Focus Seminar: Historical & Conceptual Changes of Coronary Artery Disease (1980–2020) From Focal Lipid Storage to Systemic Inflammation

Abstract

Concepts of atherogenesis have evolved considerably with time. Early animal experiments showed that a cholesterol-rich diet could induce fatty lesion formation in arteries. The elucidation of lipoprotein metabolism ultimately led to demonstrating the clinical benefits of lipid lowering. The view of atheromata as bland accumulations of smooth muscle cells which elaborated an extracellular matrix that could entrap lipids then expanded to embrace inflammation as providing pathways that could link risk factors to atherogenesis. The characterization of leukocyte adhesion molecules and their control by proinflammatory cytokines, the ability of chemokines to recruit leukocytes, and the identification of inflammatory cell subtypes in lesions spurred the unraveling of innate and adaptive immune pathways that contribute to atherosclerosis and its thrombotic complications. Such pathophysiologic insights have led to the identification of biomarkers that can define categories of risk and direct therapies, and to the development of new treatments.

Condensed Abstract

Initial concepts of atherogenesis focused on lipids. Many viewed atherosclerotic plaques as bland accumulations of smooth muscle cells that entrapped excess lipids in their extracellular matrix. This concept then expanded to embrace inflammation as providing pathways that could link risk factors to atherogenesis. The characterization of leukocyte classes and cytokines in plaques spurred the unraveling of innate and adaptive immune pathways that contribute to atherosclerosis and its thrombotic complications. Such pathophysiologic insights have led to the identification of biomarkers that can define categories of risk and direct therapies, and to the development of new treatments.

Our concepts of atherogenesis have evolved considerably with time. This review provides a chronological guided tour through the thicket of hypotheses regarding the pathogenesis of atherosclerosis (Central Illustration).

Central Illustration: Evolution of concepts of the pathogenesis of atherosclerosis.

The diagrams represent the dominant formulation of mechanisms of atherogenesis as they emerged over time (clockwise). In the mid-19^th-century Virchow and von Rokitansky fueled a heated controversy regarding the role of incorporated thrombus in atherosclerosis (top pair of diagrams.) The experiments of an Anichkov and many others lead to predominant view of atherosclerosis as primarily a lipid storage disease. This concept prevailed for much of the 20^th Century. The pioneering work of Ross and of Benditt in the 1970s emphasized the role of smooth muscle proliferation in lesion formation. The initial formulation of the “response to injury” hypothesis accorded initiating role to endothelial denudation, and did not invoke a role for inflammatory cells. Work in the 1980s and beyond used the evolving tools of immunology to define operation of both innate and adaptive immunity in atherogenesis, coming full circle to Virchow’s older observations implicating inflammation pathways in atherogenesis. Our current synthetic view of atherogenesis (center) encompasses elements of each of these pathogenic processes unraveled through the years.

The Feuding Founding Fathers

In the 1800s the pathologist Rudolph Virchow, armed with keen powers of observation and deductive reasoning, recognized lipid accumulation (“Cholesterin”), cellular proliferation and death, and elements of inflammation (“Entzündungsprozess”) in his study of human atherosclerotic lesions (Figure 1).(1) There followed considerable controversy with Karl von Rokitansky, who posited a primary role for incorporated mural thrombosis as a key pathogenic process in atherosclerosis.(2) A century later, Duguid rehabilitated the von Rokitansky notion.(3) Contemporary studies have reinforced the role of incorporated thrombus and healing of previously disrupted plaques as actors in atherogenesis. We now accord both Virchow and von Rokitansky due as pioneers in proposing mechanisms of atherogenesis: elements of both of their schemata have proven correct (Central Illustration, middle).

Figure 1. The atheromatous plaque according to Virchow.

This drawing by Virchow of a human atherosclerotic plaque shows atheromatous foam cells (a’), and a layer of proliferating cells (p) with morphologic evidence of cell division disclosed by mitotic nuclei. The figure depicts a layer of cells (h) beneath an intimal EC lining (i) on the lumenal surface. This depiction from mid 19^th century illustrates the lipid core (a, a’), smooth muscle involvement (p), a fibrous cap, and an intact endothelial monolayer. Adapted from (1).

