Novel Anti-Atherosclerotic Therapies

Abstract

Many measures can control lipid risk factors for atherosclerosis. Yet, even with excellent control of dyslipidemia, other sources of risk remain. Hence, we must look beyond lipids to address residual risk. Lifestyle measures should form the foundation of cardiovascular risk control. Many pharmacologic interventions targeting oxidation have proven disappointing. A large program tested inhibition of a lipoprotein-associated phospholipase A₂ (LpPLA₂), culminating in two large-scale clinical trials that did not meet their primary endpoints. A variety of antioxidants have not shown benefit in clinical trials. Numerous laboratory and clinical studies have inculpated inflammatory pathways in the pathogenesis of atherosclerotic events. The p38 mitogen-activated protein kinase (MAPK) inhibitor losmapimod and an inhibitor of a leukocyte adhesion molecule, P-selectin, did not alter adverse events in trials. Low-dose methotrexate, despite the promising observational studies, did not lower biomarkers of inflammation or alter cardiovascular outcomes in the cardiovascular inflammation reduction trial (CIRT). Four large-scale investigations underway will determine colchicine’s ability to reduce recurrent events in secondary prevention. The Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) showed that an antibody that neutralizes IL-1β can reduce recurrent cardiovascular events in secondary prevention. The success of CANTOS points to the pathway that leads from the NLRP3 inflammasome through IL-1β to IL-6 as an attractive target for further study and clinical development beyond lipid therapies to address the unacceptable burden of risk that remains despite our best current care in secondary prevention.

Low-density lipoprotein (LDL) lies at the root of human atherosclerosis. If humans maintained for life an LDL concentration similar to that of newborns, we would probably not face a global epidemic of atherosclerosis.^1,2 Cholesterol concentrations considered “normal” by far exceed that required to meet biological needs. Although LDL levels appear to be drifting down, concentrations in contemporary cohorts remain undesirably high from the perspective of atherosclerosis prevention.^{3, 4} Fortunately, we now possess a panel of effective tools for control of LDL. The companion article by Kastelein and colleagues presents in detail the remarkable advances in pharmacologic control of LDL that today permit driving LDL concentrations safely below those of newborn humans.

Atherosclerosis Treatment Beyond LDL

Yet, for many millions exposure to a lifetime of unnecessarily high LDL has already sown the seeds of atherosclerosis. This situation prompts the necessity of identifying targets beyond LDL to make further inroads against atherosclerotic complications. In this regard, evidence has snowballed that triglyceride-rich lipoproteins (TGRL) participate causally in atherogenesis. Rigorous clinical testing of treatments for lowering TGRL has begun. High-dose omega-3 fatty acids of pharmaceutical grade can lower cardiovascular event rates in selected individuals.⁵ Part, but not all of the benefit of eicosapentaenoic acid may accrue from an anti-inflammatory effect. A large-scale clinical trial has commenced that tests the ability of a novel selective PPAR-α modulator to improve outcomes in diabetic subjects with hypertriglyceridemia (> 200 mg/dL) and low HDL (< 40 mg/dL).⁶ RNA-based agents that lower apolipoprotein CIII and Lp(a) are likewise in development.⁷ Thus, we have come a long way in our ability to control effectively lipid risk factors for atherosclerosis. Yet, even with excellent control of LDL and TGRL, other sources of risk remain. Hence, we must look beyond lipids to expand the palette of interventions that we can offer to our patients who have already encountered a lifetime of exposure to hypercholesterolemia and dyslipidemia.

