The role of bone morphogenetic protein signaling in vascular calcification

Abstract

Vascular calcification is associated with atherosclerosis, chronic kidney disease, and diabetes, and results from processes resembling endochondral or intramembranous ossification, or from processes that are distinct from ossification. Bone morphogenetic proteins (BMP), as well as other ligands, receptors, and regulators of the transforming growth factor beta (TGFβ) family regulate vascular and valvular calcification by modulating the phenotypic plasticity of multipotent progenitor lineages associated with the vasculature or valves. While osteogenic ligands BMP2 and BMP4 appear to be both markers and drivers of vascular calcification, particularly in atherosclerosis, BMP7 may serve to protect against calcification in chronic kidney disease. BMP signaling regulators such as matrix Gla protein and BMP‐binding endothelial regulator protein (BMPER) play protective roles in vascular calcification. The effects of BMP signaling molecules in vascular calcification are context-dependent, tissue-dependent, and cell-type specific. Here we review the current knowledge on mechanisms by which BMP signaling regulates vascular calcification and the potential therapeutic implications.

Introduction

Vascular calcification is a pathological process that is broadly defined as the deposition of mineralized calcium in the form of hydroxyapatite in the vascular wall and can be divided into different types based on the mechanism of formation and location. The first type of vascular calcification arises from a process that resembles endochondral ossification with its characteristic osteogenic and chondrogenic cell populations, and the progressive mineralization of a cartilaginous matrix precursor or anlage. This type of vascular calcification occurs in atherosclerosis, targets primarily the intimal layer of the arteries, affects the stability or propensity of atheromatous plaques to rupture, and its presence is a strong predictor of risk for cardiovascular events [1]. The second type of calcification resembles intramembranous ossification, arising from the mineralizing activity of osteogenic cells without a chondrogenic anlage. This process is more common in patients with chronic kidney disease (CKD) and diabetes, forms along the medial layer of the arteries to cause stiffening and loss of compliance of the arterial wall leading to further increased blood pressure [1]. The third type of vascular calcification, calciphylaxis, results from a process distinct from ossification. Calciphylaxis most commonly occurs in patients with renal failure with dysregulated levels of calcium and phosphate, and manifests commonly as calcium deposits in the cutaneous arterioles [2]. In this review, we will focus on intimal and medial vascular calcification, and the role of bone morphogenetic protein (BMP) signaling in their pathogenesis. We will also discuss the related entity of calcific valvular disease, manifesting as ossification of cardiac valves leading to alterations of their mechanical properties, valvular stenosis and/or insufficiency, and heart failure [3]. BMPs, originally identified for their roles in promoting heterotopic bone formation[4, 5], were found to be expressed abundantly at sites of atheromatous vascular calcification[6]. The BMP ligand family has subsequently been found to play critical roles in the development and homeostasis of the vascular system[7], and are now recognized to include critical contributions to the pathophysiology of atherosclerosis, pulmonary vascular disease, and vascular and valvular calcification[8].

