A role for autoantibodies in atherogenesis

Abstract

An increased risk of cardiovascular disease (CVD) has long been recognized amongst people with autoimmune disease. It has been unclear whether this is due mainly to the ensuing treatment, particularly steroids, or whether some of this risk is due to the autoimmune process itself with subsequent inflammation. Indeed, a large body of evidence supports a role for chronic inflammation in atherogenesis, and autoantibodies have been identified as mediators in this complex inflammatory environment. Our aim is to carry out a systematic review of existing literature in order to formally establish the strength of the association between autoantibodies and atherosclerosis, amongst individuals without clinical autoimmune disease. An electronic search of five databases to June 2016 was performed by two independent reviewers. Inclusion criteria were analytical studies of adults, with at least two studies per autoantibody. Quality analysis was carried out using the Newcastle-Ottawa scale and the Cochrane Risk of Bias Quality Assessment Tool where appropriate. Where possible, studies were pooled using random effects models. Raised levels of anti-cardiolipin (odds ratio [OR] = 1.30; 95% CI: 1.15–1.49) and anti-oxidized low-density lipoprotein Immunoglobulin (Ig) G (OR = 1.25; 95% CI: 1.11–1.41), unspecified anti-cyclic citrullinated protein (OR = 3.09; 95% CI: 1.49–6.41) and anti-human heat shock protein 60 IgA (OR = 1.57; 95% CI: 1.15–2.16) were observed to increase the risk of cardiovascular outcomes. Alternatively, Anti-phosphorylcholine IgM (OR = 1.31; 95% CI: 1.14–1.50) conferred protection against CVD. Our results support an important role for autoantibodies in mediating cardiovascular events, independent of therapeutic treatments. Future research may focus on the presence of autoantibodies as markers of immune dysregulation and CVD risk.

1. Introduction

Cardiovascular disease (CVD) accounts for over 30% of deaths on a global level, translating to approximately 17.5 million deaths annually.¹ The majority of this mortality (80%) is attributed to myocardial infarction (MI) and stroke.² By the year 2030, CVD related deaths are expected to grow to more than 23.6 million.¹ CVD is also associated with significant levels of disability amongst survivors of MI and stroke. As such, CVD is a major public health concern.

The hallmark of atherosclerosis (AS) is abnormal thickening of arterial walls resulting from a number of physical and chemical insults to the endothelial cell layer working alone or together.³ Injury to the endothelium can result from trauma resulting from high blood pressure or turbulent blood flow (as is observed to occur where arteries branch), circulation of reactive oxygen species e.g. from exposure to cigarette smoke and air pollutants, chronically elevated blood glucose levels, as well as high levels of homocysteine.³ Over time plaques can grow, rupture, and thrombose, causing decreased blood flow through narrowed arteries, consequently manifesting as angina pectoris (AP), MI, or stroke.³

There have been repeated observations of an exaggerated and premature atherogenic process amongst people with autoimmune disorders that leads to their increased morbidity and mortality.^4–6 Queries have emerged as to whether this increased risk is due to treatment of the autoimmune condition, e.g. with steroids, or the disease process itself. Today, AS is considered at least partly an inflammatory disease; however, the pathways involved in vascular inflammation and the role of autoantibodies directed towards an assortment of self-antigens are still poorly understood.

A wealth of data points towards a potential role for autoantibodies both as drivers of inflammation as well as independent factors associated with CVD risk. Existing literature has largely focused on delineating the role of defined autoantigens, e.g. Heat shock protein (HSP)-60, oxidized low-density lipoprotein (OxLDL) and beta 2 glycoprotein 1 (β2GP1) and their associated autoantibodies, in the development of AS-related CVD, but several other autoantibodies have also been explored. Moreover, as in the case of anti-OxLDL, autoantibodies have been implicated in both protective and pathogenic roles, and this has yet to be properly elucidated.⁷ We present the first systematic review to our knowledge that summarizes evidence on the relationship between a range of autoantibodies and subsequent atherosclerotic related CVD mortality and morbidity amongst adults without clinical autoimmune disease.

