Abstract
Purpose of Review
Emerging data demonstrates the potential of translational applications of antibodies directed against oxidation-specific epitopes (OSE). “Biotheranostics” in cardiovascular disease (CVD) describes targeting of OSE for biomarker, therapeutic and molecular imaging diagnostic applications.
Recent findings
Lipid oxidation collectively yields a large variety of oxidation-specific epitopes (OSE), such as oxidized phospholipids (OxPL) and malondialdehyde (MDA) epitopes. OSE are immunogenic, pro-inflammatory, pro-atherogenic and plaque destabilizing and represent danger associated molecular patterns (DAMPs). DAMPs are recognized by the innate immune system via pattern recognition receptors, including scavenger receptors IgM natural antibodies and complement factor H (CFH), that bind, neutralize and/or facilitate their clearance. Biomarker assays measuring OxPL present on apolipoprotein B-100 lipoproteins, and particularly on lipoprotein (a), predict the development of CVD events. In contrast, OxPL on plasminogen facilitate fibrinolysis and may reduce atherothrombosis. Oxidation-specific antibodies (OSA) attached to magnetic nanoparticles image lipid-rich, oxidation-rich plaques. Infusion or overexpression of OSA reduces the progression of atherosclerosis, suggesting that they may be used in similar applications in humans.
Summary
Using the accelerating knowledge base and improved understanding of the interplay of oxidation, inflammation and innate and adaptive immunity in atherogenesis, emerging clinical applications of OSA may identify, monitor and treat CVD in humans.
Keywords: biotheranostic, oxidation, innate immunity, atherogenesis, molecular imaging
INTRODUCTION
In their seminal 1989 review paper entitled “Beyond cholesterol: Modifications of low density lipoprotein that increase its atherogenicity,” [1] Steinberg, Witztum and colleagues provided a scientific rationale for the “oxidation hypothesis of atherosclerosis.” This hypothesis was strongly supported by in vitro data and animal experiments in which antioxidants reduced atherosclerosis. However, the results of human clinical trials with antioxidant vitamins were mainly negative, except in selected groups of patients with clearly increased systemic oxidative stress, such as patients on hemodialysis or diabetics with haptoglobin 2-2 genotypes associated with higher hemoglobin-mediated oxidative stress. Subsequently, Witztum and colleagues developed a deeper understanding of the biological effects of oxidized low-density lipoprotein (OxLDL), and particularly the role of the innate and adaptive immune system in the response to the generation of “oxidation-specific epitopes (OSE)” (Figure 1) [2] [3]. These observations led to the appreciation of the role of OSE in inflammatory and immune reactions that defined key pathways in the development and progression of atherosclerotic lesions [2, 4, 5]. Cloning and characterization of new monoclonal antibodies against OSE greatly facilitated mechanistic and translational research of atherosclerosis. These concepts defining the role of OSE in vascular inflammation and atherogenesis have matured to allow potential clinical translation in several areas, including biomarkers, diagnostic molecular imaging and therapy of cardiovascular disease. In this review, we unify these three concepts under the term “biotheranostics”, where the target is OSE in plasma or in the vessel wall and the targeting agents are oxidation-specific antibodies. A rationale is provided why targeting OSE may not only help to understand the transition of occult atherosclerosis to clinically relevant cardiovascular disease (CVD) but also in targeting OSE to develop clinical tools to define, monitor and treat CVD in humans.