Lifestyle Modification: The Foundation of Management of Atherosclerosis Risk in Primary Prevention

For primary prevention, lifestyle measures should form the foundation of cardiovascular risk control. Although we lack sufficient data proving that lifestyle interventions improve cardiovascular outcomes in primary prevention, lifestyle modification confers many demonstrable benefits.⁸ I (P.L.) advocate placing a Pascal’s Wager on lifestyle improvements, as they entail little if any risk.⁹ Smoking cessation, weight control, and physical activity should thus precede or accompany pharmacologic intervention in primary prevention. Indeed, initiating pharmacologic anti-atherosclerotic therapy early in life presents vexing societal, ethical, and financial issues. As we validate biomarkers that indicate risk due to particular genetic variants or pathways, we might justify pharmacologic intervention on apparently well people in the future. This approach embodies the promise of personalized or precision medicine. Yet, few if any such biomarkers or interventions beyond LDL control have undergone sufficient validation for deployment in clinical practice today.

Secondary Prevention Beyond Lipids

Secondary prevention, however, has entered a new era regarding targeting non-lipid risk factors to reduce recurrent events.¹⁰ This review will consider the current state of this nascent field. The failure of elegant laboratory studies and animal experiments to translate into clinical practice has plagued this field for many years. A number of rational interventions predicated on substantial preclinical data have not realized their promise in rigorous large-scale clinical trials. We examine below some of these cases.

The Rise and Fall of Anti-Oxidant Interventions

Targeting oxidant pathways has proven disappointing. Almost all schemes of the pathogenesis of atherosclerosis invoke oxidatively modified LDL as a key instigating factor.^{11, 12} Various phospholipases can liberate lipid moieties from oxidized lipoproteins that can activate deleterious functions of vascular cells and leukocytes found in atheromata. Yet, several attempts to inhibit phospholipases to prevent the generation of these putatively pernicious mediators have not borne fruit. The largest program tested inhibition of a lipoprotein-associated phospholipase A₂ (LpPLA₂) in patients with acute coronary syndromes or in the stable phase of atherosclerosis.¹³ Despite promising signals from intravascular imaging studies in humans, reinforced by encouraging results in atherosclerotic pigs with the Lp-PLA2 inhibitor darapladib¹⁴, the two large-scale clinical trials SOLID and STABILITY did not meet their primary endpoints.^{15, 16} These results, and those from smaller studies targeting other phospholipases, have led to abandonment of this class of enzymes as therapeutic targets in atherosclerosis.

With respect to direct targeting of oxidative stress and lipoprotein oxidation, results have also proven disappointing. A variety of antioxidants have not shown benefit in clinical trials. Vitamins C and E, and beta carotene have failed to reduce events in appropriately conducted and powered clinical trials.^17–19 The powerful antioxidant succinobucol partitions into lipoprotein particles and effectively prevents LDL oxidation in vitro. Yet, the large ARISE trial did not meet its primary endpoint.²⁰ As in the case of phospholipases, enthusiasm has waned for the application of antioxidant vitamins and direct inhibitors of oxidation as therapeutic targets in atherosclerosis.

Anti-inflammatory Interventions: A New Day Dawns

Numerous laboratory and clinical studies have inculpated inflammatory pathways in the pathogenesis of atherosclerosis and its clinical complications.^21–24 This body of evidence has inspired several trials, some completed and some underway, targeting various inflammatory pathways in secondary prevention of atherosclerotic events. The mitogen-activated protein kinases (MAPKs) mediate certain actions of stressors such as oxidant stress, pro-inflammatory cytokines, and pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide.²⁵ Focused interest in the p38 MAPK emerged from a coherent series of preclinical and clinical studies using the inhibitor losmapimod. In patients with acute coronary syndromes, treatment with losmapimod decreased the biomarker of inflammation C-reactive protein measured with a highly sensitive assay (hsCRP).²⁶ A study monitoring the effect of losmapimod on fluorodeoxyglucose uptake in humans failed to meet its primary endpoint.²⁷ The preliminary phase of a large-scale clinical trial with losmapimod (LATITUDE) did not demonstrate the clinical benefit, discouraging further pursuit of this target.^{28, 29}

