Revisiting cardiovascular calcification: A multifaceted disease requiring a multidisciplinary approach

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Review

Revisiting cardiovascular calcification: A multifaceted disease requiring a multidisciplinary approach Joshua D. Hutcheson ∗,1 , Claudia Goettsch 1 , Maximillian A. Rogers, Elena Aikawa ∗ Center for Interdisciplinary Cardiovascular Sciences and Center for Excellence in Vascular Biology, Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, United States

a r t i c l e

i n f o

Article history: Received 16 April 2015 Received in revised form 1 September 2015 Accepted 2 September 2015 Available online xxx Keywords: Cardiovascular calcification Aortic valve disease Atherosclerosis Microcalcification Chronic renal disease

a b s t r a c t The presence of cardiovascular calcification significantly predicts patients’ morbidity and mortality. Calcific mineral deposition within the soft cardiovascular tissues disrupts the normal biomechanical function of these tissues, leading to complications such as heart failure, myocardial infarction, and stroke. The realization that calcification results from active cellular processes offers hope that therapeutic intervention may prevent or reverse the disease. To this point, however, no clinically viable therapies have emerged. This may be due to the lack of certainty that remains in the mechanisms by which mineral is deposited in cardiovascular tissues. Gaining new insight into this process requires a multidisciplinary approach. The pathological changes in cell phenotype that lead to the physicochemical deposition of mineral and the resultant effects on tissue biomechanics must all be considered when designing strategies to treat cardiovascular calcification. In this review, we overview the current cardiovascular calcification paradigm and discuss emerging techniques that are providing new insight into the mechanisms of ectopic calcification. © 2015 Published by Elsevier Ltd.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Cardiovascular calcification and tissue biomechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Arterial calcification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Calcific aortic valve disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mechanisms of mineral deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Calcific mineral nucleation and growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Cell phenotypes in cardiovascular calcification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2. Resident vascular wall cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.3. Cells of circulating origin contribute to ectopic calcification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.4. Tissue mimics to study cardiovascular calcification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

∗ Corresponding authors. E-mail addresses: [email protected] (J.D. Hutcheson), [email protected] (E. Aikawa). 1 These authors contributed equally to the preparation of this manuscript.

Clinical evidence demonstrates that calcium burden significantly predicts and contributes to cardiovascular disease [1–3], the leading cause of death in the United States. However, no known therapeutic strategies exist to prevent or treat cardiovascular calcification. Previous studies have focused on the cellular

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and molecular changes that drive soft tissue calcification while the physicochemical processes by which mineral forms and grows remain unknown. Cardiovascular calcification lies at the interface of physical, chemical, and biological fields, requiring a multidisciplinary approach to connect the cellular and molecular changes to the remodeling that disrupts tissue function. Pathologic changes in cell phenotype (biological) create an environment favoring calcium phosphate mineralization (chemical), leading to loss in the biomechanical function of the soft tissue (physical). Understanding the integration of these processes requires a multidisciplinary approach with the common goal to develop new therapeutic strategies to control ectopic biomineralization. In this review, we provide a holistic discussion of cardiovascular calcification connecting the tissue alterations to the disease cellular underpinnings. Section 2 overviews the biomechanical changes that result in the clinical manifestation of calcification. Recent material and chemical analyses of cardiovascular calcification are reviewed in Section 3. In Section 4 we introduce the cellular populations and phenotypes responsible for mineral deposition. Finally, we discuss the commonly used methods exploring molecular mechanisms of calcification along with the emerging technologies offering novel mechanistic insight. 2. Cardiovascular calcification and tissue biomechanics 2.1. Introduction Cardiovascular tissues function in a dynamic environment. Over the course of the cardiac cycle, cardiovascular tissues expand and contract ensuring proper blood distribution throughout the body. Appropriate biomechanical properties of these tissues depend upon the microarchitecture of the extracellular matrix [4]. Calcification disrupts this microarchitecture through the deposition of hard mineral within the soft tissues [5]. The resultant loss in biomechanical integrity can lead to both chronic and acute deleterious effects [5–7]. Specifically, two types of cardiovascular calcification significantly impair human health: arterial microcalcification and aortic valve macrocalcification [8,9]. 2.2. Arterial calcification Prospective clinical data show that the risk of cardiovascular events inversely correlates with the density of calcification present within arterial plaques as measured using computed tomography [10]. It is now well-accepted that the presence of “spotty” calcification is associated with increased cardiovascular morbidity [10,11]; however, the potential causality relationship between calcification morphology and cardiovascular risk was not made clear by clinical and histopathological studies. Recent work using finite element modeling of stress distribution within atherosclerotic plaques indicates that subcellular microcalcifications in an atherosclerotic fibrous cap promote material failure of the plaque [12–14], causing myocardial infarction and stroke. The atherosclerotic fibrous cap serves as the main barrier between the pro-inflammatory lipid pool within the plaque and the blood flowing through the arterial lumen. When this cap ruptures, platelets rapidly accumulate to form a fibrin clot during a process known as thrombosis. Local occlusion of the vessel by these thrombotic clots causes 60% of all myocardial infarction [15,16]. Each time the heart pumps during systole, increased pressure leads to expansion of the arteries, generating stresses within the fibrous cap. Plaque rupture occurs when this stress reaches a critical threshold where the collagen fiber network of the fibrous cap, can no longer remain intact [17]. Classically, weakening of the fibrous cap has been attributed to inflammation-dependent proteolytic

