The interplay of collagen, macrophages, and microcalcification in atherosclerotic plaque cap rupture mechanics

Failure of an atherosclerotic plaque is the culprit of many, often disabling or lethal, cardiovascular events, including myocardial infarction and stroke [15, 99, 144]. These acute events are associated with vascular biomechanics, and the cellular composition and matrix architecture of a plaque. The most common cause of failure of the atherosclerotic plaque is rupture, accounting for approximately 60% of events. Plaque erosion (35% of events) and calcified nodules (up to 5% of events) can also cause thrombi, however less frequently [249]. The probability of rupture is dependent on the stage of the atherosclerotic lesion. Virmani and colleagues proposed a lesion classification scheme, based on histopathological evaluation, starting with intimal thickening and progressing to intimal xanthoma, pathological intimal thickening, fibroatheroma and finally the vulnerable thin-cap fibroatheroma [268]. This last phenotype is considered as the main lesion with potential for rupture and is, therefore, named vulnerable plaque. These vulnerable plaques are characterized by a thin collagenous cap (i.e., the fibrous cap), which is often inflamed, calcified, and depleted from smooth muscle cells (SMCs) [17, 18, 81, 119, 252]. This cap is overlying a large lipid and/or necrotic core which is accompanied by inflammation and intraplaque hemorrhage [72]. Although great advancements in clinical imaging have been made to accurately image those features associated with vulnerability [58, 66], like cap thickness [108], the size of the lipid and/or necrotic core [181], inflammatory cells [85], calcifications [254], and intraplaque hemorrhages [51], phenotypic identification of the vulnerable phenotype is still found to have suboptimal predictive power for future clinical events [68, 176, 259]. Traditionally, a cap thickness of less than 65 μm is accepted to define a rupture-prone coronary plaque, while for carotid plaques, this threshold is set to 200 μm [36, 201]. However, clinical studies have shown that physical activity can trigger atherosclerotic plaque rupture, even in coronary plaques with a thickness of 70–140 μm [127]. Furthermore, carotid plaques have also been show to rupture at thicknesses up to 500 μm [201]. As such, guidelines to identify vulnerable plaques continue to be revised, with various phenotypic features of atherosclerotic plaques playing a role in creating a rupture-prone phenotype.

Years of extensive research have exposed various biochemical processes that precede and initiate plaque failure, as excellently reviewed by others [17, 20, 89, 152]. Rupture itself is a mechanical event that generally occurs at the cap when local stress levels exceed local tissue strength, which underlines the importance of understanding cap mechanics to prevent and foresee plaque failure [9, 204]. The stress distribution within the cap depends on the cap geometry, the mechanical properties of the cap constituents, and the external loading conditions (e.g., blood pressure). Nevertheless, due to the inter- and intra-cap heterogeneity, the stress distributions, as well as ultimate strength values, and failure mechanisms are substantially different. A wide range in ultimate tensile cap strength values have been reported (158 kPa [234] to 870 kPa [111]). Importantly, geometry, composition, and resulting cap stress and strength will change in a spatiotemporal manner due to the iterative interplay between the cellular populations and environmental cues, including matrix properties and mechanical loading [96, 195, 207, 260].

To improve risk assessment, it is a requisite to highlight the importance of cap mechanics in plaque biology and cap failure. This review would like to create awareness for this topic by evaluating current knowledge on the role of cap constituents in plaque cap mechanical (de)stabilization. We hereby focus on the end-stage, vulnerable thin cap, and assess the role of three components present in this phase. The first constituent included is collagen, which is the main load-bearing component of the cap [101] and often scarcely renewed in vulnerable caps due to SMC depletion and potential senescence [17, 18, 81, 252]. Second, macrophages are discussed as major cell population correlated with an enhanced rupture risk [225] by directly initiating extracellular matrix (ECM) degradation via the secretion of degrading compounds like MMPs [175]. Third, the role of microcalcifications is emphasized, as being the most abundant type of calcification present in the vulnerable plaque cap [116]. Microcalcifications are included in this review due to their increasingly named role as local stress concentrators, possibly being a cause of cap rupture [42, 52, 116, 257]. We will elaborate on the contribution of these three constituents to cap mechanics directly, their biological interplay and discuss future work that could enhance our biomechanical understanding to better identify the cap at risk of rupture.

Where the impact of mechanics is extensively studied in atherogenesis [95], its role in cap rupture is evident but less well understood. Plaques are exposed to various mechanical forces (Fig. 1A) that can induce peculiar mechanical behaviors that vary with plaque geometry, location, and composition [9, 101]. Thin caps of vulnerable plaques must possess sufficient strength to endure the force-driven stresses to which they are exposed (Fig. 1B). The most influential stress experienced by the cap is the circumferential stress induced by the blood pressure, acting perpendicular to the arterial wall, leading to deformations within the cap. The effect of blood-flow-induced shear stress on cap deformation is believed to not be substantial as circumferential stresses are orders of magnitude higher. Their influence on cap rupture can, however, not be neglected as shear stress is believed to affect cap erosion as well as endothelial function and thereby cap biological composition [84, 160]. Axial tensile stress, as a consequence of among others hemodynamic loading, differences in vessel geometry and plaque morphology, is believed to predominantly affect the rupture events upstream of the plaque where these stresses are highest [223].

