Emerging roles of fibroblasts in cardiovascular calcification

Abstract Cardiovascular calcification, a kind of ectopic mineralization in cardiovascular system, including atherosclerotic calcification, arterial medial calcification, valve calcification and the gradually recognized heart muscle calcification, is a complex pathophysiological process correlated with poor prognosis. Although several cell types such as smooth muscle cells have been proven critical in vascular calcification, the aetiology of cardiovascular calcification remains to be clarified due to the diversity of cellular origin. Fibroblasts, which possess remarkable phenotypic plasticity that allows rapid adaption to fluctuating environment cues, have been demonstrated to play important roles in calcification of vasculature, valve and heart though our knowledge of the mechanisms controlling fibroblast phenotypic switching in the calcified process is far from complete. Indeed, the lack of definitive fibroblast lineage‐tracing studies and typical expression markers of fibroblasts raise major concerns regarding the contributions of fibroblasts during all the stages of cardiovascular calcification. The goal of this review was to rigorously summarize the current knowledge regarding possible phenotypes exhibited by fibroblasts within calcified cardiovascular system and evaluate the potential therapeutic targets that may control the phenotypic transition of fibroblasts in cardiovascular calcification.

fibroblasts. All these cells can transit into osteoblast-like cells with up-regulation of osteogenic markers. [4][5][6] However, the situation is more complicated in cardiovascular calcification based on its cellular diversity.
The most common forms of cardiovascular calcification include vascular calcification, valvular calcification and myocardial calcification. Vascular calcification, with the deposition of hydroxyapatite in arterial wall, can be classified into two completely different forms: intimal calcification and medial calcification, depending on the location of calcified minerals. Valvular calcification mainly affects aortic and mitral valves, which refers to calcific aortic valve disease (CAVD) and mitral annular calcification (MAC). Last, but not the least, myocardial calcification is another form of cardiovascular calcification.
It is being recognized as a novel adverse sign of ventricular remodelling. Myocardial calcification, mostly accompanied by fibrosis, can interrupt the electrical propagation of the conduction system. It leads to arrhythmia and cardiac dysfunction. 7 Therefore, cardiovascular calcification is a serious condition affecting major components of cardiovascular system. However, the aetiology of cardiovascular calcification is still unclarified.

| THE MAIN SOURCE S OF C ALCIFIC ATION
Ectopic calcification, which refers to the mineralization of extracellular matrix (ECM) occurred in areas other than skeleton and teeth, is also named as soft-tissue calcification. It could be classified as (a) metastatic calcification, which arises in accompany with up-regulated serum calcium or phosphate; (b) dystrophic calcification, which mainly exists in chronically damaged tissues with normal serum calcium/phosphate levels; and (c) tumoral calcinosis (TC), an uncommon clinicopathological condition, which is characterized by the deposition of calcified masses in juxta-articular and a normal calcium/phosphate level in serum. 8,9 Smooth muscle cells and fibroblasts are the main players in ectopic calcification. They mediate the abnormal mineral deposition through a common process, which is an imbalance between pro-calcification stimuli and calcification inhibitors. Hydroxyapatite is the main crystal phase presented in ectopic mineralized ECM, which is predominantly constituted by calcium (Ca) and inorganic phosphate (Pi) ions. Pi plays a critical role in triggering ectopic ECM mineralization. However, simply elevating the concentration of phosphate is insufficient to induce ECM mineralization because of the existence of inorganic pyrophosphate (PPi). PPi is a strong inhibitor of ectopic mineralization which could directly inhibit the aggregation of hydroxyapatite and crystal growth. 10,11 Thus, the dynamic alterations of Pi/PPi ratio is critical in regulating the ectopic mineralization process. Most anti-mineralization and pro-mineralization factors function by altering the ratio of Pi/PPi, such as alkaline phosphatase (ALP), matrix Gla protein (MGP), ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) and pyrophosphate analogues. 12-15 ALP promotes matrix mineralization by degrading inorganic pyrophosphate and producing mineral constituents of hydroxyapatite-Pi. ENPP1 inhibits ectopic mineralization by increasing PPi supplementation. Bisphosphonate can be used as an anti-mineralization agent by mimicking PPi. 15 On the contrary, in skeleton and teeth, PPi promotes mineralization cause it can be broken down by tissue non-specific alkaline phosphatase (TNAP) to generate Pi. 14 There still exist other factors that can inhibit matrix mineralization without directly interfering the ratio of Pi/PPi, for instance fetuin-A. Fetuin-A inhibits de novo formation of hydroxyapatite crystals by forming transient-soluble, colloidal spheres (containing Ahsg, calcium, phosphate) and delaying their precipitation. 16,17 Osteopontin inhibits mineralization through directly binding to crystal surfaces and inhibiting any further propagation. 13,18 These pathways provide a strong possibility to better understand the calcification process.