degradation of collagen [18]. As collagen diminishes, the fibrous cap network becomes thinner and more likely to rupture under stress. Using material-based theories and knowledge of the properties of collagen, computational (i.e., in silico) models can predict fibrous cap stresses and the critical stress required for rupture [7]. These finite element analyses predict the stress by segmenting a plaque into discrete nodes. The stress at each node is calculated based upon the known material properties of the plaque constituents and the local geometry (e.g., curvature) at the node. By increasing the number of nodes at which these calculations are performed, the localized stresses throughout the plaque can be estimated [17]. Predictions based on modeling a homogenous collagen network in the fibrous cap suggest that plaque rupture should occur when the cap is less than approximately 65 ␮m thick [19–21], and rupture should primarily occur at the shoulders of the plaque [22]. Histopathological observations, however, indicate that 37% of cap ruptures occur at the center of the plaque, and ruptures in caps as thick as 160 ␮m have been observed [23]. Inclusion of microcalcifications in fibrous cap models, predicts rupture criteria closely match the histopathological observations [24–26]. Rigid microcalcifications do not stretch, as such fibrous cap collagen deforms around microcalcifications, amplifying stress, and increasing cap rupture risk (Fig. 1). Model data suggest that plaque rupture is a function of both fibrous cap thickness and the presence, size and orientation of microcalcifications embedded within the cap [12–14,27,28]. These findings corroborate the clinical data that demonstrate an inverse relationship between calcification density and cardiovascular events [11]. High resolution micro-computed tomography imaging identified a preponderance of small microcalcifications scattered throughout excised human coronary arteries [14]. In contrast to the increased cap stress caused by microcalcifications, large, dense calcifications may stabilize the plaque [17,29–31], although the mechanisms giving rise to these different calcification morphologies are unclear. Once an atherosclerotic plaque develops, a therapeutic goal could be a treatment tilted toward an increased fibrocalcific response. As discussed in Section 2.3, a heightened fibrocalcific response, however, must be avoided in the context of the other major site of cardiovascular calcification: the aortic valve. It should be noted that the discussion to this point has considered atherosclerotic calcification only. Atherosclerosis manifests as localized plaques at specific arterial positions [32]. Therefore, increased fibrocalcific remodeling at these locations do not appreciably compromise the integrity of the vasculature [33]. In contrast, conditions leading to elevated serum phosphate (i.e., hyperphosphatemia), in patients undergoing dialysis for chronic renal disease, lead to gross medial calcification that can severely alter arterial elasticity [34]. It is likely that in these patients, the most appropriate therapeutic goal would be prevention of the initial mineral deposition. 2.3. Calcific aortic valve disease The aortic valve is situated between the left ventricle and the aorta and controls unidirectional blood flow from the heart to systemic circulation. Unlike most other cardiovascular tissues, the motion of the aortic valve is determined predominantly by cardiac pressure and hemodynamics. An excellent review of the structurefunction relationship is provided by Sacks et al. [4]. Here, we offer a short summary of aortic valve physiology to emphasize the impact of calcification on valve function. Three thin membranous leaflets at the base of the valve recoil toward the aortic wall during systolic contraction of the ventricles, allowing blood to be pushed from the heart into the aorta. As the heart rests in diastole, the reversed pressure gradient forces these leaflets to stretch toward the center of the aortic annulus (Fig. 2A). As they stretch, adjacent leaflets meet and seal the valve orifice, preventing retrograde blood flow into the