2. Methods

An electronic search across five databases (MEDLINE, EMBASE, SCIENCE DIRECT, SCOPUS, and PUBMED) was carried out up to June 2016 to identify published observational and experimental studies investigating an association between the presence of autoantibodies and the subsequent occurrence of CVD morbidity and mortality in populations without clinical disease. All literature was identified, evaluated and extracted independently by two reviewers (R.I. and T.H.). All relevant studies underwent quality assessment and a meta analyses was carried out to evaluate the effect of each autoantibody (highest vs. lowest quartiles of autoantibody titer) on cardiovascular outcomes. Data were pooled where possible using random effects models, with measures of heterogeneity calculated and funnel plots created to check for study size effects. Details of the materials and methods utilized in this study are available in the online only data supplement.

3. Results

A total of 113 primary articles were retrieved for full text examination with 62 articles subsequently excluded (see Supplementary material online, Figure S1). Only one observational study was deemed poor quality according to the Newcastle–Ottawa Scale (NOS) (see Supplementary material online, Appendix S1).⁸ The latter was not excluded, instead we highlighted its limitations. Additionally, all identified experimental studies were deemed to be of fair quality according to the criteria utilized in Cochrane Risk of Bias Quality Assessment Tool to evaluate methodological rigor of Randomized Control Studies (see Supplementary material online, Appendix S2). Supplementary material online, Appendices S3 and S4 illustrate the quality analysis findings of the observational and experimental studies respectively. The characteristics of the 51 studies included in this review are presented in Supplementary material online, Appendix S5. A summary of the extracted information emphasizing the number of positive and negative studies for each autoantibody is presented in Supplementary material online, Appendix S6. The information contained in this section summarizes the results for the key autoantibodies whose results could be combined in a meta-analysis. This review did however additionally examine a range of other autoantibodies whose data could not be pooled. These findings are presented in the supplementary material.

3.1 Antiphospholipid autoantibodies

Our review identified 15 studies that examined the association between anti-phospholipid autoantibodies (APAs) and the subsequent occurrence of a range of AS-related CVDs amongst non-clinical samples.^9–23 A total of 10 studies identified at least one APA as a significant risk factor for future AS-related CVD, with high titers of anti-cardiolipin autoantibody (aCL) being significantly associated with future stroke and MI.^9–18 Only 11 aCL immunoglobulin (Ig) G and 4 aCL IgM studies could be combined for the purpose of a meta analyses. Overall increased aCL IgG titers were significantly associated with an increased risk of developing AS-related CVD [odd ratio (OR) = 1.30; 95% CI = 1.15–1.49]. Similarly, a significant association between increased aCL IgM titers and subsequent AS-related CVD was noted within our results (OR = 1.27; 95% CI = 1.08–1.30) with no heterogeneity observed (I² = 0%; P = 0.444). Our findings are illustrated in Figure 1.

Alternatively, based on existing literature examining the association between acL IgG and future AS-related CVD, we observed moderate heterogeneity within our results (I-squared = 52.5%, P = 0.021) and no evidence of publication bias using Egger method (P for bias = 0.177) (see Supplementary material online, Appendix S7a). Anti-β2GP1 autoantibodies were also implicated in the pathogenesis of AS-related CVD with two studies reporting a significantly increased risk of stroke and MI related morbidity and mortality^9,13; however this was insufficient to pool.

3.2 Anti-phosphorylcholine autoantibodies

Six studies examined the association between anti-PC and AS-related CVD events.^24–29 Although the former three studies utilized non-fatal CVD outcomes,^24–26 the latter three studies additionally examined the association between anti phosphorylcholine autoantibodies (anti-PC)- and CVD-related mortality.^27–29 Only two studies utilized a sample without any history of CVD at baseline.^25,27 IgM was the common autoantibody subtype measured amongst all seven studies. Moreover, all studies reported an independent association between low anti-PC IgM and an increased risk of AS-related CVD outcomes, indicating that this antibody was protective for CVD. Only five studies could be combined in a forest plot, with an overall OR of 1.31 (95% CI: 1.14–1.50) for lack of autoantibody. Moreover, no heterogeneity was observed as illustrated in Figure 2 (I-squared = 8%, P = 0.361). Interestingly, one study reported higher IgM anti-PC levels in women when compared with their male counterparts.²⁵