A hallmark of inflammation, recruitment of leukocytes, is a process that depends on endothelial-leukocyte expression. In addition to endothelial cells, platelets contain one such adhesion molecule, P-selectin. This double source points to a pivotal role of this particular adhesion molecule in acute inflammation complicated by thrombosis. Studies of acute coronary syndrome patients treated with inclacumab, an anti-P-selectin antibody, reduced troponin release but did not alter adverse events in the SELECT-ACS trial.^{30, 31}

Another agent that targets inflammation, methotrexate, has transformed the practice of rheumatology. Low weekly doses of this agent have modified the course of rheumatoid arthritis and furnish a mainstay of management of patients with rheumatoid arthritis and allied inflammatory conditions. Methotrexate appears to exert an anti-inflammatory effect by inhibiting the synthesis of purines and pyrimidines, the building blocks of DNA.³² Methotrexate causes the release of adenine nucleotides and adenosine from cells. The adenosine can engage G protein-coupled adenosine receptors, notably the A₂A receptor implicated in downstream anti-inflammatory actions.³³ Methotrexate may also inhibit the JAK/STAT protein kinase pathway, a mechanism parallel to that of losmapimod.³⁴ The Cardiovascular Inflammation Reduction Trial (CIRT) evaluated low-dose methotrexate in patients at risk for cardiovascular events.³⁵ Despite the promising observational studies, low-dose methotrexate did not alter cardiovascular outcomes in the individuals studied. Indeed, the intervention did not lower biomarkers of inflammation, and the baseline inflammation in the enrolled population was in a lower risk zone (hsCRP 1.6 mg/l).³⁶ The null results of CIRT suggests that future trials of anti-inflammatory strategies should go to pains to study individuals with sufficient inflammatory burden at baseline to benefit from the intervention.

Another agent widely used to treat inflammatory conditions, colchicine, has also shown promise in cardiovascular inflammation. Colchicine has become a standard treatment for pericarditis, avoiding the corticosteroid rebound phenomenon that plagued the use of glucocorticoids in this cardiovascular inflammatory condition.³⁷ The publication of the LoDoCo study pioneered the use of colchicine in secondary prevention of coronary artery disease events. This open label, placebo-controlled trial randomized 532 individuals with stable coronary artery disease to colchicine 0.5 mg/day vs. control.³⁸ This study showed a highly significant reduction in recurrent cardiovascular events over a 3-year follow up. A recent study used computed tomographic coronary angiography to study individuals with acute coronary syndromes who underwent treatment with colchicine 0.5 mg/day. At a one-year follow-up they found a decrease in low-attenuation plaque volume and hsCRP.³⁹ These promising preliminary studies have spawned four large-scale investigations of colchicine’s ability to reduce recurrent events in secondary prevention. Colchicine causes gastrointestinal distress sufficient to warrant discontinuation of the medication in over 10% of individuals. The precise molecular mechanism of colchicine’s anti-inflammatory effect remains incompletely understood.^{40, 41} Early work attributed colchicine’s anti-inflammatory actions to inhibition of microtubular function in inflammatory leukocytes. More recent studies have indicated that relatively high concentrations of colchicine (5 μM) can suppress activation of the NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome, by inhibiting the assembly of this macromolecular multimer.

The NLRP3 inflammasome regulates the activity of a one of its constituent proteins, the enzyme caspase-1, originally known as IL (interleukin)-1-beta converting enzyme.^42–45 The inflammasome undergoes activation by a number of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). A number of atherosclerosis-related stimuli also can co-activate the NLRP3 inflammasome. Such stimuli include cholesterol crystals, hypoxia, disturbed flow, and microbial products.⁴⁶ The augmented action of caspase-1 following NLRP3 inflammasome activation processes the inactive precursors of the pro-inflammatory cytokines IL-1β and IL-18 to the mature active forms of these pro-inflammatory mediators.