BMP and TGFβ Superfamily Signaling in Vascular Biology

BMPs are a group of ligand proteins with pleiotropic functions that extend well beyond promoting bone formation. They belong to a larger family of over thirty ligand molecules that interact with specific receptors, co-receptors and signaling modulating proteins known collectively as the transforming growth factor beta (TGFβ) signaling family. The TGFβ family of ligands consists of subfamilies including the BMPs, growth differentiation factors (GDFs), activins and inhibins, nodal, left and right determination factors, and others, named in part for their structural and functional similarities and the contexts in which they were discovered [9]. Many of the BMPs and other TGFβ ligands have been implicated in the homeostasis and pathophysiology of the cardiovascular system [7, 10, 11]. Within the vasculature, BMPs 4, 6, 9, and 10 are found in circulation and can activate receptors on vascular cells [12–16]. TGFβ is secreted in a latent form [17], while other ligands such as GDF8, GDF11, GDF15 are expressed in the circulation [18, 19]. Like other TGFβ family ligands, BMP ligand dimers engage signaling by promoting the assembly of heteromeric complexes of BMP type I and II receptors on the cell surface. Type I receptors are named activin receptor-like kinases (ALKs) 1–7, while type II receptors include BMPR2, ACVR2A, ACVR2B, TGFBR2, and AMHR2. The classic BMP ligands BMP2 and BMP4 preferentially bind ALK3 and 6 in complex with BMPR2, ACVR2A or ACVR2B, BMP6 and 7 preferentially bind ALK2 and ACVR2A, while BMP9 and 10 preferentially bind ALK1 in complex with BMPR2 or ACVR2B, but can also bind ALK2 [20, 21]. TGFβ1 signals in most cells via ALK5 and TGFBR2, whereas activins and GDFs bind ACVR2A/B in conjunction with ALKs 4, 5 or 7 [9]. The existence of heterodimeric ligands and receptor complexes with heterogenous type I receptors, as well as the spatiotemporal expression of the ligands and receptors, enable further fine-tuning of the signaling activity [10, 22]. For example, ALK2 and 3 are widely expressed, while ALK1 is more selectively expressed in endothelial cells (EC). BMPR2 and ACVR2A are also widespread, whereas ACVR2B is restricted to certain cell types including EC [23]. Upon ligand binding, the type II receptor phosphorylates and activates the type I receptor, which in turn phosphorylates and activates R-SMADs. R-SMADs form complexes with common SMAD4 (Co-SMAD4) to regulate transcription in the nucleus. Most BMPs recruit SMADs 1/5/9, while TGFβ and activins recruit SMADs 2/3. However, certain ligands such as BMP9 may activate SMAD1/5/9 and SMAD2/3 in EC [24]. In addition to this SMAD binding affinity, BMP receptors can activate non-SMAD pathways such as mitogen activated protein kinases [25]. BMP signaling can be terminated by inhibitory SMADs including SMAD6 and SMAD7, which are activated and induced by BMP signaling and switch off BMP signaling via multiple mechanisms. For example, inhibitory SMADs can recruit SMURF1 and SMURF2, E3 ubiquitin ligases that degrade the activated receptors [26]. In addition, BMP signaling is regulated by different types of co-receptors or type III receptors such as Betaglycan and Endoglin. Endoglin is highly expressed in cardiovascular tissues and participates in the receptor binding of BMP9, but can also be shed from the cell surface to function as a BMP ligand trap in circulation [27, 28]. The kinase-deficient decoy receptor BMP and activin membrane-bound inhibitor homolog (BAMBI) also sequesters ligands from type I receptors [29]. BMP-binding endothelial regulator protein (BMPER) has been reported to inhibit BMP9 signaling in endothelium [30]. BMP signaling is also regulated by antagonists, such as Gremlin and Noggin, which bind to and inhibit BMP2 and BMP4 [31, 32]. Similarly, matrix Gla protein (MGP), a secreted carboxyglutamic acid modified protein, can bind and inhibit BMP2 and BMP4 signaling [33]. The structurally diverse set of ligands, receptors and regulatory proteins provide strict regulation and specificity to BMP/TGFβ signaling by virtue of their distinct spatiotemporal expression. The need for regulation of this pathway is evident in observations from human genetics, human tissues, and disease models that show defects in BMP signaling can lead to a range of detrimental conditions.

Contributions of BMP/TGFβ signaling in congenital syndromes and animal models

Identification of mutations in genes encoding components of the BMP/TGFβ pathway in heritable human diseases and generation of animal models have provided valuable insight into the functions of BMP/TGFβ signaling. Mouse models lacking various members of this signaling pathway support critical roles in cardiovascular development (reviewed in [7, 34, 35]). Homozygous deficiency of either Eng (encoding Endoglin), Acvrl1 (ALK1) or Smad5 are embryonically lethal in mice due to cardiovascular system defects [36]. Heterozygous loss-of-function mutations in ENG or ACVRL1 in humans are associated with hereditary hemorrhagic telangiectasia (HHT) syndromes, vascular disorders characterized by arteriovenous malformations (AVMs) in the lungs, liver or brain, and hemorrhages associated with these vascular lesions [37]. HHT1 and HHT2 syndromes are caused by mutations in ENG and ACVRL1 respectively [38, 39], whereas SMAD4 mutations are reported in juvenile polyposis-HHT [40] and GDF2 (encoding BMP9, the canonical ligand for ENG and ACVRL1) mutations have been recently found in HHT5 [41, 42]. As expected, mice with heterozygous mutations in Eng or Acvrl1 develop vascular abnormalities that are reminiscent of human HHT [43–45]. AVMs are also found in Bmp10 [46] and Mgp knockout mice [47]. Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder where obliteration of the vascular lumen small pulmonary arterioles increases pulmonary vascular resistance and causes right ventricular hypertrophy and eventually failure. Mutations in BMP/TGFβ pathway genes are found in the heritable forms of PAH with reduced penetrance. BMPR2 is the most frequently mutated gene in PAH, followed by other genes such as GDF2, ENG, ACVRL1, and SMAD9 [48, 49]. Interestingly, heterozygous Bmpr2-deficient mice or mice expressing loss-of-function Bmpr2 mutations show only minimal signs of PAH without additional stimuli [50, 51]. Loeys-Dietz syndrome is characterized by cardiovascular manifestations including an aortic aneurysm caused by inactivating mutations in ALK5 or TGFBR2 [52], TGFβ ligands [53, 54] or SMAD3 [55]. Taken together, compelling evidence from multiple human disorders and animal studies highlight an important role of the BMP/TGFβ pathway in the cardiovascular system. On the other end of the spectrum, gain-of-function mutations in ACVR1 (encodes the ALK2 receptor), causes fibrodysplasia ossificans progressiva (FOP), which is a rare but disabling disorder characterized by progressive heterotopic ossification of muscle, fascia, ligaments, and tendons [56]. This mutation results in a single amino acid substitution that enables activins to induce BMP SMADs (SMAD1/5) thereby driving progenitor cells into chondrogenic and osteogenic lineages [57–60]. Genetically modified mice with activating mutations Acvr1 expressed globally or in selectively in soft tissue-resident progenitor populations such as fibroadipogenic progenitor (FAP) cells recapitulate this heterotopic ossification phenotype in skeletal muscle, ligaments, and tendons [57–59, 61]. Arguably certain forms of vascular and valvular calcification are cardiovascular manifestations of heterotopic ossification, mediated by dysregulated BMP/TGFβ signaling promoting the abnormal differentiation of vessel-resident progenitor cells into cartilage- and bone-like tissues.

