The innate immune response is a key player in the process of coronary artery and aortic valve calcification

I. F. Shlyk; M. V. Kharitonova; M. N. Morgunov; I. M. Blinov; E. D. Vasilyeva; D. Yu. Besedina

doi:10.21886/2219-8075-2024-15-4-90-98

The innate immune response is a key player in the process of coronary artery and aortic valve calcification

I. F. Shlyk, M. V. Kharitonova, M. N. Morgunov, I. M. Blinov, E. D. Vasilyeva, D. Yu. Besedina

https://doi.org/10.21886/2219-8075-2024-15-4-90-98

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Abstract

The presented literature review allows us to understand and supplement the existing ideas about the pathogenesis of coronary calciﬁcation and aortic valve calciﬁcation at the stage of formation of these changes. To study in detail the participation of the most studied immunocompetent cells of innate immunity, such as macrophages, dendritic and mast cells, neutrophils, natural killers in calciﬁcation of arteries and aortic valve, their functional role in the progression of this process. The review also presents gaps and missing data present in the study of these cell populations, the replenishment of which will contribute to the development of targeted therapy for the calciﬁcation process.

The search for literature sources was conducted in the following databases: Scopus, web of Science, MedLine, The Cochrane Library, RSCI, Springer, Science Direct — taking into account the year of publication (no more than 10 years), language of publication (English/Russian), availability of full-text publications and access to them.

Keywords

innate immune response, coronary calcification, aortic valve calcification, macrophages, dendritic cells, mast cells

For citations:

Shlyk I.F., Kharitonova M.V., Morgunov M.N., Blinov I.M., Vasilyeva E.D., Besedina D.Yu. The innate immune response is a key player in the process of coronary artery and aortic valve calcification. Medical Herald of the South of Russia. 2024;15(4):90-98. (In Russ.) https://doi.org/10.21886/2219-8075-2024-15-4-90-98

Introduction

Calcification of the arterial bed of not only coronary but also other basins is an independent predictor of a high and very high risk of fatal cardiovascular events [1]. Vascular elasticity changes during arterial calcification, which is most dangerous in the case of atherosclerotic plaques, causing their damage, rupture, and, as a result, arterial thrombosis. Moreover, the presence of arterial calcification is a powerful factor complicating carrying out interventions, including direct myocardial revascularization. Talking about the calcification of coronary arteries, it should be noted that the measurement of the calcium index (Agatston index) is a recommended study in order to assess the damage to coronary arteries by atherosclerosis and the prevalence of coronary calcification as a result of persistent inflammation, especially in people with comorbid pathology [2]. A significant amount of data has been accumulated on the study of coronary calcification; there are various theories about its occurrence, where a significant role is assigned to the immune response, which has been recently an important target for the development of targeted atherosclerosis therapy. Aortic valve (AV) calcification is also often found, most often in persons over 65 years of age with various concomitant pathologies, where the calcium deposition process has the same mechanism as in the arteries, with differences in anatomical localization and the reasons for its formation.

The purpose of this review is to reveal the available data on the role of the main cell populations of the innate immune response in the development of coronary calcification, as well as AV calcification. The characteristics of the immune response are presented from the standpoint of the anatomical classification of the calcification process, histological landmarks, the duration of the process, and the interest of certain immunocompetent cells.

In the presented review, the cited scientific works were selected using the following keywords: "coronary calcification," "aortic valve calcification," "macrophages," "neutrophils," "dendritic cells," "natural killers," "mast cells," and "innate immune response", used, among other things, in a logical combination in order to narrow the search. The following databases were used for the search: Scopus, Web of Science, MedLine, The Cochrane Library, RSCI, Springer, and Science Direct, taking into account the year of publication (not more than 10 years), the language of publication (English/Russian), the availability of full-text publications, and access to them. During the initial search, 2672 works were found, of which 59 works met the criteria for the request. Works not indexed in medical databases were not subject to review.