LDL OXIDATION
The LDL particle is exquisitely sensitive to oxidative damage due to its complex lipid-protein composition and a large number of polyunsaturated acyl chains. The mechanisms of LDL oxidation in vivo include reactions catalyzed by 12/15-lipoxygenase (12/15-LO), myeloperoxidase (MPO), nitric oxide synthases and NADPH oxidases, as well as those mediated by heme and hemoglobin (Hb) [6]. Small amounts of Hb are constantly leaking from damaged erythrocytes, particularly in the vascular regions with turbulent flow, such as arterial bifurcations and aortic curvatures, and in vasa vasorum of atherosclerotic lesions. The LDL oxidation by Hb is normally prevented by haptoglobin (Hp) binding to Hb to, but the Hp2 isoform is less effective than the Hp1 isoform [7]. Recent findings confirm that the Hp2-2 genotype is associated with an increased risk of coronary artery disease (CAD), and evidence of increased iron content, expression of oxidized phospholipids (OxPL) and malondialdehyde (MDA) OSE, apoptotic cells, and cytoplasmic blebs were found in human aortic atherosclerotic lesions [8]. Novel data was also recently published by van Dijk et al [9], showing that in human vulnerable plaques OSE become increasingly more prominent as lesions progress and rupture. OSE were particularly prominent in advanced coronary and carotid lesions in macrophage-rich areas, lipid pools, the necrotic core and in ruptured plaques. The presence of OSEs in clinically relevant human lesions provides a strong rationale to target such epitopes in plasma and in atherosclerotic plaques for clinical applications.
IMMUNE RECOGNITION OF OXIDATION-SPECIFIC EPITOPES
By analogy with microbial “pathogen associated molecular patterns” (PAMPs), OSE – the products of oxidation in lipoproteins and various cellular components – represent a class of “danger (or damage) associated molecular patterns” (DAMPs) (Figure 2) [4, 10]. The common feature of PAMPs and DAMPs is their recognition by the same “pattern-recognition receptors” (PRRs) of innate immunity. Cellular PRRs, such as scavenger receptors and toll-like receptors, are found on the cell surface and in intracellular domains of macrophages and in other cell types. In addition, there are important soluble PRRs including variants of some cellular PRRs, pentraxins, such as C-reactive protein, complement factor H [3] and natural antibodies (NAbs). NAbs can be considered immunoglobulin PRRs, having in common with cellular and soluble PRRs a limited repertoire and yet a wide range of pattern recognition. Remarkably, in normal mice and in newborn humans, as much as 15–30% of all IgM NAbs bind to OSE [11]. Among these, there is a high prevalence of IgM to MDA and related MDA- protein adducts. This suggests that removing pro-inflammatory OSE is important for host homeostasis and implies an evolutionary advantage in organisms that have high levels of OSE-specific NAbs [4].
BIOTHERANOSTIC APPLICATIONS TARGETING OXIDATION-SPECIFIC EPITOPES
The concept of “biotheranostics” as related to cardiovascular disease is derived from the preposition that one can target biological processes in the plasma or vessel wall and develop biomarker assays, therapeutic agents and diagnostic molecular imaging probes to the target. In this case the target is OSE present in circulating lipoproteins or in the atherosclerotic plaque and the targeting agents are human and murine antibodies or peptide fragments validated to detect such OSE [2].
Biomarkers
The ideal biomarker would be involved in causal pathways of atherogenesis and allow changes in clinical management when levels are measured.
A. Oxidized Phospholipid Biomarkers
A large body of work shows that measuring oxidized phospholipids on apolipoprotein B-100 particles (OxPL/apoB) fulfills many of the criteria for a clinically useful biomarker (Figure 3A, reviewed in ref [12]). Levels of OxPL/apoB, as measured with the monoclonal antibody E06, reflect vascular dysfunction and coronary calcification [13, 14], predict the presence and progression of ultrasound-measured carotid and femoral disease and angiographically-determined coronary artery disease and are elevated in acute coronary syndromes and following percutaneous coronary interventions [12]. Recently, in two parallel nested case-control studies within the Health Professionals Follow-up Study and the Nurses’ Health Study, OxPL/apoB were positively associated with risk of peripheral arterial disease (PAD) in men and women with a relative risk (RR) 1.37, 95% CI, 1.19–1.58 for each 1-standard deviation increase after adjusting for traditional risk factors (Figure 3B) [15]. Furthermore, OxPL/apoB levels, measured at baseline in a community dwelling cohort followed prospectively for 15 years, predict death, MI and stroke with hazard ratios 2.1–3.6 (Figure 3C) [16, 17]. Importantly for clinical applications, measurement of OxPL/apoB along with IgG and IgM autoantibodies to MDA-LDL, allowed reclassification of approximately 30% of patients initially in intermediate Framingham risk categories into either lower or higher risk categories (Figure 3D) [16]. Approximately one half of these patients were placed in a higher risk category and one half in a lower risk category. Additional assays to measure OxPL and assays to measure modified apoB have been described in the literature. However, a thorough comparison of the clinical predictability of cardiovascular risk by these assays has not been performed to date [18]. Due to lack of space, the reader is referred to several papers in this area for further details [19, 20].