Physiologic BMP/TGFβ signaling in vascular and valvular homeostasis

The patterning and formation of the vascular system is a critical process of embryonic development that is closely coupled to organogenesis. Vascularization can result from two mechanisms: the de novo formation of vessels, called vasculogenesis, and the development and progression of new vessels from pre‐existing ones, known as angiogenesis. Vasculogenesis occurs primarily during embryonic development and is almost absent during adulthood except in wound healing, inflammation, and the female reproductive cycle. In healthy tissues, blood vessels are formed from a combination of mechanisms that entail sprouting angiogenesis, differentiation of endothelial progenitor cells (EPCs), and vessel splitting [62]. The coordinated activity of ECs, smooth muscle cells (SMCs) and pericytes are required for this process. The earliest events in angiogenesis involve the selection of a leading migrating tip EC that invades the surrounding tissue by extending numerous filopodia. VEGF/VEGFR2 signaling triggers single EC to switch into a tip cell phenotype; these cells express Notch ligand Delta‐like 4 (Dll4), instructs neighbor ECs to become stalk cells [63]. Stalk cells trail behind the tip cells, proliferate, and form tubes; stalk cell proliferation ensures elongation of sprouting vessel [64]. Ultimately, ECs stop proliferating, acquire a quiescent phenotype, and become phalanx ECs. Finally, the newly formed vessel is stabilized by the deposition of the basement membrane and the recruitment of pericytes and SMCs [65].

The role of BMP signaling in vascular development has been illustrated by studies in knockout animal models [36]. It has been reported that BMP‐2, ‐4, ‐6 and ‐7 induce angiogenesis, EC proliferation, and migration [66]. Capillary tube formation is increased upon activation of the BMP signaling pathway by overexpression of BMPs or ID1 [67]. In contrast, BMP9 inhibits basic fibroblast growth factor (bFGF)‐stimulated proliferation and migration of bovine aortic endothelial cells (BAECs) and blocks VEGF‐induced angiogenesis [68]. BMP9 has also been reported to inhibit the migration and growth of human dermal microvascular ECs [69]. Although (high dose) BMP‐9 seems to have inhibitory effects on ECs, another report demonstrated that (low dose) BMP‐9 induces proliferation of various types of ECs in vitro and promoted angiogenesis in matrigel plug assays and human pancreatic cancer xenografts in vivo [70]

While many studies have focused on the role of ECs in the development of new vessels, it has been shown that SMCs contribute to the development of the newly formed vessels as well as in vascular disease, via the impact of BMPs on vascular SMC proliferation and differentiation [71]. BMP7 inhibits primary human aortic SMC proliferation due to stimulation with serum, platelet‐derived growth factor subunit BB (PDGF‐BB) or TGFβ1, and maintains the expression of the vascular SMC phenotype. BMP7 appears to exhibit anti‐inflammatory effects on the vasculature, and may function to maintain vascular integrity [72]. In contrast, BMP4 is expressed by ECs in response to hypoxia and promotes vascular SMC proliferation [73]. It has been demonstrated that vascular SMCs isolated from different parts of the pulmonary vasculature have different proliferation responses to BMP4, inhibiting the proliferation of SMCs isolated from the proximal pulmonary artery while inducing proliferation of SMCs isolated from distal pulmonary arteries [74]. Thus as the case with ECs, the impact of BMPs on vascular SMCs depends on the tissue and microenvironmental context.