Atherosclerosis as a Cholesterol Storage Disease and the Ascendancy of the Cholesterol Hypothesis

At the dawn of the 20^th century Nikolaj Anitchkov induced the formation of fatty lesions in the arteries of rabbits by feeding egg yolk.(4) The participation of cholesterol in atherogenesis gained foothold from these observations. Anichkov described lipid-laden foam cells in experimental lesions, the hallmark of atherosclerosis (Figure 2) (5). Adolf Windaus demonstrated cholesterol in atherosclerotic plaques.(6) The identification of a hereditary condition with elevated plasma cholesterol and premature myocardial infarction by Carl Müller established a link between cholesterol, atherosclerosis, and myocardial infarction (7).

Figure 2. A foam cell lesion in a cholesterol-fed rabbit.

This drawing by Anitchkov portrays a foam cell-rich arterial lesion from a cholesterol-fed rabbit. Note the intact endothelial monolayer (End.), the macrophages (Mf.), and the cholesterol laden phagocytes (Chol. Ph.), some with multiple nuclei. Bipolar spindle-shaped cells (likely smooth muscle) reside beneath the cluster of foam cells. Adapted from (5).

The introduction of the ultracentrifuge in biochemical investigation led to the characterization of blood lipoprotein particles. These particles consist of a core of hydrophobic lipid coated by an amphipathic cloak of phospholipids and cholesterol. These packages ferry water-insoluble lipids through the aqueous medium of the blood and extracellular fluid. The attached apoproteins direct the traffic of these particles by engaging receptors on the cell surfaces of various cells. Low-density lipoprotein (LDL) emerged as a key cholesterol carrier in human blood.

As biochemical characterization of lipoproteins and their apoproteins advanced, epidemiologic observations (e.g. the Framingham Study) established a consistent relationship between cholesterol and later LDL and the risk of atherosclerotic events.(8) Michael Brown and Joseph Goldstein elegantly identified the LDL receptor and the mechanisms that strictly regulate cellular cholesterol accumulation.(9) These findings reinforced the prevailing biochemical and epidemiologic view of atherosclerosis as cholesterol storage disease.

Atheromata as Lumps of Proliferated Smooth Muscle Cells (SMC)

SMCs accumulate in atherosclerotic lesions. Virchow deduced cellular proliferation in his observations of human atheromata. The ability to culture SMCs enabled mechanistic studies of the regulation of smooth muscle cell proliferation. The influential studies of Russell Ross pointed to the participation of platelet products, eventually identified as platelet-derived growth factor (PDGF), as a major mediator of SMC proliferation.(10) This recognition spawned a flurry of interest in the regulation of SMC division. The discovery that the simian sarcoma virus oncogene v-sis encodes the B chain of PDGF buttressed the view of atherosclerosis as a proliferative disease of SMC, akin to a leiomyoma (11).

Earl Benditt first called attention to monotypia of cell populations in human atherosclerotic lesions, reinforcing the concept of clonal expansion of SMC as a key contributor to atheroma formation.(12) Ross’ initial formulations of response to injury hypothesis, derived from Virchow’s observations, posited a denuding endothelial injury as the stimulus that provoked platelet activation and release of preformed PDGF from platelet granules (Figure 3) (13,14). Ross and Harker initially implicated hyperhomocysteinemia in this denuding endothelial injury that triggered platelet release of PDGF and unleashed SMC replication.(13) More recent data have called into question the causal role of homocysteine in atheroma complication.(15) The response-to-injury hypothesis as initially predicated by Ross postulated that the elaborate extracellular matrix produced by SMCs provided a rich filigree of fibrous proteins which could entangle lipoproteins, retarding their efflux from the arterial intima and fostering lipid accumulation.

Figure 3. The ascendancy of smooth muscle proliferation.

This drawing presents the view of Ross and Glomset in the 1970s depicting a desquamative endothelial injury with SMC migration from the media into a growing intimal lesion with mitotic figures indicating division of the SMCs. Ross and Glomset hypothesized a major role for platelet-derived growth factor in stimulating the migration and proliferation of SMCs. This formulation viewed atherogenesis as a bland phenomenon lacking inflammation or leukocytes. Adapted from (14).