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Fig. 1. Finite element analysis of microcalcification in human coronary atherosclerotic plaque. (A) Histochemical analysis shows microcalcification stained by von Kossa embedded within the extracellular matrix of the fibrous cap. (B) Finite element modeling of this microcalcification shows 5–6.4× elevated stress at the edges of the microcalcification. The intersections of the lines within the figure indicate the nodes at which the calculations were performed. Figure adapted from [12]. (C) Microcalcifications within atherosclerotic fibrous caps lead to plaque rupture and thrombus formation.

heart (Fig. 2B). The valve microarchitecture makes the valve leaflets perfectly suited to perform this task (Fig. 2C). The side of the valve leaflets proximal to the left ventricle consists of a layer of elastin fibers (known as the ventricularis) radially aligned from the base of the valve to the tip of the leaflets. These elastin fibers provide the elasticity required for the leaflets to stretch and seal the valve during diastole. On the other side of the valve leaflets—proximal to the aorta—a layer of circumferentially aligned collagen fibers (known as the fibrosa) confer tensile strength to the leaflets. The circumferential collagen alignment (i.e., perpendicular to the radially aligned elastin fibers) allows the leaflets to stretch toward the center of the valve orifice, but these rope-like fibers prevent the leaflets from prolapsing into the ventricle during diastole. A layer of glycosaminoglycans and proteoglycans (known as the spongiosa) resides between these two outer layers and provides a lubricating barrier between the elastin and collagen fibers. As evident from the involvement of aortic valve biomechanics in all of these processes it is clear that extracellular matrix components regulate appropriate valve function. Fibrocalcific remodeling prevents valve leaflets from performing their physiologic function, leading to inefficient blood distribution and a resultant increase in cardiac workload [35]. Unlike acute events resulting from arterial microcalcification discussed in Section 2.2, calcific aortic valve disease progresses slowly over the course of years to decades as fibrotic collagen accumulation and calcific deposits disrupt the

tissue microarchitecture [9]. Mineralization initiates at the base of the leaflets within the fibrosa layer and progresses throughout the leaflets in the animal models of calcific aortic valve disease [36]. Due to the absence of non-invasive therapeutic interventions, clinicians often elect to delay surgical or catheter-based valve replacement until gross changes severely hinder leaflet biomechanics [37]. In the duration, patients must cope with a reduced quality of life due to diminished cardiac function [35]. The asymptomatic progression timeline of aortic valve calcification presents a double-edged sword for the discovery and development of therapies. While the slow progression of the disease presents a long window for therapeutic intervention, the disease duration makes it difficult and costly to follow patients over the course of required randomized clinical trials [38]. Further, retrospective studies often focus on easily identifiable acute endpoints such as myocardial infarction, stroke, or death. Slowly progressing deterioration in valve function may be overlooked. As with the artery, aortic valve leaflets exhibit rapid calcification in chronic renal disease patients undergoing dialysis [39]. It is currently unclear to what extent calcification in these patients shares a common mechanism with that observed in patients with normal serum phosphate [40]. These patients may, however, present an appropriate clinical trial population to test therapeutics designed to inhibit mineral deposition. The chronic renal disease patient population has a well-defined calcification initiation point at the onset

Fig. 2. Aortic valve structure and function. (A) When the heart contracts in systole, the aortic valve leaflets are forced against the aortic wall, allowing blood to escape the heart. (B) As the heart rests in diastole, the reverse in pressure forces the leaflets to stretch toward the center of the valve orifice where adjacent leaflets meet to seal the valve. The circumferentially aligned collagen fibers prevent the leaflets from prolapsing into the ventricle during diastole. (C) A histological section of an aortic valve leaflet stained with Masson’s trichrome shows the trilaminar structure of the leaflet. The collagen fibers of the fibrosa (f) are stained yellow. The glycosaminoglycans in the spongiosa (s) are stained blue, and the elastin fibers in the ventricularis (v) are stained black.

