained its specific binding properties (Online Fig. 5). Furthermore, the secreted IK17-scFv retained its functional property to inhibit the binding of MDA-LDL and OxLDL to macrophages (Figs. 3D and 3E). Serial dilutions of plasma from mice injected with Adv–IK17-scFv displayed a dose-dependent inhibition of binding of MDA-LDL and OxLDL to J774 macrophages and at a dilution of 1:40 completely abolished binding. This degree of inhibition was much greater than that observed with equivalent dilutions of plasma from control mice injected with the Adv-EGFP vector. Although plasma of the Adv-EGFP mice also showed a slight degree of inhibition, this also occurred in plasma from wild-type mice, which is in part nonspecific and in part due to plasma proteins that may bind OxLDL (data not shown).
Plasma From Adv-IK17-scFv Injected Mice Immunostained Advanced Atherosclerotic Lesions
To demonstrate that the secreted IK17-scFv could localize to atherosclerotic lesions, a 1:20 dilution of plasma from an Adv-IK17-scFv–injected mouse was used to immunostain atherosclerotic lesions from a human brain artery (Fig. 4). The IK17-scFv preferentially stained the necrotic cores (Fig. 4A), as previously observed with affinity-purified intact IK17-Fab (23). The same dilution of plasma from Adv-EGFP–injected mice produced no specific immunostaining (Fig. 4B). The secreted IK17-scFv also localized to its own atherosclerotic lesions as documented at the time of killing in aortic sections that were obtained from mice expressing the Adv-IK17-scFv and immunostained for IK17-scFv using an anti-myc antibody. There was specific localization (brown areas) of IK17-scFv to atherosclerotic lesions (Fig. 4C). In contrast, although there was light and diffuse staining of tissue by plasma of Adv-EGFP mice, there was no specific localization to the atherosclerotic lesions (Fig. 4D).
Impact of Plasma IK17-scFv on Atherosclerosis
We evaluated the impact of the secreted IK17-scFv on the extent of atherosclerosis in the LDLR−/−/Rag−/− mice during a 16-week period of feeding the HC diet compared with mice receiving the Adv-EGFP vector under an identical protocol. Both groups of mice gained weight equally and had similar plasma cholesterol and triglyceride levels during the intervention period (Table 1). The extent of en face aortic lesion area was significantly reduced in the Adv-IK17-scFv–treated mice compared with the Adv-EGFP–treated mice (Fig. 5A), leading to a 46% reduction in lesion area (2.1 ± 0.3% vs. 3.9 ± 0.4%, p < 0.001) (Fig. 5B). Cross-sectional analysis at the aortic origin revealed only small lesions and no statistically significant difference (0.27 ± 0.05 mm2 vs. 0.23 ± 0.06 mm2, total lesion area, p = 0.62).
Effect of Adv-IK17-scFv on Macrophage Foam Cell Formation
Previously, Li et al. (21) showed that the extent of foam cell formation observed in elicited peritoneal macrophages reflected the extent of foam cell formation noted in the aorta of mice after various experimental interventions. Therefore, we elicited peritoneal macrophages from Adv-IK17-scFv and Adv-EGFP mice, as well as from LDLR−/−/Rag1−/− mice fed either chow or the HC diet. In contrast to macrophages isolated from mice that were fed a normal chow diet, macrophages isolated from hypercholesterolemic mice exhibited extensive Oil Red O droplets (Fig. 6A, chow-only diet and HC diet only). Macrophages from the Adv-IK17-scFv–treated mice showed a marked reduction of lipid accumulation (Fig. 6A, HC diet + Adv-IK17-scFv) compared with macrophages isolated from the Adv-EGFP mice or the HC diet mice (Fig. 6A, HC diet only and HC diet + Adv-EGFP). We also quantified the number of macrophages showing lipid droplets/100 cells counted. Macrophages isolated from Adv-IK17-scFv–treated mice had significantly fewer cells with lipid droplets compared with those from Adv-EGFP–treated, or HC diet–fed nontreated LDLR−/−/Rag1−/− mice (36.8 ± 7.2%, n = 4 vs. 80.5 ± 3.7%, n = 3 or 77.2 ± 5.4%, n = 4, p < 0.0001) (Fig. 6B). This observation suggests that the IK17-scFv significantly decreased foam cell formation, consistent with the demonstrated ability of the plasma of the IK17-scFv–treated mice to inhibit uptake of OxLDL by macrophages, as shown in Figures 3D and 3E.