| Roles of vascular smooth muscle cells in vascular calcification
Vascular calcification, with the deposition of hydroxyapatite in arterial wall, can be classified into two completely different forms depending on the location of minerals, including intimal calcification and medial calcification. Vascular calcification is strongly associated with acute cardiovascular and cerebrovascular events, especially combined with diabetes and chronic kidney disease. 19,20 It is closely correlated with increased all-cause and cardiovascular morbidity and mortality. 21 The walls of large and moderate arteries have three distinct layers: the tunica intima, tunica media and tunica adventitia. The intimal layer is majorly composed of endothelial cells. The media, also named the smooth muscle layer, is composed of concentric rings of smooth muscle cells and collagen, elastin fibres. The adventitia, the most complicated layer of vessel walls, is composed of fibroblasts, vascular progenitor cells and immune cells. 22,23 Intimal calcification is associated with the forming of atherosclerotic plaque. It frequently occurs in the coronary and carotid arteries, exacerbating arterial stenosis or obstruction. Coronary artery calcification is an important part of intimal calcification, which can be evaluated by overall coronary artery calcium score using non-invasive imaging. Coronary calcium score is a critical index to identify high-risk atherosclerotic CVD patients. 24,25 Medial calcification can lead to vessel stiffening and decreased compliance, occurring in extremity arteries such as tibial and femoral, causing insufficient blood supply.
Osteogenic differentiation of VSMCs is likely a common feature of intimal and medial calcification. However, stimuli that induce this process are different, and intimal calcification is often induced by disrupted lipid homeostasis, oxidative stress and inflammation. 26 Medial calcification is usually accompanied by ageing, diabetes and renal disease. 27 For vasculature, it has gained acceptance that VSMCs undergo 'de-differentiation into a synthetic phenotype and further step into osteogenic fate' during vascular calcification. The phenotypic transition of VSMCs is accompanied by the increased expression of bone markers, such as ALP, collagen 1, runt-related transcription factor-2 (Runx2), osteopontin, osteocalcin and reduced expression of VSMCs markers including SM22α, smooth muscle α-actin and smooth muscle cell myosin heavy chain. 28 The identical roles of VSMCs in intimal and medial calcification has been illustrated by others. 29 Many factors can inhibit vascular calcification by re-balancing calcium-phosphate metabolism or inhibiting the formation and propagation of hydroxyapatite crystals, such as MGP, osteopontin, fetuin-A, ENPP1 and pyrophosphate analogues. [12][13][14][15]17