Endothelial Dysfunction Supplants Denudation as an Early Event in Atherogenesis: A Revisionist View

Rudolph Altschul presciently posited a key role for the endothelium as supremely important in arterial function. Sir William Osler stated “A man is as old as his arteries.” (Did women not have arteries in Osler’s day?) Altschul reformulated this dictum by saying that one is as “old as one’s endothelium.”(16). In a slim volume entitled “Endothelium,” Altschul in 1954 discussed the difficulty of culturing endothelial cells (EC) in vitro and the conjecture that ECs could become fibroblasts in culture. This apparent metaplasia of ECs likely resulted from an overgrowth of hardier mesenchymal cells (e.g., fibroblasts, SMCs, and pericytes) that crowded out the more fastidious ECs.

Ultimate success in culturing vascular ECs gave birth to contemporary EC biology. (17) The limited proliferation of ECs in cultures frustrated the ability to do large-scale experiments. The identification of members of the fibroblast growth-factor family, and eventually of vascular endothelial growth factors, provided tools that enabled the serial propagation and passage of ECs.(18,19) These enabling innovations gave birth to a burst of interest in endothelial functions and their regulation in vitro.

Such studies unraveled tightly regulated homeostatic properties of the normal, resting endothelium that maintain vascular homeostasis.(20) These attributes include thromboresistance, and the ability to resist prolonged contact with blood leukocytes. Other studies of arachidonic acid metabolites unraveled a delicate balance between an antithrombotic prostaglandin termed prostacyclin elaborated from ECs, and a prothrombotic prostaglandin, thromboxane A2, derived from platelets. (21) The use of inflammatory substances such as bacterial lipopolysaccharide, and the advent of purified preparations of pro-inflammatory cytokines, catapulted inflammatory activation of ECs to the fore (22).

The observations of Robert Furchgott added vasodilation to the list of homeostatic endothelial function (23). The discovery of endothelial-derived relaxing factor, later recognized as nitric oxide (•NO), opened new avenues of physiologic and pharmacologic investigation. Translation to humans established that atherosclerotic arteries of humans exhibit impaired endothelial-dependent relaxation. (24) These findings led to a plethora of investigations, experimental and human, focused on the regulation of endothelial vasodilator functions. The notion of abnormal function of a morphologically intact endothelium supplanted the earlier concept of denuding injury to the endothelium as a key component of atherosclerotic lesion formation and evolution.

A Perplexing Paradox: Why do Atheromata Form Focally in Face of Uniformly Distributed Systemic Risk Factors?

Subsequent studies inverted the concept that disturbed flow elicits atherogenic endothelial functions by positing that laminar shear stress favors the expression of the palette of “atheroprotective” functions of ECs.(25) Laminar shear stress restricts pro-inflammatory, prothrombotic, and pro-oxidant properties of the endothelium. Activation of transcription factors such as Krüppel-like Factor 2 (KLF2) appear to orchestrate this suite of salubrious functions of ECs experiencing laminar shear stress (20,26).

None of the above pathophysiologic insights account for the propensity of atherosclerotic lesions to form preferentially in certain locales when exposure to traditional risk factors such as hypertension, dyslipidemia, hyperglycemia, and the products of cigarette smoking all should affect the vasculature uniformly as the luminal lining of ECs encounter the same flowing fluid phase of blood.

The union of fluid dynamic insights provided by bioengineers and endothelial biologists helped to resolve this apparent paradox. Regions of atheroma formation, for example at flow dividers or branch points within the circulation, typically encounter disturbed blood flow. Laminar shear stress (a force parallel to the endothelial monolayer) generally associates with low propensity to form atheroma in humans or in hyperlipidemic animals. Areas of flow disturbance or low-shear stress colocalize with inflammatory activation, recruitment of leukocytes, and lesion formation and complication. The molecular mechanisms of flow sensing, and the myriad properties of ECs subject to regulation by hydrodynamic conditions, as ably documented elsewhere, exceed the scope of this review (27). Thus, the merging of fluid dynamics and the study of endothelial biology helped to understand how systemic risk factors can lead to a disease with focal manifestations.