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of dialysis [39], and the accelerated mineralization circumvents the normal problems associated with the duration of aortic valve calcification. If basic mechanisms of mineral deposition are conserved, the therapeutics could be extended to the general population. 3. Mechanisms of mineral deposition 3.1. Introduction Histopathological analyses of calcified tissues and in vitro assays most commonly employ stains specific to either calcium or phosphate—the two major components of calcific mineral. These stains, however, do not offer detailed information on the type of mineral present. Detailed analysis of the mineral offers benchmarks against which the validity of cell culture and animal models may be tested. Further, as discussed in Section 2, the location and morphology of the calcific mineral is the most significant determinant of pathological alterations to tissue biomechanics. By understanding the mineral endpoints, information about the processes that led to its deposition may be ascertained. To this end, studies have employed techniques commonly utilized to assess synthetic materials to gain new insight into the mineral structure and composition in diseased cardiovascular tissues. 3.2. Calcific mineral nucleation and growth Solid state nuclear magnetic resonance spectroscopy demonstrated similarity between biomineral formed under highly controlled (bone) and pathological (atherosclerotic plaque) conditions [41]. The detected matrix-mineral atomic interface in calcified atherosclerotic plaque is very similar to that in bone and, was characterized by a predominance of glycosaminoglycans. A recent study using advanced material based analytic methods identified highly crystalline spherical particles containing calcium phosphate mineral and poorly crystalline, amorphous compact calcifications in diseased aortic valves and vascular tissues [42]. This mineral is different from the compact hydroxyapatite composing bone. The origins of these calcifying structures in both tissues remain unclear; however, evidence suggests that cellular-derived matrix vesicles contribute to the initiation of mineralization within atherosclerotic plaques by serving as nucleating sites for mineral formation [43,44]. This pathological process may be similar to physiological bone and cartilage mineralization, where resident osteoblasts and chondrocytes release extracellular matrix vesicles that serve as the nucleating foci for mineral. In bone, vesicles are enriched in calcium-binding proteins as well as enzymes such as tissue non-specific alkaline phosphatase (TNAP) that generate inorganic phosphate ions from innate sources including nucleotides and pyrophosphate [45,46]. As vesicles reach supra-physiologic levels of these ions, annexins located on the vesicle membrane may bind calcium-phosphate mineral [47,48]. These crystals expand in size, break out of the vesicle, and interact with the surrounding extracellular matrix [45]. In vitro studies have shown that cardiovascular cells can release vesicles with tissue non-specific alkaline phosphatase (TNAP) that are similar to those observed in bone biogenesis. TNAP-negative vesicles have also shown the ability to nucleate calcific mineral under high phosphate conditions [3,43]. The TNAP-positive vesicles may correspond to the osteogenic phenotypes observed in atherosclerosis, whereas TNAP-negative vesicles may play a relevant role in the hyperphosphatemia conditions associated with chronic kidney disease [44]. Nucleation and growth of calcific mineral within aortic valve leaflets remain less clear than the processes described in arterial calcification. Analyses of explanted leaflets indicate that valve

mineralization may be present as dystrophic calcification, a disorganized crystal structure that is often associated with precipitation of calcium phosphate on debris remaining after cell death, or as a bone-like organized crystal lattice of hydroxyapatite that is indicative of osteogenic processes [49]. Calcification displaying dystrophic characteristics appears to predominate in 83% of explanted leaflets; however, 13% of leaflets present bone-like structures indicative of active osteogenesis [49]. Histological analyses of the progression of valve calcification indicate that osteogenic mineral deposition may follow the initial wave of dystrophic calcification [50]. Even though markers of osteogenic phenotypes have been observed, extracellular vesicles have not been studied in the context of calcific aortic valve disease. As discussed in the following sections, a lack of clarity in the phenotypes associated with valve calcification has hindered research into the mechanisms of mineral deposition. Molecular imaging of calcific mineral deposition in mouse aortae and valves has been achieved using bisphosphonates conjugated to near infrared fluorescent (NIRF) molecules [51]. Bisphosphonates have a structure similar to the calcification inhibitor pyrophosphate [52], and is believed to bind to calcium and accumulate in mineralized crystals [53]. The accumulation of NIRFbisphosphonates in calcific regions yields detectable NIRF signal [36]. In addition to the potential for in situ plaque detection, molecular probes can be employed for pre-clinical research and ex vivo diagnostics. Bisphosphonate-based NIRF probes allow earlier detection of calcification in human plaques than light microscopy techniques currently used by pathologists [36]. This enhanced resolution may allow researchers to understand the nucleation of microcalcifications and allow pathologists to readily identify the presence of microcalcifications in subclinical atherosclerotic plaques from autopsy or tissues excised for other indications. Findings from these types of studies may provide new insight that could be used to design future iterations of optical molecular imaging strategies. In future studies, these imaging techniques may be combined with material analysis techniques to study mineral composition from the earliest stages to advanced mineral. 4. Cell phenotypes in cardiovascular calcification 4.1. Introduction Cardiovascular calcification has been considered to be a passive, degenerative process resulting from calcium phosphate waste collecting within the tissue, but recent studies have shown that the mineral forms through active cellular processes [54,55]. This new understanding offers hope that therapeutic agents may be able to reduce cardiovascular calcification development. Uncertainty remains on the cell populations and phenotypes involved in calcific mineral deposition in cardiovascular tissues, but recent studies have begun to elucidate the wide array of associated cellular processes [56]. Unlike in the context of bone, wherein hydroxyapatite mineral is deposited by well-defined cell populations in a wellorganized manner, mineral in cardiovascular tissues arises from an uncontrolled pathological environment. The complexities of this environment, including the numerous cell populations contributing to pathologic remodeling, make it difficult to ascertain causal relationships based upon endpoint analyses of diseased human or animal model tissues. Additionally, the complex cellular environment is difficult to recapitulate in a model system. Most in vitro studies focus on one type of cells. This minimalistic approach has yielded insight into mechanisms of calcification, but important intercellular and environmental interactions may be missed [57]. In this section, we overview a current understanding of the roles that various cell types play in mediating calcification (Table 1) and the methods used to study these cells in vitro.