For a detailed understanding of the presented data, it is necessary to give a histological classification of calcinosis, represented by the following types: a) calcification of the intimate layer of the arterial wall, which, according to various authors, develops as a result of cell aging, oxidative stress, hyperlipidemia, cell apoptosis, and inflammation, which is more commonly seen in patients with atherosclerosis, metabolic syndrome, and type 2 diabetes mellitus; b) calcification of media, that is, the middle layer of the arterial wall, where risk factors are cell aging, oxidative and mechanical stress, and elastase degradation, which is observed in patients with diabetes mellitus, chronic kidney disease, osteoporosis, Marfan syndrome, and connective tissue dysplasia (elastic pseudoxanthome); c) calcification of the AV leaflets, the causes of which are cell aging, hyperlipidemia, mechanical stress, and inflammation, observed in patients with arterial hypertension, rheumatic AV disease, and congenital bicuspid AV; d) calciphylaxis, in which arterioles are affected due to the presence of hyperphosphatemia, hypercalcemia, and hypercoagulation and at hemodialysis. These conditions are observed in patients with renal failure, hypo- and hyperparathyroidism, vitamin D deficiency, autoimmune diseases, and metastatic cancer [3]. As can be seen from the characteristics of the calcification process of various structures, both the arterial wall and the AV, the process of inflammation is general, which is realized by the immune response. The development of coronary calcification is based on intimate calcification, an active process that occurs as a result of dysmorphic precipitation of calcium under the influence of chondrocyte-like cells, but not osteoblast-like (as in medial calcification), and an inflammatory cascade activated primarily by macrophages with hyperproduction of various cytokines. The cascade of coronary calcification can be represented as follows: apoptosis of immature cells in the atheroma and the subsequent release of apoptotic bodies, initiating the deposition of calcium crystals, death of smooth muscle cells (SMCs) and macrophages, leading to the release of matrix vesicles, accumulation of lipoproteins in the intima, contributing to inflammation, and phenotypic modulation of SMCs into chondrocyte-like cells leading to calcium hydroxyapatite deposition [4]. These mechanisms contribute to the development of oxidative stress, inflammation, and subsequent calcification in the intima of the arteries [5][6].

Innate immunity is the first line of defense of the human body, where the main activators are molecular patterns associated with pathogens – these are PAMPs (pathogen-associated molecular patterns) and DAMPs (molecular structures associated with damage, including by own immune cells – damage-associated molecular patterns). These patterns are detected by pattern-recognizing receptors, mostly represented by Toll-like receptors (TLRs). The role of these receptors in the initiation of cardiovascular diseases has been discussed for more than a decade and is beyond doubt, since the activation of TLRs implements and supports the inflammatory response through the enhancement of the synthesis of various cytokines and chemokines through the translocation of NF- κ B into the nucleus, where it causes the expression of pro-inflammatory genes [7]. The innate immune response is realized through cell-dependent mechanisms, secreted factors, and the implementation of the adaptive immune response [8]. Most studies devoted to cardiovascular calcification have focused on the description of the innate immune response cells and their ability to release calcification inducers (intracellular vesicles) and to remodel the extracellular matrix [9]. Let us take a closer look at the role of some cell populations of the innate immune response in the development of calcification of the coronary arteries and AV.

Macrophages

Macrophages play a central role in the pathogenesis of atherosclerosis; the uptake of oxidized low-density lipoprotein by macrophages leads to the formation of foam cells and increased signal transduction through surface TLR-4 receptors [10]. TLR signaling leads to the release of proinflammatory cytokines, promoting cell adhesion and enhancing matrix metalloproteinase (MMP) release by macrophages, contributing to tissue damage. TLR-4 increases and concentrates in the area of the plaque most sensitive to rupture, thereby determining the future of the plaque (stable or unstable phenotype) [11]. Unstable plaque is associated with a pro-inflammatory mechanism that contributes to cell death and thinning of the fibrous cap [12]. Calcification is associated with inflammation, and therefore there are difficulties in differentiating the specific effects of various subtypes of macrophages. Macrophages are a unique population; they have an exceptional ability to change their functional characteristics in response to changes in the tissue microenvironment, which allows them to flexibly adapt to tasks such as tissue protection and healing, not only participate in inflammation, which is referred to as macrophage polarization [13].