Inflammation Links Traditional Risk Factors and Altered Behavior of Cells of the Artery Wall

Ross’ initial formulation of the response to injury hypothesis depicted bland proliferation of SMCs in response to denuding endothelial injury leading to platelet activation and PDGF release as summarized above (Figure 3). Yet, morphological studies of experimental hypercholesterolemia in the 1950s had identified monocyte adhesion and macrophage accumulation in forming lesions as key features of early atherosclerosis (28). These early findings gained support from ultrastructural investigations in the 1980s, and led to a renewed interest in leukocytes as mediators of altered arterial biology (29,30).

The advent of monoclonal antibody technology improved the precision of cell-type identification. Probing of human atherosclerotic plaques with such reagents revealed a picture of immune inflammation. (31) These studies localized many monocyte/macrophages in human lesions. In addition, cell-type specific antibodies detected specialized cells of the immune system, particularly CD4⁺ T cells, in the lesions. Some of these immune cells displayed signs of activation and of communication between immune and vascular cells through cytokines and cell-surface receptors (Figure 4)(32,33).

Figure 4. A simplified view of the operation of innate and adaptive immunity as thought to operate in atherogenesis.

Innate immunity initiates when macrophages (MΦ) recognize PAMPs and DAMPs binding to their pattern recognition receptors. This interaction leads to production of a host of proinflammatory molecules including cytokines (e.g. IL-1-α and -β and TNF) and small molecules such as eicosanoids. Adaptive immune reponses follow the processing of antigens (foreign or autologous) by dendritic cells (DC.) Proteolytic cleavage of protein antigens into peptides within the DC prepares the antigen for presentation on the DC surface bound to major histocompatibility complex (MHC) molecules (human leukocyte antigens, HLA in humans.) Antigen-specific immune cells recognize the nominal nominal antigen in the context of self MHC. T cells can recognize peptide-HLA complexes via specific antigen receptors. Antigen recognition in combination with signals produced by the DC prompts the T cell to activate and differentiate. Activated CD4⁺ T cells may differentiate into several cell types with different functions. Among them, Th1 cells produce interferon-γ and TNF, are highly proinflammatory, and strongly stimulate macrophage activation and vascular inflammation. Treg, on the other hand, make two anti-inflammatory and immunoregulatory cytokines, namely transforming growth factor-β and IL-10. IL-17 produced by Th17 cells activate granulocytes and stimulates collagen production, as does TGF-β. B cells recognize antigens that ligate their surface-bound immunoglobulins. The most prevalent type of B cell, the B2 cell, receives help from T follicular helper (Tfh) cells and can develop into plasma cells specialized in production of IgG antibodies. In addition, B2 cells produce cytokines that can modulate inflammation. Another B cell subset, the B1 cell, produces IgM antibodies and does not require Tfh cell help. B1 cells largely produce “natural” antibodies encoded by the germline, while the genes that encode IgG antibodies generally undergo somatic mutations to achieve greater affinity for the particular antigen with time. Single-line arrows indicate signaling molecules whereas thick arrows show cell development.

The elucidation of endothelial-leukocyte adhesion molecules, pioneered by Gimbrone and colleagues, and their regulation by inflammatory mediators, provided molecular mechanisms by which the activated endothelium could recruit blood leukocytes (17). The identification of chemoattractant cytokines and their receptors explained the entry of bound cells into the intima.(34) The expression of retention factors such as netrin and semaphorins could retard the egress of leukocytes from the artery wall, favoring their accumulation (35). Mononuclear phagocytes proliferate within the intimal lesions and can thus accentuate leukocyte accumulation within evolving lesions.(36) The recognition that intrinsic arterial cells can express chemokines and cytokines as well as respond to these mediators implicated inflammatory activation of intrinsic vascular wall cells in lesion initiation as well as in ongoing recruitment and activation of blood leukocytes.(37) (38) (39) Clinical observations reinforced the possibility that inflammation participates pivotally in precipitating the complications of atherosclerosis. The inflammatory cytokine interleukin (IL)-6 and C-reactive protein (CRP) induced by IL-6 provide sensitive biomarkers of risk for atherosclerotic events.(40,41) Studies that tracked the ability of CRP as a harbinger of future cardiovascular events, even when adjusted for traditional risk factors, supported the large body of in vitro and in vivo experimentation implicating inflammation in atherogenesis (42).