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Table 1 Cell phenotypes in cardiovascular calcification. Original cell type

Mechanism

Type of calcification

Reference

Smooth muscle cells

Osteogenic transition Release of extracellular vesicles Apoptosis Unknown Osteogenic transition Endothelial-mesenchymal transition Osteogenic transition Apoptosis Osteoclast-like cells Release of extracellular vesicles Matrix remodeling/cytokine production Pro-calcific differentiation Cytokine production

Intimal/medial Intimal/medial Intimal/medial Valvular Medial Medial/valvular Valvular Valvular

[116] [44,71,72] [117] [93–96] [75] [76,78,79] [50,91] [86–88] [101,118] [43] [36,97–99] [102,103,105] [106–108]

Myofibroblasts Endothelial cells Valvular interstitial cells Monocytes/macrophages

Circulating myeloid cells Mast cells

4.2. Resident vascular wall cells Bone-related proteins and transcription factors present within calcified atherosclerotic intimal plaques and medial calcified areas suggesting that cells within the vascular wall contribute to the calcification process. It is likely that cells of different origin contribute in different ways to intimal atherosclerotic calcification and medial calcification that are unrelated to atherosclerosis and often observed in elderly patients and patients with diabetes and chronic renal disease [58]. The concept that signaling pathways controlling bone remodeling also occur in the cardiovascular system is well accepted [59]. In the bone tissue, remodeling involves a dynamic and fine-tuned interplay among bone cells including osteoclasts, osteoblasts and osteocytes [60]. Bone cell-cell communication between bone-resorbing osteoclasts and bone-forming osteoblasts control the delicate balance and maintain bone homeostasis [60]. Various vascular cell types with multi-lineage potential exhibit morphological and biological features similar to bone cells. These cells may originate from resident cells or circulating cells with calcifying potential. However, whether vascular bone-like cells communicate in a similar way to bone cells to control mineral formation is largely unknown. Independent of the cell origin, it is likely that master transcription factors, including Runx2/Cbfa1, Msx2, and osterix (SP7), designate cells for osteoblast lineages through the induction of downstream genes such as TNAP and osteocalcin. Runx2 acts as a critical regulator of osteogenic lineage and a modulator of bone-related genes and is essential and sufficient for driving vascular smooth muscle cell (SMC)-dependent calcification [61,62]. Although numerous studies have suggested that various cell types are capable of producing a mineralized matrix [63,64], SMCs are the most widely studied cell population in vascular calcification [59]. SMCs are considered to be the major lineage source of osteogenic cells within the calcified lesion [65]. Phenotypic differentiation is a feature of SMCs [3], which are capable of differentiating into multiple lineages, including an osteoblast-like lineage, allowing for their implementation in the in vitro calcification models. Typically SMC models involve culturing human and/or animal cells in media containing high phosphate levels with or without calcium [66,67] (reviewed in depth in [68]) or media supplemented with components similar to those used in bone osteoblastogenesis studies, including beta-glycerophosphate and l-ascorbic acid-2-phosphate [69]. Additionally, several other stimuli, including dexamethasone, have been used to enhance the osteogenic differentiation of SMC (reviewed in [3]). In vitro, SMCs have been shown to contribute to calcification by undergoing apoptosis, releasing extracellular vesicles with high calcification potential, and transitioning to an osteo-chondrogenic phenotype along with an induction of TNAP activity [56]. On the other hand, the role(s) specific stimuli play in induction of calcification can change to some degree among