Atherosclerosis and vascular calcification

Atherosclerosis is characterized by the accumulation of lipid- and cholesterol-laden atheromatous plaques in the intimal layer of arteries. Low-density lipoprotein (LDL) particles deposit underneath the endothelium and become oxidized, while inflammatory cells including monocytes invade and differentiate into tissue macrophages which ingest LDL to yield macrophage-derived foam cells, resulting in the formation of fatty streaks. Inflammatory changes in the intima trigger proliferation and migration of smooth muscle cells into the intima to cause fibrous remodeling of the plaque. The formation and growth of these lesions can lead to progressive narrowing of vessels, and are at risk of acute plaque rupture and subsequent thrombosis [75]. While various inflammatory signals have been implicated in the formation of these lesions and their remodeling and stability, including [76], BMPs and other members of the TFGβ family modulate and interact with these inflammatory responses, recruit osteoblast-like and chondroblast-like cells to promote calcific remodeling and endochondral-like ossification (Figure 1). BMP and TFGβ signaling pathways may act in multiple different cell types including ECs, SMCs, myofibroblasts, dendritic cells, T cells, monocytes and macrophages [10]. These ligands and the likely receptors expressed in various target cell types are summarized in Table 1. While the relative affinity of various ligands for specific receptors, and the tissue specific expression of various receptors may be characterized, the precise identities of the critical ligand-receptor pairs for a given tissue and function have not been fully characterized in cases save for some exceptional genetic epistasis experiments. BMP2, BMP4, and osteoblast and osteoclast regulatory proteins osteopontin (OPN), osteonectin, osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-Β ligand (RANKL) were found to be differentially expressed in calcified atherosclerotic plaques [6, 77, 78], leading to the proposal that BMPs serve as the link between atherosclerotic vascular calcification with mechanisms of normal bone formation. In a microarray analysis of calcified carotid plaques from 52 patients, there was a strong association between the expression of BMP2 and BMP4 in plaques and the presence of unstable plaques [79]. Subsequent studies have implicated a causal role of BMP signaling in atherosclerosis, plaque instability, and vascular calcification [80–83]. In particular BMP2, BMP4 and BMP6 have been linked to increased plaque formation via their pro-inflammatory and pro-atherogenic effects, promoting oxidative stress, endothelial dysfunction and osteogenic differentiation [6, 84, 85].

Fig. 1.

A schematic representation of the cellular sources and targets of bone morphogenetic protein (BMP) and transforming growth factor beta (TGF-beta) signaling in the vascular wall potentially contributing to vascular calcification. Arrows represent positive regulators; Diamonds represent negative regulators.

Table 1.

A summary of the putative BMP and TGF-beta ligands, their cognate receptors, and cellular and biological targets implicated in vascular calcification associated with atherosclerosis, chronic kidney disease, diabetes, and valvular calcification, and examples of therapeutic strategies.

Calcification Type	Ligand	Likely Receptor (Type I)	Likely Receptor (Type 2)	Cell Types	Biological Effects	Potential Intervention
Atherosclerosis	BMP21−5	ALK2/ALK3/ALK6	BMPR2/ACVR2A	SMC	Osteogenic and chondrogenic differentiation	Small Molecule Inhibitors, i.e., LDN-193189, and Ligand Traps (ALK3-Fc)
				Pericytes
				Myofibroblast
				Monocytes	Infiltration and inflammation
	BMP42, 5	ALK2/ALK3/ALK6	BMPR2/ACVR2A	SMC	Osteogenic and chondrogenic Differentiation
				Monocytes	Infiltration and inflammation
	BMP66−8	ALK2	ACVR2A	EC, SMC	Osteogenic differentiation
	BMP79, 10	ALK2	ACVR2A/BMPR2	SMC	Protects against calcification
	BMP96	ALK1/2	BMPR2/ACVR2B	EC	EndMT Trans differentiation Osteogenic and chondrogenic differentiation
	TGFβ111	ALK5	TGFBR2	EC		Ligand Trap, e.g., TGFBRII-Fc
Chronic Kidney Disease	BMP23, 5, 12, 13	ALK2/ALK3/ALK6	BMPR2/ACVR2A	SMC	Osteogenic differentiation	Recombinant BMP7
	BMP45	ALK2/ALK3/ALK6	BMPR2/ACVR2A
	BMP710	ALK2	ACVR2A/BMPR2	SMC	Protects against calcification
	BMP914	ALK1/ALK2	BMPR2/ACVR2B	SMC	Osteogenic differentiation
	Activin/GDF	ALK2	ACVR2A	SMC	Osteogenic differentiation
Diabetes	BMP215	ALK2/ALK3/ALK6	BMPR2/ACVR2A	Myofibroblast	Osteogenic and chondrogenic differentiation	Ligand Trap, e.g., ALK3-Fc
	BMP415	ALK2/ALK3/ALK6	BMPR2/ACVR2A	EC
Valvular Calcification	BMP216, 17	ALK2/ALK3/ALK6	BMPR2/ACVR2A	AVICs	Osteogenic and chondrogenic Differentiation	Ligand Trap, e.g., ALK3-Fc
	BMP416, 18	ALK2/ALK3/ALK6	BMPR2/ACVR2A
	BMP916	ALK1/ALK2	BMPR2/ACVR2B
	TGFβ118−22	ALK5	TGFBR2	AVICs		Ligand Trap, e.g., TGFBRII-Fc