3.3 Anti-low density lipoprotein autoantibodies

Our literature search identified 14 studies examining the association between AS-related CVD events and a range of anti-low-density lipoprotein (anti-LDL) autoantibodies.^{10,22,30–42} The autoantibody exposures were subdivided into OxLDL autoantibodies and Malondialdehyde modified LDL (MDA-LDL) autoantibodies. The relationship between anti-LDL and primary as well as secondary AS-related CVD outcomes were equally investigated. Only five studies reported a significant association between their observed autoantibody and subsequent development of AS-related CVD outcomes.^{30,31,33,36,38} Similarly, three studies reported a reduced risk of future CV events associated with LDL autoantibodies specifically targeting apolipoprotein B-100 (ApoB-100) moiety of LDL.^39–41 Interestingly, one randomized control trial reported an association between high levels of IgM MDA-peptide 210 (MDA-p210) autoantibodies and a more rapid carotid intima media thickness progression (cIMT).⁴²

A total of seven studies were combined to examine the overall effect of OxLDL IgG on CVD risk, whilst only four studies each could be combined for MDA-LDL IgG and IgM as well as OxLDL IgM. As illustrated in Figure 3A, OxLDL IgG was observed to significantly increase the risk of AS-related CVD outcomes (OR = 1.25; 95% CI: 1.11–1.41). There was evidence of moderate heterogeneity (I² = 32.4%, P = 0.205) within the OxLDL IgG data, whilst no publication bias using Egger method (P for bias = 0.172) was observed (see Supplementary material online, Appendix S7b). Alternatively, raised anti-OxLDL IgM autoantibody levels was observed to reduce the risk of future AS-related CVD occurrence though this association failed to reach significance (Refer to Figure 3B). Similarly, anti-MDA-LDL IgG and IgM were observed to play a protective role in atherogenesis, with a reduced risk of developing AS noted across both autoantibody isotypes (Refer to Figure 4).

3.4 Anti-cyclic citrullinated protein autoantibodies

Our literature search identified three studies that examined the association between the presence of anti-CCP and the subsequent occurrence of AS-related CVD.^43–45 Two out of three studies provided evidence of a statistically significant association between these two variables, with anti-CCP positivity predicting future AS-related cardiovascular events.^43,45 All studies examined the association between anti cyclic citrullinated protein autoantibodies (anti-CCP)- and AS-related cardiovascular morbidity and mortality. One study utilized a population sample comprising individuals with and without rheumatic disease.⁴⁴ Additionally, only one study specified the anti-CCP autoantibody subtype measured.⁴³ Moreover, the latter study utilized a sample without a history of CV disease.⁴³ Interestingly, the overall effect of anti-CCP on CVD outcomes was observed to be significant as illustrated in Figure 5 (OR = 3.09; 95% CI: 1.49–6.41; I² = 0%; P = 0.787).

3.5 Anti-HSP and anti-infectious agent antibodies

Our review identified eight studies investigating the relationship between a range of anti-HSP and antibodies directed against a range of infectious agents in relation to AS-related CVD outcomes amongst non-clinical samples.^46–53 The exposures included antibodies against mycobacterial hsp65 (mHSP65), human hsp60 (huHSP60), and chlamydial pneumoniae hsp60, as well as antibodies against chlamydia pneumoniae IgA (Cpn IgA), Helicobacter pylori IgG (H.pylori IgG) and cytomegalovirus IgG. Interestingly all eight studies reported a significant association between at least one of the autoantibodies and their AS-related CVD outcome. Cpn IgA was implicated in early and advanced AS^48,50,52 although this observation was not shared amongst all studies. huHSP60 IgA was observed to predict future CV events amongst four studies,^46–48,53 whilst, two additional studies reported a role for high mHSP65 IgG as risk factors of AS development.^49,51 The remaining antibodies failed to illustrate any significant association with AS progression or MI. Only three studies could be combined in a forest plot presented in Supplementary material online, Appendix S7c1. Anti-huHSP60 IgA appear to increase the risk of subsequent CV events with no evidence of heterogeneity (OR = 1.57; 95% CI: 1.15–2.16; I² = 0%, P = 0.450).

4. Discussion

4.1 Anti-phospholipid autoantibodies

In examining a role for antibodies in AS we observed that both IgG and IgM APAs are associated with an increased risk of future CVD.