Interestingly, lipoprotein (a) [Lp(a)], which is composed of apoB and apo(a), strongly binds OxPL [21, 22]. Lp(a) is now generally recognized as a causal, independent, genetic risk factor for CVD and myocardial infarction (MI) and has become a target of therapy for reducing cardiovascular disease [23–25]. Measurement of OxPL/apoB primarily reflects the content of OxPL on Lp(a)-associated apoB particles and may account in part for the pro-atherogenic and pro-inflammatory properties of Lp(a). In fact, OxPL, OxLDL and Lp(a) trigger apoptosis in endoplasmic reticulum-stressed macrophages through a mechanism requiring both CD36 and TLR2 [26]. Macrophage apoptosis is a key process in plaque necrosis in advanced atheromata and likely contributes to plaque progression, destabilization and clinical events. Because small apo(a) isoforms are associated with high Lp(a) plasma levels, and because these lipoproteins contain the most OxPL content, the OxPL/apoB measurement is primarily a reflection of the most atherogenic Lp(a) particles [27]. Since small apo(a) isoforms are generally accepted as highly atherogenic than large isoforms but are not easily measured and not performed clinically, measuring OxPL/apoB may reflect a key biological activity of Lp(a) in predicting CVD risk. In all studies reported to date, OxPL/apoB is either a superior or equal to Lp(a) in predicting CVD risk (reviewed in ref [12]).
B. OxPL and Plasminogen
Apo(a) is highly homologous to plasminogen, duplicated itself from the plasminogen gene, sits in the opposite direction to plasminogen on chromosome 6, and has changed and remodeled by losing its protease activity, losing kringles I–III while retaining kringles IV–V, developing multiple isoforms of kringle IV-2 and binding to apoB to become the lipoprotein Lp(a). Recent studies have shown that plasminogen also binds OxPL and represents a second major plasma pool of OxPL in addition to those present on Lp(a) [28, 29]. Importantly, as opposed to OxPL on Lp(a), which predict increased cardiovascular risk, OxPL on plasminogen are associated with enhanced potential for fibrinolysis and thus may be associated with reduced atherothrombotic risk (Figure 4A) [29]. Enzymatic removal of OxPL from plasminogen resulted in a longer lysis time for fibrin clots as measured in in vitro assays. OxPL/plasminogen levels increased following acute myocardial infarction, implying that OxPL carried by plasminogen have a role in atherothrombosis (Figure 4B). Measurement of OxPL on plasminogen may provide insights into both risk of thrombosis in patients at risk of thrombotic disorders, such as MI, stroke, atrial fibrillation, pulmonary emboli and deep venous thrombosis. It may also reflect bleeding risk for patients treated with anti-coagulants and anti-platelet agents. Studies are underway to assess the potential clinical value of OxPL/plasminogen as a biomarker of thrombosis and bleeding risk.
C. Malondialdehyde-acetaldehyde (MAA) and Complement Factor H (CFH)
CFH is a very abundant plasma protein that regulates complement activation by mediating anti-inflammatory properties and protecting cells from excessive complement activation. The single nucleotide polymorphism (SNP) Y402H (rs1061170) in the short consensus repeat 7 (SCR) of CFH, present in ~35% of patients, has been previously associated with age related macular degeneration (AMD), but the underlying pathophysiology until now was unknown. The molecular defect has been defined by Weismann et al [3] by demonstrating that SCR7 as well as SCR20 of CFH are MDA and MAA adduct-binding sites (Figure 5A and 5B). Compared to individuals with wild-type CFH, plasma from patients with AMD and the rs1061170 SNP have significantly diminished ability to bind to MDA-LDL, with f heterozygous subjects having a 23% reduction in binding and homozygous subjects 52%, irrespective of the total plasma CFH levels (Figure 5C). Furthermore, CFH was shown to block the uptake of MDA-modified proteins by macrophages and mitigated MDA-induced pro-inflammatory effects, such as IL-8 secretion, in vitro and in vivo in mice (Figure 5D). In retinal pigment epithelial (RPE) cells, CFH was shown to co-localize with MDA (Figure 5E). Overall, these data suggest that the CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind pro-inflammatory MDA/MAA-protein adducts, indicating a causal link to AMD.