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While the lesions of atherosclerotic vascular calcification have histologic features that are reminiscent of orthotopic bone formation [8], the source of progenitor cells that provide the osteoblast-like and chondroblast-like cells in calcific atherosclerotic lesions is not fully established [86]. It has been proposed that pericytes, bone marrow-derived or circulating MSC, or tissue-resident MSC in the intima, media, or adventitia, or transdifferentiated VSMC may contribute to osteoblast‐like cells in these lesions, in part based on in vitro studies demonstrating the multi-potentiality of these various populations when isolated and cultured [87]. BMP signaling promotes the expression of osteoblast lineage markers including alkaline phosphatase in cultured vascular SMCs [88, 89]. BMP2 enhances the expression of the osteoblast and chondrocyte master transcriptional regulator RUNX2 to promote the mineralization of cultured human coronary vascular SMCs in a manner that was dependent on oxidative stress and endoplasmic reticulum (ER) stress [90]. Vascular smooth muscle cells (VSMCs) may also express BMP2 and BMP4 to promote monocyte infiltration and inflammation of atherosclerotic legions in a BMPR2-dependent manner [81]. BMP2 was further implicated based on the upregulation of BMP2 expression, and a downregulation of BMP2 agonists in dedifferentiated human VSMCs, potentially exerting paracrine pro-inflammatory changes in associated endothelium [91, 92]. BMPs can direct osteogenic reprogramming of vascular mesenchymal progenitors of the pericyte lineage [6]. In response to BMP2, cultured aortic myofibroblasts will co-express smooth muscle markers with osteoblast homeoprotein Msx2, suggesting that trans-differentiation of myofibroblasts into the osteogenic lineage may contribute to vascular calcification [93].

It has been suggested that vascular EC may contribute to osteogenic differentiation [94], via a process of endothelial-to-mesenchymal transition (EndMT) based on the capacity of cultured EC to co-express pluripotency and osteogenic genes in vitro after stimulation with various stimuli including BMP and TGFβ ligands, and the co-expression of endothelial and osteogenic lineage markers, and markers of pluripotency in a portion of cells which contribute to the anlage of heterotopic bone and human calcific atherosclerotic lesions [95–97]. Building on the observation that endothelial primary cilia are rarefied in calcific vascular lesions, vascular EC obtained from cilia-deficient mice were found to be sensitized to BMP-induced osteogenic differentiation in vitro, by a process that requires the β-catenin-regulated transcription factor Slug [98]. Inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) enhance BMP9-induced EndMT via a mechanism requiring downregulation of BMPR2 [99]. Further supporting a link between inflammation and BMP signaling, treatment of human coronary artery EC with oxidized low density lipoprotein (oxLDL) induces BMP2 expression via Toll-like receptors 2 and 4 [100]. BMP6 and oxidized low-density lipoprotein (oxLDL) independently and synergistically induced osteogenic differentiation and mineralization of endothelial cells, in a process that involved transcriptional upregulation of Runx2 and Msx2, and required oxidative stress [85]. In contrast, high density lipoprotein (HDL) enhances ALK1 and ALK2 expression in aortic endothelial cells, resulting in an induction of MGP, which appeared to be protective against vascular calcification [101].

The capacity of mature EC lineages to differentiate into true bone-forming cells in vivo remains a subject of debate, as early evidence for this phenomenon was based on lineage-tracing studies using Tie2-Cre [94, 102], which was found to mark in additional to endothelial cells a variety of MSC populations including fibroadipogenic progenitors that could reliably contribute to osteogenic differentiation in various heterotopic ossification models [103]: In models of the congenital heterotopic ossification syndrome fibrodysplasia ossificans progressiva (FOP), the contribution of mature EC populations to osteocytes in heterotopic bone marked when targeted by markers such as VE-Cadherin-Cre (using the Cadh5 promoter) appears to be negligible [58, 59, 103, 104].

In support of a causal role of BMP ligands in promoting vascular calcification, expression of a BMP2 transgene targeted to vascular smooth muscle accelerated aortic intimal calcification in Apolipoprotein E (ApoE)-knockout mice, which develop severe atherosclerotic disease on a high fat diet [91]. The calcified lesions in such mice were characterized by the presence of osteoblast-like cells in the intima. Conversely, inhibiting BMP signaling pathway via expression of an MGP transgene resulted in reduced vascular calcification formation in Apo-E knockout mice, whereas MGP-knockout mice exhibit spontaneous vascular calcification on a wild-type background [80, 82, 91]. Humans with homozygous loss-of-function mutations in MGP develop Keutel’s syndrome, a congenital disorder of excessive calcification of cartilaginous tissues [105]. Consistent with these in vivo observations, overexpression of MGP in multipotent mesenchymal C3H10T1/2 cells inhibited BMP2-induced osteogenic and chondrogenic differentiation, whereas deficiency of MGP enhanced these processes [106]. Similarly, depletion of MGP sensitizes endothelial cells to osteogenic differentiation in response to BMP ligands and high glucose [107]. Blockade of BMP type I receptor kinase signaling using the dorsomorphin derivative small molecule inhibitor LDN‐193189[61, 108] or sequestration of osteogenic ligands via the BMP2/4 ligand trap ALK3‐Fc in LDL receptor deficient LDLR^−/− mice inhibited high‐fat diet‐induced vascular inflammation as well as vessel wall-associated osteogenic activity and vascular calcification, confirming the role of BMP signaling in the pathophysiology of both atherogenesis and calcification, while suggesting pharmacologic BMP inhibition as a potential treatment for vascular calcification [83]. Similarly, treatment with dorsomorphin analog DMH1 reduced medial artery calcification by blocking SMAD1/5/8 phosphorylation, expression of osteoblast markers and alkaline phosphatase, and calcium accumulation in vascular SMCs [109]. Treatment of MGP-knockout mice with LDN-193189 or ALK3-Fc also inhibited aortic calcification, preventing the expression of markers of EndMT in vessels of those mice while prolonging their survival [82]. The use of complementary pharmacologic and genetic approaches are critical for demonstrating the roles of BMP signaling in vascular calcification; while MGP demonstrates antagonism of BMP signaling [80, 82, 110], it should be noted that MGP can inhibit calcification in a BMP2-independent manner in certain contexts by direct inhibition of hydroxyapatite deposition [111].