APAs are a diverse group of Igs targeting negatively charged phospholipids (CL, phosphatidyl serine, phosphatidyl inositol), protein—phospholipid complexes and plasma proteins that have no adequate anionic surface such as β2GP1 (15, 18). APAs directed against CL and its cofactor β2GP1 are best characterized.^5,7 CL is found primarily in the inner mitochondrial membrane of eukaryotic cells and plays a central role in mitochondrial bioenergetics and also appears to be of major importance in apoptosis and membrane dynamics.¹⁷ On the other hand, β2GP1, also known as Apolipoprotein H, is a plasma glycoprotein that not only binds negatively charged molecules, including phospholipids and plasma lipoproteins, but also the surface of activated platelets and membranes of apoptotic cells.¹¹

One potential pathway through which they exert their influence is by interacting with endothelial cells.¹⁵ The latter have cell surface receptors called annexin II that can attract and bind to β2GP1 which in turn attracts the APAs resulting in endothelial cell activation, increased secretion of pro-inflammatory (PI) cytokines, release of tissue factor with subsequent initiation of the coagulation cascade¹⁸ (Figure 6). Alternatively, APAs are said to promote uptake of lipoproteins with the resulting lipid laden macrophages converting to foam cells.^9,54 β2GP1 is proposed to play an anti-oxidant role in binding with OxLDL by subsequently quenching its pro-atherogenic and PI effects.⁵⁵ In this regard, studies have successfully illustrated the ability of β2GP1 to neutralize negative charges generated by oxidized LDL.^56,57 The latter complex however is highly immunogenic, and more rapidly internalized by macrophages as IgG immune complexes (ICs) in the presence of anti-β2GP1 autoantibodies.⁵⁸ Autoantibody complexes containing OxLDL and β2GP1 are therefore pro-atherogenic, resulting in increased production of lipid overloaded macrophages that in turn promotes further secretion of inflammatory mediators.^58,59 This proposed mechanism of action is illustrated in Figure 7. This notion is supported by studies illustrating the ability of β2GPI to inhibit in vitro uptake of OxLDL by murine macrophages, and conversely, the increased binding of OxLDL to macrophages by simultaneous addition of anti-β2GPI antibodies.⁶⁰ aCL autoantibodies targeting β2GP1 and anti-β2GP1 autoantibodies are implicated in this process.^58,59

It has been suggested that endothelial cell injury (perhaps caused by an initial cardiovascular event such as a stroke or CVD risk factors such as hypertension) may lead to exposure of antigens that are normally hidden within the phospholipid bilayer of the cell membrane such as CL, thus eliciting an immune response.²³ aCL can then bind to endothelial cells or β2GP1.²³ aCL may therefore be both a marker of vascular damage and a risk factor for AS events. Additionally, experimental studies have provided support for a causal role for aCL in atherogenesis. Immunization of LDL receptor deficient (LDL −/−) mice with aCL has been observed to lead to the development of anti-aCL mouse autoantibodies, that exhibit the same binding characteristics as the human aCL, and accelerated development of AS in the aortic sinus.⁶¹ Moreover, in systemic lupus erythematosus (SLE) and anti-phospholipid syndrome, aCL autoantibodies have been shown to reduce activity of plasma paraoxonase (an antioxidant enzyme circulating in plasma attached to high-density lipoproteins).⁵⁴ The latter is postulated to result in diminished natural anti-oxidant mechanisms in turn allowing for the oxidative conversion of LDL to OxLDL in subendothelial spaces.⁵⁴

4.2 Anti-PC autoantibodies

Anti-PC has been shown to be a protection marker for AS development and CVD, in different populations consisting of healthy individuals, acute coronary syndrome (ACS) patients, as well as patients with SLE or rheumatoid arthritis (RA).^24–29,62 Moreover, passive immunization with a monoclonal anti-PC IgM antibody in a murine model of native aortic and vein graft AS as well as active immunization of Apolipoprotein E (ApoE) knockout mice with PC resulted in reduced AS.^63,64