This study demonstrates that infusion of the IK17-Fab into LDLR−/− mice inhibits atherosclerotic lesion formation. Furthermore, expression of Adv-IK17 in vivo as a functional single-chain antibody (IK17-scFv) also led to inhibition of foam cell formation and decreased atherosclerosis. These data support the hypothesis that a major mechanism by which oxidation-specific antibodies inhibits atherosclerosis is by blocking OxLDL binding to macrophages, supporting an important role for SR-mediated uptake of OxLDL in the pathogenesis of atherosclerosis. Furthermore, these results suggest the potential of oxidation-specific antibodies in treating cardiovascular disease in humans.
Although atherogenesis is a highly complex process, it is strongly related to elevated plasma LDL cholesterol levels. Once native LDL penetrates the intima, it binds to the glycoprotein matrix, which prolongs its half-life and renders it susceptible to a variety of modifications (1,24). Among these, the generation of OxLDL has been widely studied and considered important, as OxLDL is recognized by a variety of SRs on macrophages, leading to unregulated uptake. Indeed, although excellent experimental studies in genetically targeted mice have supported the importance of SR-A and CD36 in atherogenesis (25–27), other such studies have provided opposing data and conclusions (6,28). Aside from differences in experimental conditions (4), the nature of the modified lipoprotein ligands for the SRs induced by diet may be an important variable in such studies (27). In addition, such SRs are innate “pattern recognition receptors” and may well have other vital functions (14,29). Deleting them via genetic targeting may have disrupted other important properties involved in immunity and atherogenesis, and/or led to unknown compensatory functions that may complicate the interpretation of their importance in the uptake of OxLDL. Therefore, inhibiting the uptake of OxLDL with oxidation-specific antibody fragments lacking immunologic properties other than their ability to recognize their respective epitopes allows one to examine the importance of SR-mediated uptake of OxLDL in atherogenesis.
We previously characterized IK17-Fab, the first cloned human antibody to OxLDL (23). It binds to an epitope different from that recognized by E06 (7), one contained on both OxLDL and MDA-LDL, but like E06, it has the ability to block the uptake and degradation of OxLDL by macrophages. To date, we have not fully characterized the exact epitope to which IK17 binds, although it appears to be a complex MDA-type structure, present in both the lipid and protein moieties of OxLDL and MDA-LDL. Importantly, IK17-Fab immunostains atherosclerotic lesions in mice, rabbits, and humans, especially the necrotic core (23,30,31), and has been used to successfully image lesion formation in vivo in LDLR−/− and apoE−/− mice (15,31). It thus targets relevant epitopes in lesions and would be strategically positioned to inhibit OxLDL uptake by macrophages in vivo. Indeed, in this study, we demonstrated that either the passive transfer of IK17-Fab to LDLR−/− mice or the adenoviral-mediated expression of IK17-scFv in LDLR−/−/Rag1−/− mice inhibited atherosclerosis progression. To our knowledge, neither IK17-Fab nor the IK17-scFv has any immunologic properties of intact antibodies other than the ability to bind to oxidation-specific epitopes and thus inhibit uptake of OxLDL by macrophages. Furthermore, they achieve this inhibition by binding to SR ligands on OxLDL and not by interfering directly with SR functions. We believe that these data strongly support the hypothesis that SR-mediated uptake of OxLDL plays an important role in atherogenesis, particularly in the setting of the HC diet used in these studies (28). Indeed, the demonstration that in vivo foam cell formation in peritoneal macrophages was inhibited by the expression of IK17-scFv provides direct evidence to support this hypothesis.