The inflammation in atherosclerosis that drives the IL-1–Il-6–CRP axis can derive from multiple sources (37). Two principally different, although interconnected, immune responses contribute to the inflammatory response in atherosclerosis (Figure 4). The primitive and swift innate-immune response recognizes only hundreds of structures through binding to pattern-recognition receptors (e.g. the Toll-like receptors) by damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs). The phylogenetically more recent adaptive immune response, in contrast to innate immunity, recognizes many millions of structures by unique receptors on each T or B cell clone. Among the triggers to inflammatory and immune activation in atherosclerosis, oxidatively modified lipoprotein constituents have received much attention (43). Yet, no anti-oxidant intervention has improved outcomes in human atherosclerotic patients.

If monocytes and macrophages constitute the foot soldiers of the innate-immune response, T lymphocytes comprise the commander that issues orders to the more multitudinous mononuclear phagocytes. Early observations supported elaboration of the mediator interferon gamma by plaque TH1 cells, yielding activation of the plentiful mononuclear phagocytes within the atheroma, as gauged by expression of class II histocompatibility molecules.(33) This pathway presented a prototype of a command issued by the adaptive officers to the innate immune troops of our host defenses. T cells may also dampen immune responses. Regulatory T cells (Treg) can mute inflammation by elaborating the immunomodulatory cytokine transforming growth-factor beta (TGFB) (44) as well as the anti-inflammatory IL-10. Several other cell types can also produce these two mediators, including certain B cells and macrophages, and in the case of IL-10, also Th2 cells.

This ensemble of observations begged the question of what instigates inflammation and the immune response in atherosclerosis. Indeed, inflammatory signaling could provide a series of mechanisms by which traditional risk factors alter the functions of arterial wall cells. For example, atherogenic lipoproteins, particular remnant particles or triglyceride-rich lipoproteins, correlate with concentrations of the inflammatory biomarker CRP (45). Thrombin and other activation-coagulation factors, and mediators elaborated by activated platelets, can elicit inflammatory functions from leukocytes and intrinsic vascular wall cells alike (Figure 5) (46).

Figure 5. Thrombosis begets inflammation.

Platelets not only contribute to thrombus formation through well understood pathways but when activated also release pre-formed proinflammatory mediators from their granules. Thrombin activates platelets to produce the proinflammatory mediators such as RANTES, IL-6, and to exteriorize CD40 Ligand, and also stimulates SMCs to proliferate and activates ECs. Adapted from (47).

Thrombosis Begets Inflammation

Platelets not only contribute to thrombus formation through well-understood pathways but when activated also release pre-formed pro-inflammatory mediators from their granules. Thrombin stimulates platelets to elaborate pro-inflammatory mediators such as RANTES and interleukin-6 (IL-6) and to exteriorize CD40 ligand, and also stimulates SMCs to proliferate and to activate ECs (Figure 5).(47) Mediators of hypertension, including angiotensin II, may also elicit immune and inflammatory responses and help to mobilize leukocytes from hematopoietic organs (48).

LDL particles can activate both innate and adaptive immunity. Modifications of LDL can confer the capacity to activate pattern-recognition receptors in macrophages and other cells. Furthermore, cholesterol released from endocytosed LDL can crystallize and co-activate the NLRP3 inflammasome, a process that generates the mature proinflammatory cytokines IL-1β and IL-18. Ubiquitous preformed “natural” antibodies recognize phospholipids derived from LDL. Finally, oligopeptide fragments of the LDL protein, apoB100, can act as autoantigens to initiate T cell activation and adaptive immune responses.(33) (49) Depending on the local milieu in the artery and its draining lymph nodes, such responses can act either to promote macrophage activation and inflammation, or to dampen it (regulatory immunity). These findings raise the exciting possibility that activating adaptive immunity to LDL under conditions that favor regulatory immunity by vaccination could tame atherogenesis (33).