Other BMP receptors, co-receptors, and antagonist molecules may regulate vascular calcification. Expression of BMPR2, the receptor for classic osteogenic ligands BMP2 and BMP4, is decreased in patients with advanced coronary atherosclerosis and is protective against atherosclerosis in animal models [112], suggesting a context-specific role of BMPR2-mediated signaling. Endoglin is known to be overexpressed in SMCs in human atherosclerotic plaques but its functional significance remains unclear [113, 114]. Expression of BMP regulatory molecule BMPER is localized to neointimal and medial SMCs in calcified atherosclerotic plaques [115]. In support of a protective role, compound Bmper haploinsufficient, ApoE knockout mice exhibit more severe atherosclerosis and increased aortic endothelial BMP signaling activity as compared to ApoE knockout controls [116]. Silencing of BMPER expression in SMCs promotes osteoblast-like differentiation by upregulating osteoblast markers and downregulating contractile SMC markers [115]. Inhibitory SMAD6 appears to play a protective role, as SMAD6 knockout mice develop vascular and valvular calcification [117]. A nonsynonymous C484F mutation in SMAD6 decreases its capacity to inhibit an osteogenic response to BMP signaling [118].

In addition to the role of BMPs, TGFβ ligands also contribute to atherosclerosis and vascular calcification. TGFβ signaling was originally thought to be protective against atherosclerosis by promoting stable lesions, but recently increased expression of TGFβ ligands, receptors, and phosphorylated SMAD2/3 was demonstrated in atherosclerotic lesions [113, 119–121]. Similarly, increased expression of TGFβ was found in the coronary arteries of individuals with plaque and complicated legions compared to healthy patients [122, 123]. Mechanistically, it is thought that TGFβ signaling may promote the synthesis of extracellular matrix (ECM) in VSMC of conduit arteries to promote the growth and calcification of atherosclerotic legions [113, 124]. TGFβ also drives EndMT together with oxidative stress and hypoxia, resulting in a fibroblast-like phenotype with altered balance between collagen and matrix metalloproteinases [97].

Chronic kidney disease and vascular calcification

A major risk factor for vascular calcification is chronic kidney disease (CKD), attributed to the loss of renal clearance and homeostatic regulation of products of metabolism and electrolytes. Animal and human studies demonstrate that vascular calcification in CKD is largely associated with oxidative stress, uremia, and hyperphosphatemia. Medial calcification is the most prevalent vascular pathology in CKD patients, and the deficiency of inhibitory factors produced by arterial smooth muscle cells appear to be important drivers—inhibitors that include pyrophosphate (PP_i) and MGP. In addition to its role in antagonizing BMP signaling, MGP functions to bind and clear calcium ions and inhibit hydroxyapatite crystal growth [125], an activity that is weakened by excess phosphate ion concentration [126], suggesting a mechanism by which the function of MGP might be attenuated by the hyperphosphatemia found in renal failure [111]. The protective activity of MGP also requires it be carboxylated in a vitamin K-dependent process. While both the active (carboxylated) and uncarboxylated forms of MGP are upregulated in renal failure [111], the fraction of carboxylated MGP vs. total MGP was diminished in the plasma of dialysis patients with calciphylaxis, a life-threatening vasculopathy characterized by sub-acute calcification of small-to medium-sized arterial vessels in patients with advanced CKD, particularly those on hemodialysis [127]. In hemodialysis patients with calciphylaxis there is evidence of increased activation of the BMP signal transduction pathway, with increased levels of phosphorylated SMAD 1/5/9, ID1, ID3, and RUNX2 in cutaneous vessels [128] (Table 1). Thus, multiple activities of MGP including regulation of BMP signaling and inhibition of mineralization contribute to its protective effects in CKD-associated medial calcification.