Intimal Intimal/valvular Intimal/medial/valvular Valvular

different SMC models. Furthermore, differences could exist among induced and/or inhibited pathways through varying culture media compositions, making it difficult to fully integrate interpretations between independent SMC studies. Of note, differences in osteogenic related gene expression and mineral composition occur in mesenchymal stem cells due to both the phosphate concentration and source (inorganic or organic) [70], a finding that may be relevant to SMC calcification models given the similarities of bone and vascular calcification. Calcifying SMCs release extracellular matrix vesicles that serve as initial mineral nucleation sites and have been implicated as an important component of the vascular calcification process [52]. These extracellular vesicles are released from SMC in response to disturbed intracellular calcium homeostasis [44] and an osteogenic environment [71]. Calcifying extracellular vesicles released from calcium/phosphate stimulated SMC, which mimic chronic renal disease, exhibit an exosomal phenotype [72] and do not show increased TNAP activity [44]. SMC that undergo osteogenic transition release TNAP-enriched extracellular vesicles [71] suggesting that SMC release extracellular vesicles with different pro-calcific features dependent on the environment. Osteogenic transition of vascular SMCs can be induced by bone morphogenetic proteins, inflammation, oxidative stress, or high phosphate levels, and leads to a unique molecular pattern marked by osteogenic transcription factors [56]. A recent transcriptomics study coupled with bioinformatics analysis revealed that calcifying SMC keep their own identity while using mechanisms that bone osteoblasts use for mineralization [73]. Principal component analysis showed that calcifying SMCs and osteoblasts have distinguishing characteristics in terms of global gene expression. This finding may suggest that it is possible to therapeutically target vascular calcification without affecting physiologic bone remodeling. In addition to well-studied SMCs, evidence suggests that adventitia-derived cells contribute to ectopic medial calcification since surgical resection of the adventitia prevents medial artery calcification in rats [74]. Adventitial myofibroblast cells can be diverted to an Msx2-dependent osteoblast lineage, possibly contributing to medial calcification observed in diabetes and end-stage renal disease [75]. Vascular and valvular endothelial cells serve as the cellular barrier between the blood and underlying tissue constituents. Endothelial cell dysfunction leads to the recruitment and infiltration of the leukocytes as discussed in Section 4.3. In addition, endothelial cells may act as a source of multipotent cells contributing to cardiovascular calcification in states of hyperglycemia or lack of calcification inhibitors [76]. Endothelial cells participate in endothelial–mesenchymal transition (EndMT), an important process in the pathobiology of fibrotic and calcific tissue injury responses [77]Endothelial cell can act as osteoprogenitors via [119] EndMT and thereby promote valve calcification [78].

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Fig. 3. Comparison of in vitro calcific nodules from VICs and SMCs. (A) VIC calcific nodules have been shown to form in vitro through a cell death-mediated mechanism wherein necrotic VICs (red fluorescence) are surrounded by a ring of apoptotic VICs (green fluorescence) forming discrete calcific structures. Scale = 100 ␮m. Figure adapted from [87]. (B) This is in contrast with intact SMCs (arrow) that deposit mineral within a matrix in vitro as shown by alizarin red S staining (*).

Further, endothelial cell phenotype is associated with osteofibrogenic predilection during arteriosclerosis via Wnt signaling [79]. Dkk1 enhances EndMT in aortic endothelial cells, whereas Wnt7b and Msx2 signals preserve endothelial cell phenotype. Understanding the cellular contribution to the progression of calcific aortic valve disease has proven to be challenging in vitro [80]. Valvular interstitial cells (VICs) are the most abundant cell type in the aortic valve but are comprised of a poorly characterized heterogeneous cell mixture. Various VIC phenotypes have been identified in the leaflets [81]. Quiescent fibroblast-like VICs can become activated myofibroblast-like VICs during development and in various pathological conditions [82,83]. Cells with osteoblastlike features have also been identified in vivo [36]. Studies have demonstrated the ability of VICs to undergo osteogenic differentiation [50]; however the mineralization capacity of these cells remains questionable [84]. In vitro, VICs readily undergo activation to the myofibroblastic phenotype [85], and studies have shown that in the presence of the cytokine transforming growth factor-␤1 and/or mechanical strain, these myofibroblast-like VICs form calcific nodules through an apoptotic-dependent mechanism [86–88]; however, the relevance of this process to the in vivo situation remains uncertain [89]. These in vitro calcific nodules also appear phenotypically different than those deposited by SMCs in an osteogenic culture environment (Fig. 3), and physicochemical differences have been observed in nodules produced by VICs under various stimuli [90]. Osteogenic markers of VICs has been achieved in vitro using other media compositions [91], and future studies of mineral composition and the mechanisms of mineral