The M0 phenotype corresponds to resting macrophages. After antigenic stimulation and activation, they differentiate to a greater extent into two main phenotypes: M1/classically activated macrophages or M2/alternatively activated macrophages [14].

The M1 phenotype is more often polarized by cytokines produced by Th1 lymphocytes, such as IFN-γ, TNF-α, and IL-1, as well as by lipopolysaccharides [15]. The M2 macrophage phenotype, in turn, is divided into three subtypes – M2a, M2b, and M2c. M2 polarization is induced by essential cytokines, including anti-inflammatory ones, including IL-4, IL-13, IL-10, IL-33, and TGF- β [14][16]. IL-4 and IL-13, which are Th2-lymphocyte cytokines, directly trigger the activation of M2 macrophages by activating the STAT6 signaling pathway via the IL-4 receptor α (IL-4R α). Herewith, IL-10 regulates M2 polarization by stimulating STAT3 through the IL-10 receptor. Besides, phagocytosis of apoptotic cells causes M2 polarization, characterized by the release of anti-inflammatory mediators together with prostaglandin E2 [16]. In M1 macrophages, glycolysis mainly occurs, suppressing the oxidation of fatty acids and increasing their uptake. This metabolic shift leads to intracellular accumulation of fatty acids, promoting the formation of foam cells, a characteristic feature of late-stage plaques. In contrast, M2 macrophages are mainly involved in the metabolism of oxidative phosphorylation, mediating anti-inflammatory and repair processes usually observed in early-stage plaques [17]. During experimental studies on the reproduction of the macrophage response in microcalcified media, a phenotypic shift similar to M2 was noted at hyperphosphatemia. Macrophages had an increased ability to absorb phosphate and enhanced hydrolysis of arginine, which affects the formation of calcium crystals in the plaque, as well as the secretion of higher levels of adenosine triphosphate (ATP) and pyrophosphate (PPi), thereby inhibiting calcium phosphate deposition [18]. PPi is formed by the hydrolysis of extracellular ATP by the enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (eNPP), forming PPi hydrolysis products and adenosine monophosphate. Thus, PPi inhibits calcium phosphate precipitation, preventing the formation of hydroxyapatite and promoting its dissolution [19]. During incubation of macrophages in enriched phosphate-calcium medium, extracellular vesicles were released, and expression of interleukin-6 (IL-6) was increased in M1 phenotype cells; in M2 phenotype cells, induction of arginase-1 expression was decreased, which indicated polarization of macrophages in the M1 phenotype under these conditions [20]. Increased extracellular content of Ca²⁺ alone can induce the activity of NLRP3 (cryopyrin-cytosolic protein, Nod-like receptor of the NALP family, the main component of the same type of inflammasome (NLRP3 inflammasome), involved in the activation of caspase 1 and 5, leading to intracellular processing and formation of IL-1 β and IL-18 active forms) [21]. Moreover, calcium phosphate crystals can be actively absorbed by macrophages, cause polarization of macrophages into the pro-inflammatory phenotype M1 through phagocytosis, and also activate the NLRP3 inflammasome complex with the release of IL-1 β [22]. The release of the IL-1 β in response to phagocytosis of cholesterol crystals and activation of NLRP3 leads to the recruitment of neutrophils and early formation of atherosclerosis [23]. However, this pro-inflammatory response to calcium phosphate particles can be inhibited by co-incubation with fetuin A, enriched by Gla (GRP – γ-carboxyglutamic acid), which is a Ca²⁺-binding amino acid that is necessary for the functioning of calcium-binding proteins and acts as a natural inhibitor of calcification [24]. Stimulation of THP-1-derived macrophages by hydroxyapatite nanoparticles, a natural mineral form of calcium phosphate, can itself also cause expression of GRP and Gla matrix protein (MGP), a potent vitamin K-dependent inhibitor of vascular calcification synthesized by vascular smooth muscle cells (VSMCs) and chondrocytes [25]. In addition to the generally recognized M1 and M2 phenotypes, additional macrophage phenotypes, including Mox and M (HB), contribute to the complex dynamics of vascular calcification. [26]. The transition of macrophages to the Mox subpopulation, which makes up about 30% of macrophages in plaques, is facilitated by oxidized phospholipids. Mox macrophages release proinflammatory cytokines such as IL-1β and COX-2 (cyclooxygenase 2). Erythrocytes and hemoglobin cause activation of M(HB), a macrophage subtype capable of producing anti-inflammatory mediators such as IL-10, thereby interfering with the progression of plaque formation. Notably, CD163 + M(HB) macrophages represent a distinctive alternative subtype localized in sites of intra-plaque hemorrhage. These macrophages can activate the NF-κB pathway in VSMCs, having an inhibitory effect on calcification [27]. The more data on macrophages is accumulated, the more conflicting assumptions arise about their role in varying degrees of stability of atherosclerotic plaques, as well as in macro and microcalcified lesions. Previously, it was assumed that an unstable plaque with a high content of pro-inflammatory macrophages contains numerous macrocalcifications, but now a number of authors show a greater prevalence of