CANTOS Established Inflammation as a Therapeutic Target in Atherosclerosis

The Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) assessed the ability of an antibody that neutralizes IL-1β in reducing recurrent cardiovascular events in individuals who had sustained an acute myocardial infarction at least 30 days before enrollment.⁴⁷ This study included individuals who, despite treatment with guideline-directed medical therapy including highly effective doses of statins, had an hsCRP > 2.0 mg/L, the approximate population median. This study showed a 15% reduction in the primary endpoint of major adverse cardiovascular events.⁴⁸ Pre-specified analyses of individuals who achieved a greater than median reduction in hsCRP after administration of the antibody, canakinumab, showed a 26% reduction in events and an over 30% reduction in cardiovascular and all-cause mortality.⁴⁹ Responders to canakinumab gauged by achieving an IL-6 concentration below median showed a similar magnitude of benefit.⁵⁰ CANTOS demonstrated the effectiveness of targeting inflammation in atherosclerosis. This study also validated the inflammasome pathway as a promising avenue for further anti-inflammatory interventions.

As expected from prior experience, and in keeping with interference with a host defense mechanism, canakinumab therapy associated with a small but statistically significant increase in fatal infections in CANTOS. The knowledge gained regarding the risk factors for and the types of infectious complications in the nearly 7,000 patients exposed to canakinumab in CANTOS can inform management to reduce this risk. Yet, future studies should aim to develop anti-inflammatory or pro-resolving therapies that entail less perturbation of host defenses.^{22, 51}

Beyond CANTOS

Work published in the 1980s suggested a hierarchical scheme for a pro-inflammatory cytokine cascade whereby IL-1 would induce IL-6, the principal mediator of the acute phase response in hepatocytes (Figure).^52–54 The marker of the acute-phase response, hsCRP, thus reports on this pathway. The recognition that the inflammasome regulates caspase-1 activity suggested a mechanism for triggering the IL-1 to IL-6 to CRP pathway. Targeting different elements of this pathway will provide further opportunities to build on the success of CANTOS to develop anti-inflammatory interventions for atherosclerosis. There are several programs targeting small-molecule inhibitors of the NLRP3 inflammasome.^{45, 55}

The inflammasome–IL-1β–IL-6 pro-inflammatory pathway.

The NLRP3 inflammasome undergoes activation by a number of atherosclerosis-related stimuli including co-activation by cholesterol crystals, hypoxia, and disturbed flow. The activated inflammasome unleashes the activity of caspase-1, the converting enzyme that processes pro-IL-1β and pro-IL-18 to their mature pro-inflammatory forms. IL-1β can in turn strongly induce production of IL-6, the major mediator of the acute phase response. IL-6 signaling through the canonical receptor on hepatocytes unleashed the acute phase response. Among acute phase reactants, fibrinogen and plasminogen activator inhibitor play causal roles in atherothrombosis. Therapeutic interventions exist or are under development to inhibit this pathway at all levels as shown on the left of the figure. IL-1 alpha, the sibling cytokine to IL-1 beta shares many of its actions, but has distinct biochemical and cell biological properties, and doesn not depend on the inflammasome for activation. CRP denotes C-reactive protein.

Downstream of the inflammasome, IL-6 signaling furnished another attractive anti-inflammatory target. Strong and concordant human genetic studies support the causality of IL-6 signaling in atherothrombosis in humans.^{56, 57} Targeting the IL-6 pathway has complexities, however. IL-6 can signal through its canonical receptor, instigating inflammatory pathways. This receptor signals through the gp130/Jak/Stat pathway. Accordingly, in experimental atherosclerosis in mice, IL-6 promotes lesion formation. IL-6 can also signal through an alternative “trans” pathway, whereby it binds to a soluble form of the canonical receptor and activates gp130.^58–60 IL-6 trans signaling may retard experimental atherosclerosis. Experimental ablation of IL-6 can augment experimental atherosclerosis. The clinically used anti-IL-6 antibody, tocilizumab, consistently increases plasma triglycerides in patients.⁶¹ Hence, this therapy may produce an atherogenic dyslipidemia that could promote atherosclerosis and balance any beneficial effect of neutralization of IL-6. In sum, the success of CANTOS points strongly to the potential benefits of targeting the NLRP3 inflammasome pathway, opening a fertile field for future investigation.

