PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circ Res. Author manuscript; available in PMC Aug 19, 2012.
Published in final edited form as:
PMCID: PMC3167074
NIHMSID: NIHMS315813
THE REGULATION OF VALVULAR AND VASCULAR SCLEROSIS BY OSTEOGENIC MORPHOGENS
Kristina I. Boström,* Nalini M. Rajamannan, and Dwight A. Towler§
*Department of Medicine, Division of Cardiology, David Geffen School of Medicine at UCLA, 10833 LeConte Avenue, Los Angeles, CA 90095
Department of Medicine, Division of Cardiology, Northwestern University Feinberg School of Medicine, 300 East Superior Street, Chicago, IL 60611
§Department of Medicine, Division of Endocrinology, Department of Developmental Biology, Washington University in St. Louis, 660 South Euclid Avenue, St. Louis, MO 63110
Please address correspondence to any author by email at: Kristina I. Boström MD, PhD, kbostrom/at/mednet.ucla.edu, Nalini M. Rajamannan MD, n-rajamannan/at/northwestern.edu, Dwight A. Towler MD, PhD, dtowler/at/im.wustl.edu
Vascular calcification increasingly afflicts our aging, dysmetabolic population. Once considered only a passive process of dead and dying cells, vascular calcification has now emerged as a highly regulated form of biomineralization organized by collagenous and elastin extracellular matrices. During skeletal bone formation, paracrine epithelial-mesenchymal and endothelial-mesenchymal interactions control osteochondrocytic differentiation of multipotent mesenchymal progenitor cells. These paracrine osteogenic signals, mediated by potent morphogens of the BMP and Wnt superfamilies, are also active in the programming of arterial osteoprogenitor cells during vascular and valve calcification. Inflammatory cytokines, reactive oxygen species and oxylipids – increased in the clinical settings of atherosclerosis, diabetes, and uremia that promote arteriosclerotic calcification – elicit the ectopic vascular activation of osteogenic morphogens. Specific extracellular and intracellular inhibitors of BMP-Wnt signaling have been identified as contributing to the regulation of osteogenic mineralization during development and disease. These inhibitory pathways and their regulators afford the development of novel therapeutic strategies to prevent and treat valve and vascular sclerosis.
Keywords: BMP, Wnt, inflammation, osteoblast differentiation, vascular calcification
Arteriosclerotic calcification increasingly afflicts our aging and dysmetabolic population1. Vascular calcification was once thought to be a passive process of dead and dying cells. However, elegant studies by the bone and cartilage biologist H. Clarke Anderson in the 1970s identified alkaline phosphatase (ALP) - containing matrix vesicles as sites of atherosclerotic and medial vascular mineralization2, 3; this provided intriguing evidence that actively regulated osteogenic processes were participating in vascular mineralization. Another bone biologist and physician-scientist, Marshall Urist, had identified a “morphogenetic matrix for differentiation of bone tissue” earlier in that decade4, and in 1988 John Wozney, Vicky Rosen and colleagues cloned and characterized the first genes encoding bone morphogenetic proteins, including BMP25. In 1993, Boström, Demer and colleagues first demonstrated the expression of BMP2 in calcified human atherosclerotic plaques – and the capacity of BMPs to direct osteogenic programming of vascular mesenchymal progenitors of the pericyte lineage6. Thus, in a span of ~2 decades, a picture emerged in which the mineralizing pathobiology of vascular calcification is painted in part by secreted osteochondrogenic factors -- polypeptides also responsible for bone morphogenesis during skeletal development7 and fracture repair8.
Since 1993, the notion that osteochondrogenic morphogens participate in the biology of arterial calcification has been robustly confirmed and extended to encompass newly-discovered regulators of skeletal development9. In this manuscript, we review the regulation of valvular and vascular sclerosis by osteogenic morphogens. We highlight mechanistic insights afforded by these studies that provide the foundation for novel therapeutic approaches to treat arteriosclerotic valve and vascular disease.