Sites of inflammation remote from the arterial wall may provide systemic stimuli that can produce “echoes” within the prepared “soil” of the abnormal arterial intima (Figure 6).(38) Cytokines elaborated by visceral adipose tissue or other ectopic fat deposits (e.g. the periarterial collection of fat that often enrobes human arteries) can augment local cytokine production within arterial wall cells and experimental lesions. Niduses of infection such as bronchitis, periodontitis, chronic urinary tract infections, or cutaneous lesions can elaborate PAMPs including endotoxins that can impinge upon leukocytes lying await within evolving atheromata to elicit a round of enhanced local production of inflammatory mediators (Figures 4 & 6) (50). Smoking cigarettes associates with increased chronic bronchitis and other pulmonary infections that may augment thrombosis.

Figure 6. The expanded cardiovascular continuum.

Risk factors beget atherosclerosis that can cause thrombosis leading to tissue injury such as acute myocardial infarction. The sympathetic stimulation that myocardial infarction engenders can activate the bone marrow to release leukocyte progenitor cells that can reside in the spleen and migrate to the atherosclerotic plaque. The infarcted myocardium also leads release of such pro-inflammatory cytokines as IL-1β, able to propagate inflammation to remote sites, including the atheroma itself. Hence, both systemic and local inflammation can impinge on the prepared soil of the plaque leading to local inflammatory activation exacerbating the inflammatory state. Thus, the cycle of inflammation can perpetuate leading to recurrent events and aggravated atherothrombosis. Adapted from (38).

This concept that systemic or remote inflammation can elicit “echoes” locally within the artery wall, originally posited in the early 1990s, has gained renewed attention from two sources of experimental evidence (38). First, signaling incited by acute myocardial injury can aggravate inflammation and leukocyte recruitment and activation within experimental atheromata in mice (Figure 6) (51). Second, the concept “trained immunity” has heightened recognition that a second encounter with a pro-inflammatory stimulus can yield an accentuated response (52,53). While long accepted in the concept of adaptive immunity, the anamnestic response intrinsic in the concept “trained immunity” may apply particularly to atherosclerosis. These various considerations provide additional links between systemic inflammation (the fluid phase of blood) or localized inflammation in extra-arterial sites and accentuated atherogenesis, or in eliciting properties of preformed plaques (the solid state of the lesion itself) that can trigger their thrombotic complication (Figure 7).

Figure 7. Thrombosis: The ultimate complication of atherosclerosis.

Arterial thrombi cause acute coronary syndromes and many ischemic strokes. The thrombotic potential of a plaque depends on the production of tissue factor by macrophages or SMCs within the intima, the “solid state” of the plaque. Plasminogen activator inhibitor (PAI-1) production by lesional cells in response to inflammatory stimuli inhibits these promoters of fibrinolysis. ECs can express thrombomodulin which can limit thrombosis, but inflammatory stimuli and regulation by KLF-factor 2 can limit thrombomodulin expression by ECs reducing their baseline anti-thrombotic effects. ECs can also produce tissue type and urokinase-type plasminogen activator (tPA and uPA) that can activate the fibrinolytic system. When inflammatory stimuli augment its production, PAI-1 inhibits these promoters of fibrinolysis. Thus an inflammatory state augments the thrombogenicity of the plaque by boosting tissue-factor expression, mutes the intrinsic anticoagulant effect of the normal endothelial monolayer, and combats fibrinolysis (an effect that stabilizes clots). The consequencing of a given plaque disruption, be it fibrous cap rupture as depicted here, or superficial erosion (not shown), depends not only on the solid state of the plaque but also on the fluid phase of blood. Microparticles that bear tissue factor may also contribute to the extension of thrombosis due to plaque disruption. Adapted from (79).

The recent recognition of the link between clonal hematopoiesis and increased cardiovascular risk provides a new and previously unsuspected link between systemic inflammation and aggravation of atherosclerosis. With age, somatic and acquired mutations in bone marrow stem cells can yield clones of leukocytes in peripheral blood that bear mutations in the genes that drive acute leukemia. By the eighth decade of life, over one in ten individuals will harbor such clones of mutant leukocytes in peripheral blood (54) The presence of these clones augments the risk of developing acute leukemia, a condition that generally requires the accumulation of several mutations within the same clone. Yet, the toll of increased mortality associated with clonal hematopoiesis far outstrips deaths attributable to acute leukemia. Cardiovascular events account for much of this gap in mortality.(55) Most individuals who bear these mutations will never develop acute leukemia, hence the designation clonal hematopoiesis of indeterminate potential (or CHIP). Several experimental observations provide evidence that increased operation of the inflammatory pathway that involves the production of active IL-1β via the inflammasome participates causally in the aggravation of atherosclerosis associated with certain mutations that cause CHIP (TET2 and DNMT3A) (55,56). Another mutation that causes CHIP, JAK2, augments lipid core accumulation in experimental atheroma, and actives integrins involved in leukocyte recruitment. (57–59) The risk for atherosclerotic events conferred by CHIP appear independent of traditional risk factors for atherosclerosis (60).