Evidence from animal models and human diseased tissues suggests that certain osteogenic BMPs are expressed medial calcific lesions associated with CKD. In a uremic rat model of CKD-associated vascular calcification due to adenine and hyperphosphatemia, early development of vascular calcification was correlated with overexpression of BMP2, BMP4, along with ALK3 and MGP in the tunica media of the aorta [129]. In addition, BMP2 and BMP4 were increased in the serum in a manner that correlated strongly with aortic calcium content, suggesting utility as biomarkers of calcification associated with CKD. In support of this a concept, two studies found elevated BMP2 in the serum of patients with CKD [130], with BMP2 expression in one study correlating with vascular stiffness and markers of oxidative stress [131]. In the latter study, high phosphate and BMP2 induced mineralization and expression of Runx2 and Msx2 in VSMC, in a ß-catenin dependent fashion, linking BMP2, hyperphosphatemia, and Wnt signaling in the pathogenesis of vascular calcification in CKD [130].

The impact of BMPs in CKD-associated vascular calcification may be ligand- and context-specific (Table 1) (Figure 1). BMP7 is expressed in healthy adult kidneys, contributes to their normal development and homeostasis, and exerts protective effects in models of kidney fibrosis [132–135], leading to the hypothesis that BMP7 might be protective in models of vascular calcification associated with CKD. In chronic kidney disease, circulating BMP7 is reduced due to kidney fibrosis, causing phenotypic remodeling and proliferation of SMCs and medial thickening, which synergizes with elevated phosphate levels to result in intramembranous calcification. Systemic administration of BMP7 is protective against renal failure [136], and in a mouse model of uremia combined with an atherogenic LDLR knockout background, BMP7 normalized the vascular calcification and overexpression of osteocalcin that were seen in uremic animals to equal to or lower than those seen in non-uremic control animals [137]. In the vessels of nephrectomized rats with uremia, and uremic patients undergoing renal transplantation, overexpression of BMP antagonist Gremlin was observed in association with calcific lesions of the vascular media, suggesting a regulatory role of Gremlin in this process [138]. In another study which combined uremia with LDLR-knockout mice, BMP7 not only attenuated vascular calcification, but also corrected hyperphosphatemia associated with uremia, and stimulated orthotopic skeletal phosphate deposition while simultaneously preventing vascular calcification by direct action on vascular smooth muscle cells, suggesting this single intervention could correct aspects of renal osteodystrophy and vascular calcification [139, 140]. BMP9 has also received attention in CKD-associated vascular calcification. BMP9 was elevated in the circulation of children with CKD, and increased calcium content, alkaline phosphatase (ALP) activity, and expression of osteogenic markers, in a manner dependent on ALK1 and SMAD4 canonical signaling [141].

Given the clinical and in vitro data implicating BMP/TGFβ signaling in CKD-associated vascular calcification, therapies targeting this pathway have been investigated. Similar to the efficacy demonstrated in models of calcific atherosclerotic disease, administration of the BMP type I receptor kinase inhibitor LDN-193189 in a partial nephrectomy mouse model of CKD was found to abolish endothelial dysfunction and vascular smooth muscle cell osteogenic differentiation, both of which were dependent on the activation of phosphatase-and-tensin homolog (PTEN) in the endothelium and smooth muscle [142], providing a mechanistic link between CKD and associated medial calcification.

Administration of RAP-011, a multifunctional BMP/activin/GDF ligand trap based on the extracellular domain of ACVR2A, in a murine model of uremia and LDLR deficiency abrogated aortic vascular smooth muscle dedifferentiation, osteoblastic transition, and neointimal calcification [143]. Inhibition of ACVR2A ligands with RAP-011 also diminished circulating and renal expression of Wnt antagonist Dickopf1 (Dkk1), suggesting that the modulation of Wnt signaling downstream of BMP/activin/GDF signaling is involved in the development of medial calcification. These findings build upon similar work describing elevated expression of Dkk1 in the circulation of CKD, and that neutralization of Dkk1 prevents vascular calcification [144]. RAP-011 was similarly able to prevent hydroxyapatite deposition in the aortic media and adventitia in a mouse model of Alport syndrome, abrogating aortic expression of osteoblast markers Runx2 and Osterix, providing further support for the activity of ACVR2A ligands in the pathogenesis of medial calcification [145]. While the critical ligands responsible for this therapeutic effect are not known, they could include activins A and B, GDFs 8 and 11, and BMPs 6 and 7, or a combination thereof.

Diabetes and vascular calcification

Diabetes, characterized by an inability to produce or respond to insulin in the regulation of glucose, is another condition marked by increased vascular calcification and cardiovascular mortality. The burden of vascular calcification is a predictor of cardiovascular and overall mortality in patients with diabetes [146]. Animal models testing the impact of high fat, diabetogenic diets in atheroprone LDLR knockout mice found enhanced vascular calcification corresponding with upregulated aortic expression of Msx2, which synergizes with BMP2 to promote osteogenic differentiation of myofibroblasts [147], prompting the hypothesis that BMP2-Msx2 signaling promotes calcification in diabetic vascular disease by inducing differentiation of myofibroblasts into an osteogenic lineage [93].