deposition are needed to clarify the relevance of different VIC phenotypes to the tissue changes observed in calcific aortic valve disease [92]. One reason for the lack of clarity could arise from the fact that many mechanistic studies of valve disease rely on VICs derived from porcine, bovine, and ovine origin. No commercially available source of human VICs currently exists; therefore, tissues obtained from local abbatoirs often offer the most convenient source for VIC isolation and study. Given the current absence of defining VIC characteristics in the human aortic valve, unrecognized cross-species differences may further confound studies into the molecular mechanisms associated with pathological differentiation of this heterogeneous population. In addition, a population of SMCs, distinct from the myofibroblast VIC phenotype associated with valve disease, has been observed in human aortic valves [93–95], and markers of SMCs are prevalent in calcified regions of valve leaflets [96]. Though these valvular SMCs have received relatively little attention compared to VICs, they may provide an answer to potential species differences and inconsistent cell culture models of valve disease. The SMCs may lack persistence in culture due to a higher proliferative rate of VIC populations. Future studies may attempt to isolate different cellular populations directly from the human valve interstitium to investigate the role of each phenotype in contributing to calcific aortic valve disease. 4.3. Cells of circulating origin contribute to ectopic calcification In vitro studies [97], ex vivo analyses [98], and in vivo molecular imaging [36,99] have established the concept of an

Fig. 4. Association between inflammation and calcification in cardiovascular tissues. (A) Fluorescence reflection imaging of an excised apoE−/− mouse heart and aorta reveals co-localization of inflammation and calcification within the cardiovascular tissues. (B) Longitudinal intravital molecular imaging of apoE−/− mouse carotid artery shows that inflammation precedes the onset of calcification. Figures adapted from [36].

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Fig. 5. Extracellular vesicle-mediated calcification in chronic renal disease. Pro-inflammatory macrophages release vesicles that can nucleate mineral in high phosphate conditions. The mineral is nucleated on a calcium-binding complex of phosphatidylserine-annexin 5-S100A9 within the vesicles. Figure adapted from [43].

inflammation-dependent calcification in both valves and vessels (Fig. 4). Calcification in these tissues follows the accumulation of macrophages, and end-stage calcification is concomitant with a reduction in inflammation [36]. These observations suggest that inflammation is involved in the initiation and propagation of calcific mineral deposition [100]. Macrophages are involved the early, pro-inflammatory phase of calcification by releasing extracellular vesicles containing a phosphatidylserine-annexin V-S100A9 complex that facilitates mineral nucleation (Fig. 5) [43]. These procalcific macrophages exhibit a pro-inflammatory M1 macrophage phenotype [43]. However, the role of macrophage polarization in cardiovascular calcification remains largely unexplored. Monocytes-macrophages may act as precursor of osteoclast-like cells within the vessel wall. The presence of osteoclast-like cells in calcified areas of vessels indicate that this is likely to be a significant component of the vascular calcification process; however the contribution of osteoclastogenesis remains debatable and evidence that vascular osteoclast-like cells exhibit resorption activity in vivo is lacking. A link between calcifying vascular SMCs and osteoclastlike cells has been demonstrated by Byon et al. Elevated Runx2 in calcifying SMC promoted the expression and secretion of RANKL resulting in the migration and differentiation of bone marrowderived macrophages into multinucleated tartrate-resistant acid phosphatase-positive osteoclast-like cells [101]. Recently, a novel concept emerged that circulating cells harboring osteogenic potential can home to atherosclerotic lesions and contribute to intimal [102] and aortic valve calcification [103]. A distinct subpopulation of circulating myeloid cells expressing osteocalcin and bone alkaline phosphatase (OCN(+)/BAP(+)) has procalcific activity in vitro and in vivo, especially in subjects with type 2 diabetes [104] and are correlated with the extent of necrotic core and calcification in patients with angiographically non-obstructive coronary artery disease [105]. An in vivo genetic fate mapping study revealed that marrow-derived cells from the circulation also contribute to the early osteo-chondrogenic differentiation in atherosclerotic vessels [65]. Similarly, circulating levels of osteocalcin-positive endothelial progenitor cells (CD34+/OCN+) were found to increase with progression of aortic valve stenosis, and CD34+/OCN+ cells were observed to accumulate in calcific aortic valve tissue [103]. Another circulating cell, the mast cell, has also been observed in stenotic aortic valves. Mast cell accumulation exhibits a positive correlation with the extent of valve calcification [106–108]. A direct causal role for mast cells in calcific aortic valve disease remains elusive; however, these cells may serve as