In study 1, we injected purified lipopolysaccharide-free IK17-Fab into LDLR−/− mice. The impact of IK17-Fab infusion was limited by the development of murine anti–IK17-Fab antibodies during the latter part of the intervention period, and, indeed, at the end of the study, the titer of murine anti-IK17-Fab antibodies correlated directly with lesion formation, suggesting that depletion of IK17-scFv was associated with greater lesion formation. Despite these limitations, lesion formation was inhibited by 29%, supporting the hypothesis that macrophage SR uptake plays an important role in early lesion formation. This conclusion was strongly supported by the findings in study 2, in which a biologically functional IK17-scFv was generated and expressed in plasma for a period of 16 weeks. The IK17-scFv is a linear protein in which the antigen-binding domains of the antibody, the respective CDR3 regions of the VH and VL genes, are joined by a short synthetic linker that allows appropriate folding of the expressed protein to affect the biological properties of the intact IK17-Fab. IK17-scFv lacks any of the effector properties of an intact antibody other than its ability to bind to its target epitope. Sustained expression of IK17-scFv was accomplished by biweekly injections of Adv-IK17-scFv into LDLR−/−/Rag1−/− mice. Not only was en face lesion formation inhibited substantially, by 46%, but foam cell formation was inhibited in peritoneal macrophages isolated from these mice. Inhibition of foam cell formation was not seen in LDLR−/−/Rag1−/− mice injected in an identical protocol with the same adenoviral vector but bearing the EGFP gene.
It should be noted that in study 2 we maintained a small cohort of LDLR−/−/Rag1−/− mice on the same HC diet but performed no interventions. The plasma cholesterol levels in these mice were lower than in both groups of Adv-injected mice and at time of killing, the extent of atherosclerosis was similar to that of the mice injected with the Adv-IK17-scFv. This suggests that the repeated injections of adenovirus in this protocol increased both plasma cholesterol levels and extent of lesion formation. Because cholesterol levels of Adv-EGFP– and Adv-IK17-scFv–injected animals were the same throughout the study, this demonstrates that despite the apparently increased atherogenic stress caused by the repeated adenovirus administration, IK17-scFv decreased lesion formation. Another caveat is that the IK17 interventions only decreased lesion formation in the en face analyses and not at the aortic root. We do not know the reasons for this. In our previous study with immunization of LDLR−/− mice with MDA-LDL, we observed the opposite (i.e., decreased lesion formation at the aortic root, but not distally). In those studies, we used a high-fat, HC diet and plasma cholesterol levels exceeded 1,500 mg/dl. In the current studies, we used a cholesterol-enriched diet that does not have any added fat and produces more modest hypercholesterolemia and almost devoid of very low density lipoprotein. High very low density lipoprotein cholesterol levels observed in a high-fat, HC diet have been associated with enhanced lesion formation at the aortic origin (32). With the HC diet used in our current study, we observed smaller lesions at the aortic root, and, in particular, in the LDLR−/−/Rag−/− mice, the aortic root lesions were only ∼10% of those seen in the LDLR−/− mice.
These studies not only provide evidence to support an important role for SR uptake of OxLDL in macrophage foam cell formation, but demonstrate the potential therapeutic efficacy of interventions that target OxLDL uptake by macrophages, such as oxidation-specific antibodies or antibody fragments. We and others previously showed that increasing the titer of E06/T15 antibodies directed to oxidized phospholipid by immunization strategies (11,13) or direct infusion (12) inhibited atherosclerosis progression, and the current study and data of others (33,34) have shown that direct infusion of a Fab or an IgG1 to MDA-type oxidation-specific epitopes also limits lesion formation.
In the current paper, we also show the feasibility of using gene transfer techniques to deliver into plasma an antibody fragment capable of influencing macrophage foam cell formation. The adenovirus-encoding IK17-scFv produced a significant level of transgene in the plasma 4 to 5 days post-injection, which displayed the identical binding properties as its parental IK17-Fab (23). It maintained its ability to bind and block the uptake of OxLDL by macrophages in culture and immunostained oxidation-specific epitopes in atherosclerotic lesions. Most importantly, it decreased foam cell formation in peritoneal macrophages and substantially inhibited the progression of atherosclerosis. Therefore, this gene transfer experiment demonstrates that an scFv gene can be delivered and expressed in tissues, leading to the secretion into plasma of a biologically active antibody fragment.