In support of a link between diabetes and enhanced BMP signaling, there is increased expression of BMP2 and BMP7 in the vasculature associated with early in the development of autoimmune diabetes in NOD mice [148], while aortic expression of BMP4 is seen to increase as diabetes progresses in the db/db murine model of type 2 diabetes [149]. Mechanistically, hyperglycemia in a murine model of type 1 diabetes (Ins2^Akita/+) activates BMP signaling in endothelial cells and SMAD1/5/9 signaling in the aortic wall in vivo. It also enhances expression of BMP2, BMP4, ALK1, and ALK2 in the aortic endothelium, which all correspond with osteogenic gene expression and medial calcification, and are abrogated by expression of an MGP transgene [107, 150] (Figure 1).

The activation of BMP2 signaling in the vasculature may reflect the synergy of multiple metabolic insults in promoting vascular calcification. Vascular calcification in response to high doses of cholecalciferol (Vitamin D₃) was analyzed in the ob/ob murine model of insulin resistance and obesity vs. C57BL/6 controls. Intrarenal vascular calcification and diminished renal function in the ob/ob mice versus controls was observed—with enhanced BMP2 expressed in the renal vessels exclusively in the ob/ob mice [151], concordant with previous observations that VSMC from this metabolic syndrome mouse model are sensitized to osteogenic trans-differentiation and mineralization in response to BMP2 stimulation [152].

Role of BMP signaling in valvular calcification

The cardiac valves are highly organized structures composed of leaflets containing three distinct cellular layers: the collagenous fibrosa layer oriented opposite to the blood flow, the proteoglycan-rich middle spongiosa layer, and elastin fiber-rich layer facing the blood flow. The fibrous and calcific remodeling of these ECM layers serve as pathological hallmarks of calcific valve disease, a highly prevalent and morbid form of cardiovascular disease. While the mechanisms by which valvular calcification proceeds remain incompletely understood, recent evidence suggests that it occurs via the highly-orchestrated process the resembles heterotopic endochondral ossification, with osteoblast-like and chondroblast-like cells resembling those seen in orthoptopic bone formation, and a similar contribution of inflammatory signaling and BMP signaling.

Similar to observations in vascular calcification, BMPs, in particular the osteogenic morphogens BMP2 and BMP4, are overexpressed in ossified regions of human calcified valves by myofibroblasts and pre-osteoblasts in areas densely infiltrated with B- and T-lymphocytes [153]. TGFβ, a known regulator of vascular smooth muscle cell calcification and osteoblastic differentiation, has also been demonstrated to localize in calcified aortic valves (AVs) [154, 155]. Within calcified human aortic valves, TGF-β1 and BMP2 induce the pro-osteogenic activation of interstitial cells (AVICs), as evidenced by the upregulation of micro-RNA (miR)-486, which inhibits SMURF2 expression and leads to the downregulation of miR-204 [156]. In the endothelial cells of calcified human AV fibrosa, expression of phosphorylated SMAD1/5/9 is substantially increased relative to cells from uncalcified valvular fibrosa, while expression of phosphorylated SMAD2/3 remains relatively unchanged [157], consistent with BMP signaling activation in calcified AV fibrosal cells (Figure 1). Canonical BMP signaling pathways are also activated in calcific AVs derived from mice lacking Klotho, a critical regulator of fibroblast growth factor 23 (FGF23) and phosphate homeostasis [158]. In these mice, endothelial AV cells demonstrate increased SMAD1/5/9 phosphorylation and heightened expression of osteochondrogenic genes ALP, Runx2, and Osteopontin prior to the appearance of calcific nodules [158].

Conclusions

Vascular calcification recapitulates many features of endochondral and intramembranous ossification, including the requirement for coordinated activation of members of the BMP/TGFβ family of signaling molecules for the recruitment of osteogenic and chondrogenic progenitors for the development of calcific cardiovascular disease. As in the development of orthotopic bone, various BMP/TGFβ ligands, receptors and modulators exert context- and tissue-specific, temporally-dependent effects on the recruitment and activation of cellular progenitors for generating vascular calcification, and the synthesis of ECM and induction of mineralization. This marked sensitivity to context may result in overlapping or opposing function of various ligands, depending on the complement of receptors expressed and absence or presence of co-signaling in the responding cell type. Further elucidation of the spatiotemporal expression and activation of the BMP/TGFβ family of signaling molecules in relationship to hyperlipidemia, inflammation, hyperglycemia, and metabolic dysregulation via systems biology approaches and in authentic models is needed to define the precise manner in which these signals are integrated to manifest these diverse types of pathology, and to design therapeutic interventions that are beneficial in these specific conditions.