macrocalcification in a stable plaque, which, as a rule, is denser and enriched by anti-inflammatory macrophages [28]. Microcalcification (1–5 μm) is mainly observed at the early stages of the formation of atherosclerotic plaques, which are more prone to rupture [19]. Currently, it has been shown that mechanisms of the initiation of macrophage-induced microcalcification include apoptosis, release of extracellular vesicles and inflammatory mediators by macrophages, and osteogenic transdifferentiation of VSMCs. In the formation and mutual transformation of microcalcification and macrocalcification, these mechanisms are crucial. Macrophage apoptosis acts as an essential factor promoting plaque calcification, where apoptotic bodies generated by macrophages act as nucleation centers for calcification [29]. In the early stages of atherosclerosis, macrophage apoptosis helps to reduce the damage area and reduce inflammatory reactions. However, at later stages, macrophage apoptosis progresses into secondary necrosis, increasing plaque calcification [30]. As mentioned above, extracellular vesicles are membrane microparticles in the extracellular matrix that interact with matrix proteins; extracellular vesicles induce Ca⁺⁺flow and initiate the calcification process [31]. The accumulation and aggregation of extracellular vesicles trigger the formation of calcium hydroxyapatite particles, promoting vascular calcification [32]. The role of extracellular vesicles in vascular microcalcification is confirmed in patients with diabetes mellitus, where it has been noted that under conditions of elevated glucose levels, macrophages polarize M0 into the predominant M1 phenotype and demonstrate increased expression of micro-RNA-32 both in macrophages and in their extracellular vesicles, which contributes to increased formation of osteogenic cells from VSMCs [33]. In addition, an increased level of galectin-3 stimulates in macrophages movement of extracellular vesicles, derived from VSMCs, to the inner layer of blood vessels, which leads to calcification development [34]. Proinflammatory macrophages, which are activated by lipid oxidation products and calcium phosphate crystals, secrete cytokines such as TNF-α, IL-1β, and IL-6. Hydroxyapatite particles upon calcification stimulate the release of TNF-α from macrophages, forming a closed circle. The induction of Pro-IL-1β, its subsequent processing and release are linked to the development of extensive plaque calcification and changes in the Rac2 level (Ras-related C3 botulinum toxin substrate 2), which initiates the secretion of proinflammatory cytokines and phagocytosis of apoptotic cells [35]. As a member of the IL-1 superfamily, the proinflammatory cytokine IL-18. along with IL-12, participates in immune inflammatory responses and promotes calcification and fibrosis through the activation of the non-selective cation channel TRPM7 [36]. By increasing the level of TNF-α, M1 macrophages stimulate the expression of carbonic anhydrase I (CA1) and carbonic anhydrase 2 (CA2) in VSMCs, thereby promoting atherosclerotic calcification [37]. The phenotypic transition and calcification process of VSMCs are influenced by inflammatory cells via matrix metalloproteinase-9 (MMP-9), as was shown in single-cell sequencing and animal studies [38]. At the same time, the lack of cartilage oligomeric matrix protein, a natural inhibitor of calcification in VSMCs, leads to increased osteogenic transdifferentiation and, as a result, increases calcification [39]. An equally important role is assigned to IL-6, which acts as a marker affecting mortality in patients with coronary artery disease, which contributes to the calcification of VSMCs, transforming them into osteoblasts through the IL-6/STAT3/JMJD2B pathway [40]. There are some differences in the formation of macrocalcifications (≥ 50 μm), when macrophages and VSMCs release extracellular vesicles, which are foci of the origin of microcalcifications, which ultimately merge into macrocalcifications [41]. Currently, there are few studies on how microcalcification transits to macrocalcification, as well as how the macrocalcification environment influences macrophages. The study of the progression from microcalcification to stable macrocalcification is of clinical importance due to the instability of microcalcification and its potential relationship with cardiovascular mortality. Next, we will consider the contribution of macrophages to medial calcification occurring in small and medium arteries in diabetes, chronic renal failure, and aging [42]. In the study of atherosclerotic plaques in patients with diabetes mellitus, macrophages showed an increase in the expression of the CD40⁺ molecule in calcification zones, as well as in patients with uremia [43]. There is an interesting fact about the regulatory role of vitamin D receptor activation, which promotes the transition of the macrophage procalcium phenotype to anticalcium, making it possible to conclude about a possible target in the treatment of calcification [44]. Experiments conducted in a controlled environment in individuals with uremia demonstrated the ability of calcium ions to interact intracellularly in macrophages with aldosterone, leading to the calcification of VSMCs and stimulation of neighboring VSMCs in the involvement of calcium salt accumulation and acceleration of vascular calcification [45].