MDA and MAA adducts are also present in atherosclerotic lesions from patients with cardiovascular disease, and are particularly prevalent in vulnerable plaques and necrotic cores [9]. It was also demonstrated that CFH and MDA co-localize in human atheroma from patients with acute coronary syndromes (Figure 5F), suggesting that it may also play a role in mitigating the effects of MDA/MAA in plaque destabilization. Clinical studies are now being carried out to address this hypothesis.
Molecular Imaging
The presence of OSE in the vessel wall, and in particular in high-risk human lesions, provides the rationale to develop imaging approaches to detect such OSE [9]. Molecular imaging of OSE may provide a means to detect and monitor such lesions and assess the efficacy of established or experimental therapies. Three unique MRI based approaches have been developed to non-invasively image OSE, using gadolinium, manganese or specifically-targeted, lipid-coated, iron oxide nanoparticles (as opposed to dextran coated nanoparticles which are taken up non-specifically by macrophages) with attached murine (MDA2, E06) or human (IK17) oxidation-specific antibodies (Figure 6) [30–32]. MDA2 is a murine IgG monoclonal antibody that binds to MDA-lysine epitopes present on modified LDL or other MDA-modified proteins. IK17 is a fully human Fab or single-chain Fv (scFv) fragment that binds advanced MDA and MAA epitopes. E06 is a natural murine IgM monoclonal antibody cloned from apolipoprotein E–deficient (apoE−/−) mice that binds to the phosphocholine head group of oxidized, but not native phospholipids. These antibody targeted nanoparticles bind OSE in plasma or in the vessel wall, are then internalized by macrophages and provide excellent images of experimental atherosclerotic lesions in vivo (Figure 6). For the Mn-micelles, when this nanoparticle binds to extracellular OxLDL and is taken up by macrophages, free Mn is released intracellularly resulting in ~10X increase in relaxivity and visualization of macrophages, thus becoming an indirect macrophage-targeting agent. Because these nanoparticles accumulate in macrophages, they may not only provide a means to quantify plaque burden, but also to potentially predict plaque instability.
In an extension of these studies, transgenic zebrafish were generated to express a temperature-inducible, enhanced green fluorescent protein (EGFP)-labeled, IK17-scFc-EGFP construct. Feeding a high-cholesterol diet supplemented with a red fluorescent lipid marker to transgenic zebrafish larvae resulted in vascular lipid accumulation. After heat shock–induced expression of IK17-scFv-EGFP, time-dependent vascular accumulation of IK17-specific MDA epitopes could be observed [33].
Overall, these studies suggest that very early lesions, as in the zebrafish, as well as moderately advanced lesions containing macrophages, as in LDLR−/− and apoE−/− mice, can be visualized non-invasively. Ultimately, molecular imaging approaches targeting OSE may be useful in the diagnosis of high risk patients, surveillance of plaque progression and plaque instability, testing of novel therapeutic agents, assessing effect of established therapies and perhaps providing guidance on more aggressive therapy.