Immune activity also links vascular inflammation to lipid metabolism. Immune cells in the liver and adipose tissue modulate the synthesis and catabolism of lipoproteins. Cytokines, antibodies, and cell-cell interactions can mediate these processes. Whereas inflammatory activation tends to inhibit clearance of lipoproteins from the circulation, regulatory immunity favors this process (33). Therefore, inflammation synergizes with and amplifies metabolic risk factors during atherogenesis.

A Synoptic Synthesis of Atherogenesis

Current evidence supports the validity of key elements of most or all of the concepts of atherosclerosis elaborated by investigators through the decades discussed above (Central Illustration). A synoptic view of atherosclerosis incorporates elements from each of these pathogenic schemata, often construed as competing, but in our view complementary.

Few dispute today the causal role of LDL in human atherogenesis. Levels of this lipoprotein that exceed those found in newborn humans and in many animal species likely prove permissive for atherosclerosis. A lifetime exposure to higher concentrations of LDL furnishes a foundation for this disease. The concept that incorporation of mural thrombi explains atherogenesis has regained legitimacy: the healing of disrupted atheromata after plaque disruption probably provokes a round of smooth muscle migration and proliferation. Indeed, thrombosis can result from inflammation (Figure 5) but can also promote inflammation, as platelets release preformed inflammatory mediators upon activation, and thrombin can directly and indirectly promote this process (Figure 6). The augmented production of extracellular matrix by stimulated SMC can expand intimal lesions and promote their growth. Thus, elements of the concepts of Virchow, von Rokitansky, Benditt, and Ross all have achieved increased support as our understanding of the complex pathogenesis of this disease increases. Recent experiments have renewed interest in SMC proliferation and in metaplasia of SMC to macrophage-like cells, and have expanded the appreciation of heterogeneity of the cells involved in atherosclerosis (61).

The advent of single-cell RNA sequencing and mass-spectrometric analysis of proteins in plaque cells has markedly expanded appreciation of the heterogeneity of mesenchymal cells and leukocytes within the atheroma, as well as ECs themselves.(62,63) (64) The recent publication of “atlases” of these various cell types into increasingly more restricted populations highlights the complexity of the pathogenesis of atherosclerosis. In some ways, however, the sorting of cells into ever more categories may provide more confusion than illumination. The ever finer subtyping of cell populations in experimental atheromata recalls the scholasticism of medieval academics who hotly debated “How many angels can dance on the head of a pin?” Recognition of cellular subtypes and rigor in their definition is highly useful, but perhaps only up to a point. We could thus conclude, in the extreme, that there are as many cell types as there are cells, a reductio ad absurdum. This view, albeit possibly or even likely true, may have limited utility.

Of importance to clinicians and patients is not an infinitesimal calculus of cell types and a myriad of mediators, but rather how to modulate the disease and its consequences. We should resist seduction by technological advances to subscribe to an increasingly reductionist view of immune and inflammatory pathways. The experimental conditions of many contemporary studies of experimental atherosclerosis use conditions of exaggerated hypercholesterolemia that limits their translation to human disease, particularly in an era of increasingly effective LDL lowering therapies (65,66).

Frontiers for Future Advances

How can the field move forward most productively and harness the technical and experimental advances to provide new inroads to combat atherosclerotic disease? The Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) established in humans the relevance of inflammation biology to atherosclerotic events (67). This study pointed to one pathway susceptible to manipulation by therapeutics existing and in development that can target inflammation. Low-dose methotrexate studied in the Cardiovascular Inflammation Reduction Trial (CIRT) neither improved the cardiovascular outcomes assessed nor reduced biomarkers of inflammation linked to the IL-1β pathway.(68) The null results of this trial underscores the notion that the modus of manipulation of inflammation matters, as does the patient population targeted (73). The success of CANTOS, while CIRT was negative, heightened interest in the NLRP3 inflammasome–IL1β–IL6 pathway as a potential drug target.(37) Multiple possibilities exist for targeting this pathway and others that contribute to inflammation and atherosclerosis (69,70).