In addition to IL-1β, its sibling isoform, IL-1α, exerts many of the same biological activities.⁶² Yet the cell biology and biochemistry of IL-1α differ considerably from that of IL-1β. IL-1α does not require activation by the inflammasome to exert biological activity.⁶² IL-1α signals through contact or acts at short distances, as it associates primarily with the surface of cells. Dying cells can liberate IL-1α locally. Experimental evidence supports a causal role for IL-1α in experimental atherosclerosis.^{63, 64} An antibody that neutralizes human IL-1α is available for clinical use.^{65, 66} Thus, IL-1α, in addition to IL-1β, merits consideration as an anti-inflammatory therapy in human atherosclerosis.

Vaccinations and Other Immunotherapies for Mitigation of Atherosclerosis

The recognition of the involvement of adaptive immunity in experimental atherosclerosis has prompted several efforts to harness these insights for therapy in humans. Experimental observations showed that IgM antibodies (natural antibodies) that recognize epitopes associated with oxidized LDL or certain infectious agents such as Pneumococci can limit atherosclerosis.⁶⁷ This observation has prompted the development of several strategies for stimulating endogenous antibodies that might mitigate atherogenesis in humans. Among various approaches, using immunogenic epitopes of the major apolipoprotein of LDL, apolipoprotein B, has received attention from several groups.^68–70 Promising animal experiments have laid the groundwork for potential clinical investigations. Immunization experiments in mice generally use congenic strains that have a limited haplotype of the major histocompatibility complex that governs the afferent limb of adaptive immunity. Thus, extrapolation of the experimental results to humans with a much more diverse human leukocyte antigen system than inbred mice could prove challenging. Nonetheless, the hope for developing an anti-atherosclerosis vaccine strategies merits continued evaluation. Other immune therapies under clinical consideration include the use of very low-dose IL-2 to stimulate the activity of regulatory T cells (Treg), (Low-Dose Interleukin-2 in Patients with Stable Ischaemic Heart Disease and Acute Coronary Syndromes, LILACS, NCT03113773).⁷¹ These cells secrete transforming growth factor beta (TGF-β), an anti-inflammatory and pro-fibrotic cytokine. An early phase clinical study currently underway is testing the safety of administration of low-dose IL-2 to individuals with acute coronary syndromes and seeking evidence for anti-inflammatory action. This pioneering study may be the first of several to harness protective adaptive immune responses to moderate atherosclerosis.

Clonal Hematopoiesis: A Newly Recognized but Potent Cardiovascular Risk Factor

Recent observations have identified a heretofore unsuspected cardiovascular risk factor that will likely identify yet further novel approaches for targeting anti-atherosclerotic treatments and point to new pathways for therapeutic intervention. With human aging, bone marrow stem cells can acquire somatic mutations that confer a proliferative advantage. These mutated stem cells can give rise to clones of leukocytes that circulate in peripheral blood. The acquisition of further mutations by such a clone can lead to myelodysplastic syndrome and, ultimately, acute leukemia.⁷² The prevalence of such clones exceeds 10% in septuagenarians and continues to increase with age.⁷³ As would be expected, individuals with these clones of mutant leukocytes have a higher incidence of acute leukemia and mortality due to hematologic malignancy. Yet, the increased mortality due to leukemia does not account for a striking increase in mortality in carriers of these clones. Cardiovascular disease makes up this gap in mortality.^{73, 74} CHIP associates with worsened outcomes for three major cardiovascular threats: myocardial infarction, stroke, and ischemic heart failure.^{74, 75} Individuals with CHIP do not have leukocytosis. Although this condition may be regarded as pre-malignant, most people with CHIP will not develop leukemia. This condition therefore carries the designation Clonal Hematopoiesis of Indeterminate Potential (CHIP).