In our initial gene transfer experiment in wild-type LDLR−/− mice, there was the rapid extinguishing of the adenovirus-mediated expression of IK17-scFv, which we thought secondary to adaptive immune responses. However, we were surprised that the Adv-IK17-scFv was also rapidly extinguished after 2 weeks even in LDLR−/−/Rag1−/− mice, which lack adaptive responses. This most likely involved inactivation of the cytomegalovirus promoter by hypermethylation (22). If one were to apply such gene transfer techniques to humans, the use of alternative promoters and the use of fully human antibody fragments would limit such inadequacies. The adenoviral vector used in this study was selected to afford high titers to allow demonstration of the feasibility of this approach, and obviously more appropriate vectors would be needed for the long-term expression in other animal models or in eventual application to humans. The recent reports of the long-term expression of gene products in humans using lentiviral vectors (35,36) offers promise that one day vectors will be developed that will allow safe and sustained gene transfer, which could then be used to express antibodies such as the IK17-scFv described in this report.
Immunohistochemistry was performed only in the aortic root where the lesions were very small and there were no differences in treatment effect in the extent of atherosclerosis. Because the remaining aorta was used for extensive processing for Sudan staining and en face atherosclerosis quantitation, it was not available for immunohistochemical analyses to assess differences in cellular, matrix, and oxidation-specific epitope content in regions of the aorta where a treatment effect was present. Future studies will focus on changes in plaque characteristics and atherosclerosis regression in response to human oxidation-specific antibodies.
Our data demonstrate the feasibility of direct infusion of human oxidation-specific antibodies to inhibit lesion uptake of OxLDL by macrophages, which presumably plays an important role in macrophage activation and participates in the induction of macrophage apoptosis and death (30,37,38). Oxidation-specific antibodies such as IK17 and E06 have recently been shown to strongly immunostain human vulnerable and ruptured plaques (30). Oxidized phospholipids are also released from clinically relevant human lesions in significant quantities and can be captured by distal protection devices after percutaneous coronary and carotid interventions (39). One might envision the infusion of oxidation-specific antibodies in the setting of unstable or acute coronary syndromes to rapidly stabilize plaques or use before percutaneous coronary, carotid, and peripheral interventions to neutralize released proinflammatory oxidized lipids (40). In addition, these experiments provide a template for using the biologically functional scFv as a reagent targeting the vulnerable plaque for imaging approaches or to mediate delivery of therapeutic agents or drugs (15,16). If this approach is translated to humans, we anticipate its initial application to be by direct infusion of an appropriate dose of the antibody, similar to other antibody therapeutics for inflammatory conditions and cancer treatment. Finally, one might also envision a therapeutic approach leading to a more sustained elevation of titers of oxidation-specific antibodies, which could be accomplished by a variety of techniques, including gene transfer techniques, once safe and effective vectors are developed as well as the use of vaccine approaches.
The authors thank Joe Juliano, Karen Bowden, and Florence Casanada for their technical assistance in these studies.
For a supplemental Methods section and figures, please see the online version of this article.
These studies were supported by National Institutes of Health grants R01 HL086559 (to Dr. Witztum), PO1 HL0888093 (to Drs. Witztum, Tsimikas, and Hartvigsen), and R01 HL087391 (to Dr. Li); by a grant from the Fondation Leducq (to Drs. Witztum and Tsimikas); and by AHA Scientist Development Awards to Drs. Hartvigsen and Shaw. Drs. Tsimikas, Shaw, and Witztum are named as inventors on patents and patent applications for the potential commercial use of antibodies to oxidized LDL held by the University California, San Diego. Dr. Tsimikas has served as a consultant to Quest and is a director of and has equity interest in Atherotope, Inc. Dr. Witztum has served as a consultant to Isis Pharmaceuticals, Regulus, and Amira, and is a director of and has equity interest in Atherotope, Inc. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Jawahar L. Mehta, MD, served as Guest Editor for this paper.
- Abbreviations and Acronyms
- adenovirus-expressed enhanced green fluorescent protein
- adenovirus-expressed IK17 single-chain Fv fragment
- copper-oxidized low-density lipoprotein
- high cholesterol
- IK17 single-chain Fv fragment
- low-density lipoprotein
- low-density lipoprotein receptor knockout
- malondialdehyde-modified low-density lipoprotein
- oxidized low-density lipoprotein
- phosphate-buffered saline
- polymerase chain reaction
- Rag 1 knockout
- scavenger receptor
- Received April 10, 2011.
- Revision received June 9, 2011.
- Revision received July 11, 2011.
- American College of Cardiology Foundation
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