local reservoirs for transforming growth factor-␤ and angiotensin II [106,107], two factors previously associated with aortic valve remodeling. Clarification of the roles of these circulating cells in cardiovascular calcification may reveal new therapeutic targets outside of the commonly studied pathways associated with tissue resident cells. 4.4. Tissue mimics to study cardiovascular calcification Cardiovascular calcification has been observed in the vascular media layer, as in chronic renal disease, the intimal layer of atherosclerotic plaque, and in cardiac valves [64]. It is currently unclear whether or not calcification in these different locations shares common pathologic characteristics. If such is not the case, distinct models may be required for mechanistic and therapeutic studies. Having in vitro models that accurately reflect the human disease pathology(s) established through a critical assessment of the cellular components within calcification systems will help to avoid contradictory or disconnected results due to differences in experimental design. Basic cell culture models can provide valuable mechanistic information; however, the non-physiological nature of cell culture can influences cell behavior [80]. For example, VICs spontaneously differentiate into a pathologic myofibroblast-like phenotype when grow on tissue culture plastic [85], making the study of mechanisms associated with valve calcification difficult in normal culture conditions. Further, in the arterial wall, endothelial cells have been shown to affect SMC phenotype through the paracrine release of biochemical factors and microRNAs that control expression of SMC proteins [109]. These complex intercellular interactions are difficult to model in a two-dimensional culture setting. Animal models offer the ability to study diseases in a more natural three-dimensional environment; however, animals are often not amenable to understanding the complex molecular underpinnings of human disease and lack the controllability of in vitro models. To address these problems, in vitro screening platforms engineered with humanderived cells to mimic aspects of tissue can be used along with in vivo animal models and ex vivo analyses to understand the mechanisms of cardiovascular calcification [110]. A hybrid hydrogel system of methacrylated hyaluronic acid and methacrylated gelatin was recently shown to maintain VICs in a quiescent state until activated with exogenous cytokines (Fig. 6) [111]. Similarly, three-dimensional hydrogel-based models have been used to study interactions between valve endothelial cells and VICs [112],

Please cite this article in press as: Hutcheson JD, et al. Revisiting cardiovascular calcification: A multifaceted disease requiring a multidisciplinary approach. Semin Cell Dev Biol (2015), http://dx.doi.org/10.1016/j.semcdb.2015.09.004

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Fig. 6. Three-dimensional tissue mimics to study calcification. (A) VICs were embedded in a hydrogel that is polymerized by ultraviolet light to form a three-dimensional cell culture. (B) Hemotoxilin and eosin staining of a histological section of a three-dimensional hydrogel shows VICs embedded within the porous structure. (C) The optical clarity of the hydrogels allows for direct monitoring of cell processes and calcification. Figure adapted from [111].

vascular endothelial cells and SMCs [113], and SMCs and macrophages [114,115]. As use of these systems become more widespread, studies may be able to fill the remaining knowledge gaps between cell culture mechanisms and in vivo endpoints. 5. Conclusions The relationship between cellular phenotypes responsible for fibrocalcific processes within cardiovascular tissues remains poorly understood. Numerous cell types contribute to ectopic calcification at different stages of the progression. In order to develop strategies to prevent calcification before the “state of no return” we need to understand the contribution of each cell type to the calcification process. Additionally, despite many overlapping risk factors it is now postulated that vascular and valve calcification represent two distinct pathologies mediated by at least two different cell types. Future studies of valve and arterial calcification may need to recreate aspects of the in vivo situation in order to assess the importance of intercellular communication and interactions between cells and the extracellular matrix. This requires a detailed understanding of the tissue to be modeled including cell populations, extracellular matrix components, leaflet biomechanics, and true pathologic endpoints. Calcification research, therefore, requires a truly multidisciplinary approach in order to connect these various aspects of the disease. References [1] Vliegenthart R, et al. Coronary calcification improves cardiovascular risk prediction in the elderly. Circulation 2005;112(4):572–7.

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Please cite this article in press as: Hutcheson JD, et al. Revisiting cardiovascular calcification: A multifaceted disease requiring a multidisciplinary approach. Semin Cell Dev Biol (2015), http://dx.doi.org/10.1016/j.semcdb.2015.09.004