Mutations in only a handful of genes cause CHIP. The most common mutations, TET2 and DNMT3A, encode enzymes involved in regulating the methylation of DNA. Thus, these variants likely lead to epigenetic changes that alter transcription.⁷⁶ Experiments in atherosclerosis-prone mice have demonstrated that introduction of a CHIP mutation, Tet2, accelerates atherosclerosis.^{74, 77} These experiments support the causality of CHIP in atherosclerotic cardiovascular disease rather than this condition merely serving as a marker of aging. The augmented cardiovascular risk conferred by CHIP mutations does not depend on classical risk factors. Moreover, CHIP mutations do not appear to associate with elevations in the traditional marker of inflammation hsCRP. The leukocytes from mice engineered to harbor Tet2 lack of function show increased expression of a number of pro-inflammatory cytokines and chemokines, including IL-1β and IL-6.

The discovery that CHIP associates with cardiovascular disease, and the absence of a tight correlation with classical risk factors or hsCRP, points to the existence of leukocyte-mediated and possibly pro-inflammatory pathways that can aggravate atherothrombosis beyond those currently recognized or tracked by CRP. For example, another CHIP mutation in a Janus kinase (JAK2) associated with myelodysplastic syndrome and polycythemia vera, particularly the JAK2^V617F mutation, appears to promote thrombosis by sensitizing mutant granulocytes to formation of neutrophil extracellular traps (NETs).⁷⁸ Atherosclerosis-prone mice bearing the Jak2^V617F mutation develop larger lipid cores within atheroma, and activate the inflammasome/IL-1β pathway. A great deal of recent work implicates NETs in venous and arterial thrombosis.^{79, 80} Inhibitors of JAK2, notably the JAK1/2 blocker ruxolitinib, have approval for clinical use. Targeting carriers of this CHIP mutation with this small molecule therapeutic provides an example of using a genetic variant to allocate a specific therapy, the ultimate goal of precision medicine.

A Bright Future for Taming Atherosclerosis

In conclusion, primordial and primary prevention provide a key to stemming the worldwide spread of the atherosclerosis epidemic. With the rise of obesity and insulin resistance and diabetes, we may be losing rather than gaining ground in prevention of atherosclerosis with lifestyle intervention. Nonetheless, we should redouble our efforts to implement lifestyle intervention before pharmacologic intervention in primary prevention. In secondary prevention, however, we stand on the threshold of an exciting era of innovation. Novel therapies enable us to plumb the depths of LDL lowering. Several interventions to target triglyceride-rich lipoproteins, now considered causal in cardiovascular disease, are under investigation. Intervening on inflammation has proven promising, particularly the components of the inflammasome pathway. The recent recognition of CHIP promises to reveal previously unsuspected pathways independent of classical risk factors that promote atherogenesis and that may provide new biomarkers for allocation of therapy and identify new targets for therapy. In the war against atherosclerosis, we perhaps find ourselves at the turning point that Sir Winston Churchill recognized during the conduct of the second World War when he stated, “Now this is not the end. It is not even the beginning of the end. But it is, perhaps, the end of the beginning.”

Highlights.

• In addition to lipids, novel therapies that target inflammatory pathways may address risk of atherosclerotic risk.

• Not all blockers of inflammation appear capable of reducing atherosclerotic events, trials targeting oxidation, phospholipases that generate pro-inflammatory lipids, p38MAP Kinase, and administration of methotrexate have not met their endpoints.

• The ability of higher doses of eicosapentaenoic acid to improve cardiovascular outcomes in individuals with hypertriglyceridemia may result in part from an anti-inflammatory action.

• Neutralization of interleukin (IL)-1 beta has succeeded in improving cardiovascular outcomes in statin-treated patients stable post myocardial infarction with residual inflammation, pointing to the inflammasome to the IL-1 beta to IL-6 pathway as presenting several attractive targets for further study.