Paracrine signals control skeletal osteogenesis
Bone and cartilage are the two best recognized tissues of the vertebrate skeleton, the former arising in intimate association with the vasculature10. The skeleton contains two major anabolic cell types, the osteoblast and the chrondrocyte11. True bone formation or “ossification” occurs via two distinct mechanisms. Membranous ossification describes the mineralization that occurs in neural crest-derived craniofacial bones12, 13. Membranous ossification does not require a preceding cartilaginous template, but occurs directly within a type I collagen –based extracellular matrix elaborated and organized by osteoblasts. By contrast, most bones of the body mineralize via endochondral ossification. This second process requires an initial, avascular type II collagen-based extracellular matrix template deposited by chondrocytes. Subsequent chondrocyte hypertrophy at the growth plate, type X collagen deposition, VEGF-driven vascular invasion, chondrocyte apoptosis and cartilaginous matrix mineralization ensues. This is followed by osteoclast-mediated matrix resorption and osteoblast-mediated bone deposition (again, type I collagen based) that replaces the calcified cartilage template12, 13.
During skeletal development, paracrine epithelial-mesenchymal interactions control the osteogenic programming of skeletal anlage, best characterized during odontogenesis and endochondral ossification of the appendicular skeleton14. As first elucidated in studies of limb morphogenesis, proximal-distal, anterior-posterior, and dorsal-ventral morphogen gradients15 interact to control skeletal patterning, growth and ossification16. For example, reciprocal interactions between epithelial FGF4 / FGF8 in the apical ectodermal ridge (AER) and mesenchymal FGF10 maintain limb bud outgrowth. Ectodermal Wnt3a signaling is required for AER FGF8 production. Sonic hedgehog (Shh) production by posterior mesenchyme called the zone of polarizing activity (ZPA) maintains AER function via gremlin, a negative regulator of BMP signaling. Limb BMP4 tone provides a powerful ventralizing signal that is modulated by gremlin (preserves the AER) and by Wnt7a elaborated from the dorsal ectoderm. Dynamic expression of Dkk1 in ventral limb bud, anterior and posterior mesenchyme, and the AER antagonizes Wnt3a and Wnt7a signals that maintain FGF8 and Shh expression17. Thus, a complex 4-dimensional interplay between Wnt, BMP, FGF, Hedgehog, Dkk1 and other paracrine signals controls the growth, lineage specification, and differentiation of osteochondroprogrenitors in limb bud mesenchyme16. Likewise, paracrine PTHrP signals at the mineralizing growth plate control the temporospatial timing of endochondral bone formation via negative regulation of Indian Hedgehog (Ihh), and the FGF- and VEGF-dependent vascularization required for endochondral ossification18 as mentioned above. Again, in the craniofacial skeleton, membranous bones and teeth form without the requirement of a preceding calcified cartilage template13. Whether arising from endochondral or intramembranous processes, once true bone has formed paracrine BMP and endocrine PTH tone maintains osteogenic Wnt/β-catenin signals that support post-natal skeletal anabolism, mechanical integrity, and fracture repair8, 9, 19.
Transcriptional mediators of osteochondrogenic differentiation
Sox9-positive mesenchymal condensations demarcate the skeletal morphogenetic field11, 20. Chondrogenic programming requires coordinated actions of L-Sox5, Sox621 and HIF22. Robust osteogenic programming of mesenchymal progenitors requires gene regulatory programs directed by at least six DNA binding transcription factors – Runx2 (a.k.a. Cbfa1), Msx1 and Msx2, LEF1, NFATc1, and Osx 11. β-catenin, a transcriptional co-adapter that promotes transcription directed by the TCF/LEF transcription factor family, is indispensible for osteoblast differentiation as well 23 and maintains the elaboration of osteogenic transcriptional programs (Figure 1). Runx2 11 has all the attributes of a ‘master gene’ differentiation factor for the osteoblast lineage and bone matrix gene expression in both membranous and endochondral bone. During embryonic development, Runx2 expression precedes osteoblast differentiation and is restricted to mesenchymal cells destined to become osteoblast 11(Figure 1). In addition to its critical role in osteoblast commitment and differentiation, Runx2 appears to control osteoblast activity, the rate of bone formation by differentiated osteoblasts, in concert with ATF411. In the skull, additional signals provided by the homeodomain proteins Msx1 and Msx2 are critical for intramembranous bone formation – a process that that cannot proceed even in the presence of an intact Runx2 gene when Msx expression is lacking24. Down-stream of Msx, Runx2, and β-catenin genomic programs is osterix (Osx), a zinc finger transcription factor required for matrix mineralization in concert with NFATc125 (Figure 1). Importantly, the expression of Msx1, Msx2, Runx2, and Osx during osteogenic differentiation is critically dependent upon bone morphogenetic protein (BMP) signaling11. Moreover, BMP2 signaling in mesenchyme maintains the specification of osteoprogenitors required for post-natal skeletal integrity8.