PC is a biologically important epitope in PI oxidized phospholipids (OxPLs) such as platelet activating factor-like lipids (PAF-like lipids) and lysophosphatidylcholine.²⁸ PC is also a major active component in bacteria including streptococcus pneumonia and in apoptotic cells.²⁵ Natural IgM PC antibodies exist in humans but little is known of their clinical relevance. The latter are postulated to react to PC on bacteria, OxLDL and apoptotic cells but not to those on unoxidized phospholipids or viable cells.²⁸ Researchers have reported a homeostatic function for anti-PC autoantibodies. The latter are said to inhibit uptake of OxLDL by macrophages through scavenger receptor, cluster of differentiation (CD).^25,62. Moreover, they are believed to inhibit the PI effects of PAF-like lipids generated during LDL oxidation by inhibiting PAF-induced endothelial cell activation.^25,57,65 The latter associations are reported to occur independent of traditional risk factors including high sensitivity C reactive protein.²⁷ As a result, anti-PC is considered not to be a measure of system inflammatory burden, but rather a measure of a subject’s ability to cope with inflammatory burden.²⁸

4.3 Anti-LDL autoantibodies

Modification of LDL may occur through a variety of ways and generates an array of neoantigens capable of inducing autoantibody driven immune responses.⁶⁶ LDL contains phospholipids, free cholesterol, cholesterol esters, triglycerides and apolipoportein B that are all susceptible to modification.⁵⁹ Oxidized phospholipids and aldehyde modified peptide sequences are the major LDL targets for the immune system.³¹ Though anti-LDL autoantibodies represent one of the most extensively studied group of autoantibodies in relation to AS, their role in the development and progression of this disease has remained controversial. The latter is largely due to the inconsistent reports regarding their association with AS-related CVD severity and risk largely attributed to a poor understanding of the heterogeneous nature of oxidative modification as well as the varied effects resulting from different classes and subclasses of anti-LDL autoantibodies.

As has been observed in our review, both a protective as well as a pro-atherogenic role has been described amongst existing literature for this group of autoantibodies. In general, data suggests IgG autoantibodies are pro-atherogenic whereas IgM autoantibodies are protective. Our results support the notion that OxLDL IgG autoantibodies may play a role in promoting an atherosclerotic process (Refer to Figure 3A). This is consistent with the existing knowledge that highlights the predominant isotype of OxLDL autoantibodies as IgG of subclass 1 and 3, which is classically considered PI.⁶⁷ In particular, anti-OxLDL IgG autoantibodies form ICs with modified LDL that are reported to be pro-atherogenic and PI.^67,68 The latter promote macrophage uptake of LDL-IC via interaction with Fc gamma receptor 1, with subsequent macrophage activation and release of, PI cytokines such as IL-1 and tumor necrosis factor (TNF), proteolytic enzyme metalloproteinase and oxygen active radicals as well as advancing macrophage transformation into foam cells.^67,68 Moreover, the PI effects of anti-OxLDL IgG autoantibodies are believed to be partly responsible for thinning of the plaque fibrin cap and its ultimate rupture leading to ACS or stroke.⁶⁷ Interestingly, human LDL-IC have been shown to induce cholesterol ester accumulation in a number of macrophage like cell lines to a much higher degree than OxLDL, even when OxLDL concentrations were about 10 times greater than the amount of OxLDL contained in the OxLDL-IC used as control.⁶⁹ Alternatively, IgM OxLDL autoantibodies reportedly signify a natural immune response against pro-atherogenic oxidized LDL.^70,71 EO6 is currently the best characterized prototypic monoclonal IgM OxLDL autoantibody and is derived from a panel of B cell hybridomas from the spleen of atherosclerotic apolipoprotein deficient (ApoE−/−) mice.^70,71 The latter recognizes the lipid and protein moiety of OxLDL, binding specifically to OxPLs containing the PC headgroup (but not native unoxidized PC) and initiate a series of events including inhibiting OxLDL uptake by macrophages.^70,71 The protective feature of IgM OxLDL autoantibodies was also represented in our findings (Refer to Figure 3B). These proposed mechanisms are illustrated in Figure 7.