As cap constituent material properties affect the stress distribution throughout the cap and determine the strength of the cap, this section specifically elaborates on the variations in collagenous matrix properties, macrophages, and microcalcification seen within human atherosclerotic plaque caps (Fig. 1C) and the impact thereof on cap mechanics and stability (Fig. 1D). Cap mechanics and stability are hereby described on the mesoscale by terms such as tissue stiffness and strength as defined in Table 1. For an in-depth description of the complexity of plaque biomechanics, nano- and micro-scale mechanics of the collagenous matrix, as well as the effect of collagenous matrix properties on SMC-driven matrix formation and degradation, we refer the reader to the following literature [9, 39, 80, 84, 193, 203, 223, 255, 265].

While collagen, macrophages, and microcalcifications each distinctively affect cap mechanics, as discussed in “Cap components: role in cap mechanics”, they cannot be considered as independent contributors. Their continuous interplay affects plaque cap development and ultimate rupture risk (Fig. 2). This section sets forth on the current knowledge regarding their interplay. Other cellular players that are generally accepted to be involved in fibrous cap destabilization, like T cells and SMCs, are occasionally mentioned. Figure 2 provides an overview of the most important reviews describing their interplay with the three compositional parameters elaborated on in this review.

Throughout this review, we emphasized that the presence, characteristics, and iterative interplay of collagen, microcalcification, and macrophages have clear implications for tissue mechanics and consequently the risk of rupture. We believe that enhanced mechanistic insight into how biological processes change cap mechanical properties is a prerequisite for identifying the cap at risk of rupture.

In view of cap mechanics and the interplay between collagen, macrophages, and microcalcification, it can be concluded that much is still largely unknown (Fig. 3). Regarding collagen, emerging studies focus on the mechanical properties of collagen using a bottom-up approach studying nanoscale collagen mechanics [80], to be able to link this to mesoscale. In addition, increasing attention is being paid to the differences in global and local mechanical behavior [57, 241]. With respect to microcalcification, in-depth histological or micro-CT analyses of patient material, assessing microcalcification clustering, particle shape and density, are lacking. In addition, there is limited knowledge about microcalcification surface topography, which might greatly influence macrophage functionality [132] and consequently the collagenous matrix. Concerning macrophage heterogeneity, more athero-specific subsets are found to exist, mainly due to advances in single cell analysis [60], that provides a plethora of compounds affecting plaque properties and preventing rupture risk. The generally accepted statement that primarily pro-inflammatory macrophages are involved in rupture is obsolete as more atheroprotective subsets have been correlated with plaque vulnerability [19, 211]. A better distinction regarding macrophage phenotype and function should be made in relation to the environment to better understand macrophage-driven cap weakening. Furthermore, the impact of other ECM components (e.g., elastin, GAGs) [221, 230], or immune cells (non-macrophages) [61] needs to be assessed.

To fill the knowledge gaps, a holistic multidisciplinary approach is needed where the fields of immunopathology, vascular biology, biomechanics, tissue engineering, and biomedical engineering are combined. Many of the experimental studies discussed utilize animal, 2D in vitro or numerical models. Although informative and required, tissue content and mechanics are known to substantially diverge between animals and humans [97] and direct translation of 2D in vitro or numerical work to human cap mechanics is complex. To improve translatability and to validate computational approaches, more sophisticated 3D in vitro platforms are required that increasingly resemble pathophysiology, wherein human cells are exposed to various environmental factors simultaneously in a controlled manner (i.e., a multifactorial 3D in vitro model) [128]. Examples of such models include the collagen hydrogel that was utilized by Hutcheson et al. to mimic structural features of the atherosclerotic plaque to study microcalcification formation [106]. In addition, Mallone et al. bioengineered atherosclerotic plaques to investigate etiopathogenesis [158]. Our group recently made use of tissue engineering concepts to scrutinize relationships between tissue composition, the presence of microcalcifications, and mesoscale mechanical properties [109, 261]. Similar model systems can also be used to study patient variation [45, 121], as differences in age, sex, and co-morbidity are likely to affect tissue mechanics [91, 121]. Moreover, computational approaches combining models that describe blood flow and plaque deformation simultaneously could potentially provide new insights in the role of biomechanical factors in atherosclerosis [216].

To ultimately confirm findings and stratify them to patients, advanced imaging techniques are needed. Non-invasive imaging modalities including CT and MRI are currently the most widely used diagnostic tools in the clinic. However, invasive imaging techniques such as optical coherence tomography (OCT) and intravascular ultrasound (IVUS) can achieve higher resolution, and fusing these invasive techniques provides even further advancements for biomechanical modeling [90]. Recent hybrid imaging systems, such as PET–CT or PET–MRI can facilitate the detection of microcalcification and inflammation, to be able to create patient-specific modeling approaches and evaluate local mechanical properties [233]. Furthermore, specific tracers or diffusion tensor imaging can be used to visualize cap components, such as collagen type, maturity [192], and alignment [8, 239, 240].

Where this review elaborates on how cap compositional parameters affect mechanics, it is important to also obtain better mechanistic insight in how cell functionality and matrix properties are determined by the physical cues in their environment (i.e., the mechanobiology). It was shown that hemodynamic loading can determine macrophage phenotype [94] (add ref) and matrix content [27], among others by changing collagen’s susceptibility to degradation [77, 78]. It is, thus, likely that hemodynamic loading also influences other cap components such as microcalcifications, possibly altering their shape, size or topography.

Taken together, the creation of more complex, multifactorial 3D in vitro model systems as well as advancements in imaging techniques are required to better assess the impact of individual cap components as well as their reciprocal interplay on cap strength and vice versa. Obtained knowledge will ultimately serve as input to help in the diagnosis, prevention, treatment, and design of new therapies against atherosclerotic cap rupture.