| Roles of fibroblasts in ectopic calcification
Ectopic soft-tissue calcification is also a characteristic sign of many diseases, such as Werner's syndrome (WS) and pseudoxanthoma elasticum (PXE). Werner's syndrome, a rare autosomal recessive disorder, caused by mutations in the WRN gene, is often accompanied by extensive subcutaneous calcification, 30 whereas pseudoxanthoma elasticum is caused by the ABCC6 gene mutation and characterized as progressive calcification of elastic fibres in skin, eyes and the cardiovascular system. 31 Besides, gammaglutamyl carboxylase (GGCX) syndrome, caused by GGCX gene mutation, also possess PXE-like symptoms, with calcium deposits in vessel walls and elastic fibres. 32 In the occurrence of dermal ectopic calcification, fibroblasts are concerned as principal candidates. 33 Dermal fibroblasts treated with pro-calcifying medium for 3 weeks exhibited evident calcium deposits. 34

| EMERG ING ROLE S OF FIB ROB L A S TS IN VA SCUL AR C ALCIFIC ATION
Vascular calcification is practically in parallel with the progression of atherosclerosis. Calcified lesions in atherosclerotic plaques most likely appear at the late stage of atherosclerosis. According to the diameter of calcified particles, calcified lesions are divided into macroscopic calcification and microcalcification. Macroscopic calcification, which is defined as calcified particles >50 µm in diameter, 44 makes plaque more stable. μCalcs (microcalcification) seems to be harmful when it is embedded in the fibrous cap, which can increase the vulnerability of plaque by increasing local stress over 2-5 times. 45,46 Once the local stress grows to over 300 kPa, plaque rup- plaque areas in rabbit models of abdominal aorta balloon injury, implying a critical role of adventitia in atherosclerosis. 48 As the main cells in adventitia, adventitial fibroblasts (AFs) play an important role in the pathological progression of atherosclerosis. In response to many cytokines and growth factors (ie ROS, transforming growth factor -beta1 (TGF-β1), tumour necrosis factor-α (TNF-α), fibroblast growth factor-2 (FGF-2), osteocalcin), [49][50][51][52] AFs can trans-differentiate into myofibroblasts and migrate towards the lumen, contributing to the neointima formation in injury-induced and graft-induced atherosclerosis. 53 Besides, Karamariti et al revealed that the transplanted AFs can migrate to the intima and trans-differentiate into smooth muscle cells in mouse wire-induced femoral artery injury model. 54 In atherosclerotic lesions, fibroblasts can be derived from endothelial cells through endothelial-to-mesenchymal transition (EndMT). By using lineage-tracking system, Evrard et al revealed that EndMT is common in atherosclerotic lesions. Higher incidence of EndMT transition is closely related to a higher risk of plaque rupture in human. 55 The vasa vasorum (VV) provides nutrients and oxygen to adventitia and the outer part of media of large arteries, and removes waste products. AFs might play a critical role in regulating the expansion of vasa vasorum in atherosclerotic disease. The potential mechanism might be associated with AF-secreted pro-inflammatory cytokines and pro-angiogenic growth factors, including interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). 56,57 Li et al reported that AF-derived VEGF plays a pivotal role in the increase of VV count. 58 Studies have discovered that the neovessels of VV contribute to plaque progression by delivering inflammatory cells to the plaques, and the leakage of immature VV is responsible for intraplaque haemorrhage. A recent study suggested that lysed erythrocytes promoted the osteoblastic differentiation of VSMCs in a NO-dependent manner. 59,60 Furthermore, the early calcified human atherosclerotic plaques contained large amounts of platelets due to plaque neovascularization by leaky vessels, blood extravasation and haemorrhage.
In patients with carotid artery stenosis, levels of osteocalcin content in circulating platelets and total osteocalcin release after activation were significantly increased. Furthermore, the overexpression of osteocalcin dramatically accelerated the mineralization of vascular  commonly believed that MAC is another form of atherosclerosis. 76,77 MAC is also discovered to increase the incidence of arrhythmias in similar to myocardial calcification. 78 In patients with severe myocardial infarction, mitral valve thickness is always witnessed, leading to mitral valve regurgitation, a severe complication. 79 Besides, multiethnic cohort studies revealed that MAC is robustly associated with CVD. 80,81 The initiating process of CAVD involves leaflets' fibrotic changes, and granules of mineralization occur mainly in the fibrotic region of leaflets. 82 According to the diverse morphologies, valve cells can be divided into several cell populations. Valve interstitial cell is recognized as the major cellular origin of calcific valve, which is capable of undergoing osteogenic differentiation in response to the microenvironmental and mechanical cues. 83 However, recent study showed that valve-derived stromal cells (VDSCs), as uniform spindle-shaped fibroblasts, exhibiting potent proliferating ability and expressing both mesenchymal and osteogenic markers, may potently contribute to valve calcification. 84 In addition, valve ECs also showed the potential of osteogenic differentiation via EndMT process. 85,86