C. Therapeutic Approaches
The potential therapeutic use of oxidation-specific antibodies for CVD is being evaluated in pre-clinical and early phase studies. For example, immunization of LDLR−/− mice with pneumococcal vaccine (containing phosphocholine epitopes) caused an increase in circulating E06 levels that is associated with a reduction in atherosclerosis progression [34], potentially by inactivating or clearing relevant OSE. Deletion of IgM antibodies in mice is associated with higher risk of atherosclerosis progression [35, 36]. Several studies in experimental models have suggested that direct infusion of oxidation-specific antibodies results in lower rate of progression of atherosclerosis or enhanced regression of established lesions [37, 38]. Finally, high levels of IgM antibodies are associated with atheroprotection in epidemiological studies [16, 39, 40] although this is not always independent of traditional risk factors.
In recent work, the human antibody IK17 was either infused intraperitoneally as a Fab or overexpressed with an adenoviral vector as single-chain Fv fragment, resulting in significantly reduced atherosclerosis progression (Figure 7A–B) [41, 42]. Importantly, mechanistic information from these studies demonstrated reduced binding of OxLDL/MDA-LDL to macrophages and significant reduction in foam cell formation (Figure 7C). Furthermore, sustained overexpression of IK17 in a zebrafish model of hypercholesterolemia, induced regression of oxidized lipid deposits in the vascular wall (Figure 7D–E)[33].
This suggests that if these antibodies were to be used clinically, they would have the potential to acutely decrease OxLDL uptake and cholesterol accumulation in macrophages and potentially result in rapid plaque stabilization by preventing pro-inflammatory effects of foam cells. This is consistent with data from animal models showing that during dietary-induced regression, removal of OSE, such as OxPL and MDA epitopes, from the vessel wall is one of the first events that occurs, even before physical regression of the atheroma as a whole [43, 44]. Along with this loss of OSE, there is the concomitant presence of features of plaque stabilization, such as gain of smooth muscle cells and collagen and loss of macrophages, and reduced oxidative stress [45]. A clinical Phase II trial with an humanized IgG antibody that presumably bound modified apoB was reported to show no difference in FDG-PET uptake of the carotid arteries, but the details have not been published to date. Studies of such antibodies with clinical endpoints have not been performed to date.
V. CONCLUSIONS
Novel paradigms are now emerging to translate the advances in fundamental knowledge of the interaction of oxidative pathways, the immune system and the resulting inflammatory responses to OSE into clinical practice. Encompassed by the concept of biotheranostics, OSE biomarkers show strong associations with both progression of CAD/PAD and in predicting future events, suggesting that they may be used to complement the existing clinical armamentarium. Emerging diagnostic molecular imaging approaches targeting OSE, if translated to humans, may provide unique information about plaque burden as well as plaque activity, and therapeutic use of oxidation-specific antibodies may allow more refined approaches targeting bioactive, pro-inflammatory OSE to treat active CVD or diminish future risk of events.
KEY POINTS.
Oxidation of lipoproteins generates a variety of oxidation-specific epitopes that are immunogenic, pro-inflammatory, pro-atherogenic and plaque destabilizing.
The innate immune system recognizes oxidation-specific epitopes as danger associated molecular patterns (DAMPs) and uses innate pattern recognition receptors, including soluble receptors, such as IgM natural antibodies and complement factor H, to bind, neutralize and/or clear such DAMPs.
Oxidation-specific epitopes can be targeted in plasma or in the vessel wall for “biotheranostics”-, i.e. biomarker, therapeutic and molecular imaging diagnostic applications.
Development of assays to measure oxidized phospholipids on lipoproteins and plasminogen provide insights into atherothrombosis and can be used clinically to predict new cardiovascular events
Oxidation-specific antibodies attached to nanoparticles can be used for magnetic imaging of lipid-rich, oxidation-rich plaques and be directly infused to reduce the progression of atherosclerosis.
Acknowledgments
This study was supported by NIH grants HL055798, HL093767 and HL088093.
Footnotes
Disclosures
Dr. Tsimikas is a co-inventor and receives royalties from patents owned by the University of California for the commercial use of oxidation-specific antibodies, is a consultant to Quest, Sanofi, Genzyme, Regeneron and ISIS and has received investigator-initiated grants from Pfizer and Merck. Dr. Miller is a co-inventor of a patent owned by the University of California for the use of the hypercholesterolemic zebrafish model and has received an investigator-initiated grant from Merck.
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