Interestingly, both IgG and IgM anti-MDA-LDL autoantibodies were observed to infer protection against AS (Refer to Figure 4). Notably, research that has assessed the association between baseline levels of these autoantibodies and AS have spawned conflicting observations that have ultimately emphasized the importance of considering autoantibody antigenic targets.^39–42 One important antigenic epitope that is linked to AS is ApoB-100. The ApoB-100 peptides 45 (amino acids 661-680) and 210 (amino acids 3136-3155) both in their native and aldehyde modified form have been identified as important targets for immune responses against LDL.^39–42 Studies that have highlighted their specific autoantibody antigenic targets have specified MDA-LDL autoantibodies against the latter ApoB-100 peptides to be associated with less severe AS and a lower risk of future CV events amongst both men and women.^31,39–41 Moreover, experimental studies involving the treatment of AS prone mice with human recombinant IgG recognizing the MDA peptide 45 (MDA-p45) epitope that resulted in reduced aortic plaque area and plaque inflammation have provided further support for an inverse association between these autoantibodies and AS.⁷² Furthermore, immunization of ApoE knockout mice with MDA-p45, resulting in an increase in specific IgG, associated with a decrease in aortic plaque area and plaque content of inflammatory cells has provided support for the notion that the p45 sequence of ApoB-100 may be a potential target for immunomodulatory treatment for AS.⁷³ IgM autoantibodies against modified LDL are suggested to play a role in clearing OxLDL from the circulation highlighted by studies reporting an inverse association between IgM MDA ApoB-100 autoantibodies and plasma OxLDL.⁶⁹ Similarly, animal studies investigating the effect of hypercholesteroleamia on immune effector responses have highlighted an association between anti-atherogenic transforming growth factor beta (TGFβ) and IgG2b MDA-LDL autoantibodies.⁷⁴ In this regard, ApoE−/− mice fed with a high cholesterol diet showed increased levels of plasma TGFβ followed by elevated IgG2b MDA-LDL autoantibodies.⁷⁴ Increased TGFβ1 levels were proportionally correlated to IgG2b MDA-LDL autoantibody titres as well as the level of plasma cholesterol.⁷⁴ Though the exact clinical significance of these parallel relationships is yet to be elucidated, TGFβ is associated with decreased oxidative stress, inflammation and adhesion protein expression further suggesting an anti-atherogenic role for MDA-LDL autoantibodies.⁷⁴ Likewise, low levels of IgG autoantibodies specifically targeting native peptide 210 and 45 of ApoB-100 have also been linked to an increased risk of future CV events.^39–41

It is important to note that anti-OxLDL autoantibodies are observed to be present in healthy individuals as well as in patients with AS-related CVDs.^70,71 Moreover, LDL with minor modifications is reportedly present in the circulation, whereas more severely oxidized LDL have been observed inside AS plaques.⁷⁵ Subsequently, antibodies to modified LDL may potentially also be an indicator of the degree of immunogenicity of modified lipoproteins which depends largely on the degree of modification of LDL.^67,75 Anti-LDL are a noteworthy group of autoantibodies based on the fact follow up studies have illustrated the ability of elevated anti-LDL autoantibodies to predict the development of AS-related CVDs whilst experimental data has also indicated that anti-LDL antibodies may be protective.⁷¹ Collectively, the sum of published data is contradictory and there remains a need to better evaluate their role in health and disease whilst accounting for the influence of idiotype, class, and subclass of autoantibody.

4.4 Anti-CCP autoantibodies

Anti-CCP are characteristically present in RA, and associated with its distinctly higher inflammatory activity,⁷⁶ although all studies included here were in populations with no clinical disease. The latter recognize a citrullinated arginine residue that is a product of a common posttranslational modification due to the action of peptidylarginyl deaminase enzymes, released by human macrophages and neutrophils. Citrullination of proteins is believed to be a natural component of the inflammatory process within AS plaques as in synovia.⁴³ It seems probable that citrullinated proteins are ordinarily cleared by innate scavenging mechanisms that circumvent the adaptive immune response. As such, their presence in the context of an immune disorder may highlight interference with the latter process, in turn resulting in autoantibody generation.⁴³

4.5 Anti-HSP and anti-infectious agent antibodies

The term, HSPs refers to 24 stress proteins and cognates sequestered in low levels inside the cells, sometimes being expressed on the cell surface by nucleated cells in response to environmental stress or injury including changing temperature, oxygen tension, and infection, as a means of protecting themselves from unfavourable conditions.^{46,47,49,77,58} Classification of HSPs is based on molecular weight with HSP60, HSP65 and HSP70 being the most extensively studied.⁴⁷ Bacterial HSP have a high sequence homology with their human counterparts (75% at the amino acid level).⁴⁷ The phylogenetically conserved nature of HSP expression suggests a key role for these molecules in cellular protection.⁴⁶