| FIB ROB L A S TS IN MYO C ARD IAL C ALCIFIC ATION
Pathological cardiac calcification is often observed in the aged and in patients with end-stage renal disease who have disturbed calciumphosphate metabolism or for unknown reasons. 87,88 With calcium deposited in the myocardium, heart can be the only organ with significant calcium deposition. The deposition of calcium in myocardium is often associated with poor outcome, such as arrhythmia or sudden cardiac death. 87 Cardiac calcification has been proved as a predictor of poor outcome following myocarditis or myocardial infarction. 87,89 However, in myocardial calcification, few is known regarding the identification of cellular source. 90 Heart is a highly organized structure that contains several types of cells. Adult mammalian heart is mainly composed of cardiomyocytes, fibroblasts, endothelial cells (ECs) and leucocytes. Fibroblasts constitute 20-30% of total non-cardiomyocytes in the heart. 91 They form the cardiac scaffold and play a critical role in heart development by producing extracellular matrix and expressing various cytokines. 92 In response to pressure overload or ischaemic injury, the heart undergoes a dynamic remodelling process, producing a multitude of ECM proteins, such as collagens and fibronectin. The deposition of osterix, bone sialoprotein and osteopontin). In vivo, three murine models of C3H strain (high-dose steroids, cryo-injury and ischaemic injury) were created. Murine hearts exhibited calcium hydroxyapatite deposition in injured regions where there was fibrosis. In border region, genetically labelled CFs expressed osteoblast markers and Runx2, the master osteogenic transcription factor. This report suggests that cardiac fibroblasts possess a high degree of plasticity and can adopt an osteogenic phenotype. Notably, some strains of mice like B6 strains failed to exhibit hydroxyapatite deposition post-heart injury. In the meantime, few osteogenic markers were witnessed in labelled CFs of those strains, suggesting that osteogenic fate of CFs is thick with mouse strains.
Researchers also found the expression of ENPP1 was up-regulated after injury, and inhibition of ENPP1 with small molecules could dramatically decrease ectopic cardiac calcification and preserve cardiac function. This seems to be contrary to the previous opinion that This review, for the first time, systemically summarizes the emerging roles of fibroblasts in cardiovascular calcification and discusses the potential therapeutics targeting fibroblasts in cardiovascular calcification. It is noteworthy that the lack of specific markers to identify fibroblasts makes it difficult to explore the precise features of fibroblasts. The markers identifying fibroblasts, such as fibroblast-specific protein-1 (FSP-1), collagen 1a1 and transcription factor 21 (TCF21), are not uniformly specific. They are also expressed on VSMCs, ECs and immune cells. 100 Periostin, a newly recognized marker of myofibroblasts, is expressed in adult heart tissues only after injury. Further study identified that periostin + myofibroblasts in heart are originated from TCF21 + tissue-resident fibroblasts. 97 Since the complex of fibroblast plasticity, more specific markers need to be explored, a combination of two or more fibrotic markers is recommended to label fibroblasts in tissues. Future studies will further our knowledge of fibroblasts in different diseases.

ACK N OWLED G EM ENT
This study was supported by the National Natural Science Foundation of China (No. 81670259 and No. 81870203 to Meixiang Xiang).

CO N FLI C T S O F I NTE R E S T
The authors declared no conflict of interest.