Macrophage participation in the calcification of the vascular bed involves not only the activation of various factors but also their effect on endogenous calcification inhibitors. It is known that there are several endogenous mechanisms preventing ectopic calcification. Macrophage-induced inflammation causes several types of vascular cells including SMCs, endothelial cells, and pericytes to undergo phenotypic changes, leading to a change in the expression of factors, which modulate calcification [46]. Inflammatory cytokines produced by macrophages simultaneously initiate loss of VSMCs, expression of calcification inhibitors such as MGP, osteopontin, and PPi, and acquisition of inducers such as osteoprotegerin [45][47].

Thus, the analysis of various literature sources has shown that there is no holistic understanding of the multifaceted role of macrophages in the calcification of not only vessels but also the AV with the involvement of their molecular mechanisms, and therefore, it requires further investigation.

Dendritic cells

Another cell population of the innate immune response is dendritic cells (DCs), which initiate an antigen-specific response through their antigen-presenting function to cells of the adaptive immune system. DCs are found in turbulent flow regions in both atherosclerosis-affected and calcified valves; they support the fact that DCs play their own role in cardiovascular calcification. In arteries affected by atherosclerosis, DCs are located in the intima layer in the shoulder region of the fragile plaque and co-localize with T-cell clusters, leading to plaque destabilization and subsequent calcification [47]. Upon the activation of the CD86⁺ receptor of the SPP1 signaling molecule expressed by DCs, the DC function is simulated, which affects the progression of AV calcification by increasing the synthesis of IFN-γ, which, together with IL-17, mediated DC migration and T-cell activation in AV and changed the Th17/Treg ratio [48]. The presented data about the role of DCs are only few, and it requires further clarification and experimental work.