Our review identified a potential pro-atherogenic role for infection with Cpn, as well as the presence of autoantibodies targeting human HSP60 and mycobacterial HSP65 antigens.^47–53 Atherosclerosis resulting from bacterial infection has been linked to immune reactions targeting the HSP60/65 antigens.⁷⁸ Immune responses to microbial agents are believed to induce AS by causing autoimmunity to endogenous HSP60 due to molecular mimicry and enhancing HSP specific T helper (Th) 1 immune responses.⁷⁸ The latter has been observed in animal studies of normocholesteroleamic rabbits and LDL receptor deficient mice immunized with mycobacterial HSP65 as well as mice infected with H.pylori.^79–81 Moreover, anti-HSP60 autoantibodies are observed to correlate strongly with human IgA Cpn and IgG H.pylori.⁵⁰ Anti-HSPs have been shown to mediate endothelial cytotoxicity by inducing secretion of PI cytokines such as TNFα, interleukin (IL)-1, IL-6, IL-12, promotion of monocyte adhesion to endothelial cells and the recruitment of inflammatory cells into vascular tissues.^47,58,77

The latter process is postulated to occur in individuals with borderline hypertension who are reported to have significantly higher anti-HSP65 and circulating anti-huHSP60 antibody levels compared with normotensive subjects.⁴⁶ High pulsatile shear stress has been demonstrated to induce HSP60 in endothelial cells.⁴⁶ Human serum HSP65 autoantibodies have also been shown to react with recombinant form of human HSP60 and similarly result in endothelial cell cytotoxicity.⁷⁸

One particular animal study demonstrated a role for epitope binding specificity of antibodies in determining their involvement in AS development.⁸¹ In the latter study, administration to Apo E deficient mice of a specific monoclonal antibody II-13 that recognizes amino acid residues 288–366 of HSP60 effectively induced AS lesions via endothelial cell damage followed by an increase in leukocyte attachment and accumulation of macrophages and smooth muscle cells in lesion.⁸¹ Contrastingly, administration of monoclonal antibody ML-30 that binds amino acid 315–318 of HSP60 did not have cytotoxic effects in vitro.⁸¹ The latter study provides further support for the cytotoxic role of anti-HSP autoantibodies in atherogenesis.

5. Conclusion: a role for autoantibodies in atherogenesis

This review uncovered an increasing body of evidence that points towards a dual role for autoantibodies as both risk and protective factors for AS. Moreover, animal studies have provided mechanistic insights into the role of autoantibodies in atherogenesis. Notably, the association between autoantibodies and AS-related cardiovascular events was observed amongst individuals with no diagnosed autoimmune conditions, strengthening the case for an atherogenic role of these antibodies in their own right, not just due to subsequent treatment with immunosuppressive agents.

There are still some limitations. First, it was a common observation that blood was drawn at a single time point several years prior to the cardiovascular event. As antibody prevalence may change over time, these studies may underestimate the association between autoantibodies and AS-related cardiovascular event occurrence, i.e. bias towards the null. Additionally, the nature of antigens recognized by specific autoantibodies remains a topic of much debate. Antigenic targets of autoantibodies observed in autoimmune disease appear to differ from the same autoantibodies resulting from an infection.^16,17 Other limitations include failure to account for medications, low prevalence of autoantibodies amongst control groups, relatively small number of events in some studies and variations on the basis of selecting study participants as well as small sample sizes and short follow ups.^{8,10,14,16,43}

In order to build on existing knowledge, it is recommended that future research account for such factors as assay variability, isotypes, and cutoffs for labelling presence or absence of antibodies. Nevertheless, the indication that some autoantibodies have protective effects raises the potential to harness these as therapeutic agents.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Acknowledgement

I would like to acknowledge my supervisors for their ongoing support that made this research possible.

Conflict of interest: none declared.

Funding

The study was funded by the University of Newcastle Postgraduate Research Scholarship courtesy of the Collaborative Research Network (CRN) for Mental Health and Wellbeing—Scholarship.

References