CIRT enrolled a population with lower inflammation than CANTOS, as gauged by hsCRP concentration (~1.5 vs ~ 4.1 mg/L). This situation underscores the notion that employing biomarkers that reflect the presumed mechanism of action of the intervention can serve both to guide the design of clinical trials and the deployment of therapies in the clinic in an informed and rational manner. Several ongoing trials are evaluating colchicine as a therapy for reducing recurrent atherosclerotic events as suggested by the LoDoCo study (71,72). Another trial underway will test the hypothesis that low-dose IL-2 will skew the adaptive immune response toward Treg and thus limit inflammation in individuals with established atherosclerosis (73). Very recent authoritative reviews also discuss these therapeutic aspects (69,70,74).

Beyond LDL, triglyceride-rich lipoproteins comprise a continued contributor to residual cardiovascular risk. Lowering of this class of lipoproteins may have contributed to the striking reduction in cardiovascular events in the REDUCE-IT trial that employed as an intervention pharmaceutical grade eicosapentaenoic acid ethyl esters.(75) Agents in clinical evaluation currently target several other factors associated with hypertriglyceridemia as additional inroads against this atherogenic lipoprotein class.

Observational epidemiologic and contemporary human genetic studies underscore the causal role of lipoprotein(a) [Lp(a)] in human atherosclerotic risk. Novel therapies that target apolipoprotein (a) production have also entered clinical evaluation and provide promise for those in whom elevated concentrations of this highly atherogenic lipoprotein can contribute to risk (76).

The recognition of CHIP as a contributor to atherosclerotic risk raises several interesting therapeutic possibilities. First, CHIP individuals might merit intensified lifestyle or pharmacologic intervention not predicated on traditional risk factors. Thus, the presence of CHIP might serve to target therapy, much as in the JUPITER trial hsCRP identified a group of individuals without substantial elevation of LDL who nonetheless benefitted from statin therapy (77).

In addition, individuals bearing particular CHIP mutations may show susceptibility to specific interventions, following the model of targeted therapies in current oncologic practice. Thus, individuals who bear JAK2, TET2 or DMT3A mutations might benefit from therapies directed towards components of this pathway, e.g. approved the IL-1β or JAK2 inhibitors (78).

Thus, we possess a number of tools that can enable the goal of translating advances in the fundamental mechanisms of atherosclerosis derived from laboratory studies to our patients. Such progress promises to enable further advances in the management of risks for atherosclerotic events, which remain a leading cause of morbidity and mortality worldwide.

Bullet Points.

• Concepts of the pathophysiology of atherosclerosis have changed considerably over the years.

• Many traditionally regarded atherosclerosis as mere lipid buildup on the arterial wall.

• Advances in vascular biology permitted experimental study of endothelial and smooth muscle cells.

• The concept then prevailed that atheroma arise from bland excessive proliferation of smooth muscle cells.

• Dysfunction of an intact endothelium replaced denuding injury as key in atherogenesis.

• Recent concepts invoke inflammation as a key mediator between risk factors and artery wall cells.

• Advances in understanding inflammation in atherosclerosis has led us to novel therapeutic strategies.

Abbreviations

CHIP

clonal hematopoiesis of indeterminate potential

LDL

low-density lipoprotein

PDGF

platelet-derived growth factor

•NO

nitric oxide

KLF2

Krüppel-like Factor 2

(IL)-6

Interleukin-6

CRP

C-reactive protein

DAMPs

damage-associated molecular patterns

PAMPs

pathogen-associated molecular patterns

CIRT

Cardiovascular Inflammation Reduction Trial

Treg

regulatory T cells

CANTOS

Canakinumab Anti-inflammatory Thrombosis Outcomes Study

SMC

smooth muscle cells

TGFB

transforming growth factor beta

EC

endothelial cell