Mast cells

Mast cells (MCs) are participants in the first line of defense against pathogens and the source of mediators such as proteases and cytokines. There are activated and resting MCs. The main effector mechanism of MCs is mediated by specific proteases, tryptase, and chymase released during their degranulation and leading to elastin degradation [44]. It is assumed that the damaged side of the AV valves can be the area of MC migration from the blood circulation to the site of inflammation. MCs can be a secondary phenomenon in relation to the degeneration and calcification of AV valves, since this role is primarily played by macrophages accumulating in large numbers inside stenosed AVs, which secrete cytokines that activate valve myofibroblasts and trigger their osteoblastic transdifferentiation, and also promote the migration of monocytes into developing AV stenosis [49]. There is evidence that the production of MC protease is associated with aortic stenosis, and an increased amount of MCs is associated with the severity of aortic stenosis, as evidenced by the detection of a large number of degranulated MCs in the calcified valve [49]. Finally, the secretion of MC chymase can promote the conversion of angiotensin I to angiotensin II, which is associated with the thickening of valve leaflets in mice, their remodeling, and calcification. In turn, cathepsin G produced by MCs causes elastin degradation, and tryptase destroys endostatin, namely an antiangiogenic molecule, in the cells of AC patients, leading to neovascularization, aggravating AV stenosis [47]. Both activated and resting MCs are able to cause reprogramming of VSMCs and transition to a pro-inflammatory osteochondrocyte-like phenotype. In experimental work, it was shown that both MC subpopulations were about 8% (3% resting, 5% activated) of all immunocompetent plaque cells. Histopathological studies conducted on human cell samples showed that activated MCs were present in unstable plaques, especially calcified plaques, in the area of the shoulders, prone to ruptures and associated with neovascularization, with signs of hemorrhage and thrombosis [50]. Moreover, this ability of MCs is realized in connection with macrophages found in large numbers in atherosclerotic plaques at different stages of atherosclerosis. However, the role of MCs in the development of calcinosis is not unambiguous; their inhibitory effect has been shown in experimental work through the production of osteoprotegerin, maintaining high levels of pyrophosphate [51].

Neutrophils

Neutrophils are the most common white blood cells in the systemic circulation that are part of the innate immune response. The involvement of neutrophils in atherosclerosis is due to various effector mechanisms, including the release of granules, phagocytosis, and the formation of neutrophil extracellular traps (NETs), the inducers of which in the cardiovascular system are hemodynamic forces [52]. NETs, represented by chromatin subunits, trigger the clotting process by attracting platelets and leading to thrombus formation. Through platelet activation, neutrophils promote valve leaflet calcification during high shear stress, causing osteogenic differentiation in interstitial valve cells [53]. In addition, platelets express and release osteocalcin and TGF-β, enhancing their participation in mineralization [54]. Myeloperoxidase and neutrophil elastase, being components of NETs, stimulate macrophages and promote the secretion of cytokines, including IL-1β, as well as ROS [55]. Kopytek et al. confirmed the correlation of NET content in stenosed valves with the severity of the disease, which suggests the contribution of neutrophils to this pathogenic process [56]. The role of neutrophils in the development of arterial calcification is mediated (by attracting immunocompetent cells to the atherosclerotic plaque) and is of greater importance when it is ruptured. The direct mechanisms of neutrophils affecting arterial calcification are not well understood.

Natural killer cells

Natural killer (NK) cells are cytotoxic cells that act by releasing various cytotoxic molecules and exhibit immunoregulatory function. It is noted in the literature that a significant number of NK cells are present in atherosclerotic plaques, but their relation to the development of calcification of both arteries and the AV has been little studied [57]. It is assumed that after activation by stress signals, NK cells release cytotoxic molecules, granzymes, and perforins, or act through death receptor ligands such as FasL and TNF-associated ligands inducing apoptosis of immunocompetent cells [58]. On the other hand, it can be assumed that the indirect function of NK cells is the release of cytokines such as IFN-γ, TNF, and IL-10, potentially modulating the inflammatory response in the atherosclerotic plaque and AV [59]. However, further studies are needed to provide evidence for these hypotheses in vitro and in vivo.

Conclusion

Analyzing the literature sources, it can be concluded that the pathogenesis of calcification of the coronary arteries and AV, as the main cause of cardiovascular disease complications, is really very relevant. The currently available data characterizing the cellular component of innate immunity as the primary link that forms the response to damage are incomplete and require further study in terms of interaction with the adaptive immune response in order to create targeted therapy for coronary and aortic calcification.

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