Figure 1
Figure 1
The relationship of chondrocyte and osteoblast lineages to skeletal and vascular osteoprogenitors
BMPs: Prototypic osteogenic morphogens
BMPs are secreted polypeptides, a subgroup of the transforming growth factor (TGF) β superfamily of growth factors. BMPs were first identified as a bioactivity in demineralized and pulverized bone powder capable of inducing ectopic endochondral bone formation in muscle26. Over the ensuing years, more than 15 distinct BMP family members have been identified (BMP system is extensively reviewed elsewhere)27, 28. The BMPs are secreted in an active form, with function modulated by extracellular antagonists27 such as noggin, chordin, gremlin, cross-veinless 2 (CV2, also referred to as BMPER), cartilage oligomeric matrix 2 (COMP2)29, and matrix Gla protein (MGP; Figure 2). BMPs elicit their effects through activation of receptor complexes composed of type I and type II Ser/Thr kinase receptors, to which the BMPs bind independently. There are seven type I receptors, termed activin receptors-like kinase (ALK1 to ALK7), which determine the specificity of the BMP signal in concert with five type II receptors including the BMP type II receptor (BMPR2) 30, 31. When the ligand binds, the type II receptors phosphorylate and activate the type I receptors, which propagate the signaling by phosphorylating transcription factors referred to as Smads (Figure 2). Receptor complexes may also contain so-called type III co-receptors 28 which further modulate signaling. The BMP type I receptors (ALK1, ALK2, ALK3 and ALK6) activate the receptor-activated (R)-Smads, Smad1, Smad5 and Smad8, whereas the activin and the TGFβ type I receptors (ALK4, ALK5 and ALK7) phosphorylate Smad2 and Smad331, 32. Activated Smads assemble into complexes together with the common mediator (co)-Smad4, which translocate to the nucleus and modulate gene expression in concert with Runx2, TCF/LEF and other factors. This process is regulated in part via inhibition by the inhibitory I-Smads, Smad6 and Smad7 32(Figure 2). BMPs can activate non-Smad signaling pathways as well. Studies by Rawadi, Baron and colleagues first identified one of these non-Smad signals to be a paracrine Wnt/β-catenin relay that promotes alkaline phosphatase expression and matrix biomineralization in conjunction with and parallel to BMP/Smad signaling 33, 34(Figure 3).
Figure 2
Figure 2
The essentials of osteogenic BMP signaling in arteriosclerotic calcification
Figure 3
Figure 3
The essentials of osteogenic Wnt signaling in arteriosclerotic calcification
Wnts: Osteogenic modulators and mediators of BMP action
The Wnts are a family of secreted, lipid-modified polypeptide ligands35 that classically signal via activation of heterodimeric receptor complexes containing LRP5, LRP6, or LRP4 and a GPCR co-receptor of the Frizzled (Fzd) gene family36 (Figure 3). LRP co-receptor complexes with ROR2 are clearly also important, and reviewed in detail elsewhere37. In bone formation and skeletal homeostasis, Wnt10b, Wnt7a, Wnt7b, Wnt3a, and Wnt1 have emerged as important, although other ligands are certain to contribute34. With canonical or classical Wnt signaling mechanisms, nuclear accumulation of β-catenin ensues38. β-catenin is a transcriptional co-adaptor indispensible for Runx2- and LEF1- mediated programming of osteogenic differentiation23. Collaborative interactions with BMP- and TGFβ- regulated Smad complexes also occur39, 40. Even in the presence of skeletal osteochondrogenic transcription fractor expression (Runx2, Msx1/2, LEF1, Osx), robust elaboration of osteogenic programs is impossible in the absence of activated Wnt/β-catenin signaling23. The canonical pathway sequentially utilized a casein kinase 1 / dishevelled signaling cascade to phosphorylate GSK3 and inhibit the β-catenin destruction complex, thus increasing cellular β-catenin levels and nuclear accumulation41. Additionally, a “non-canonical” pathway also requiring dishevelled but proceeding via protein kinase C – delta activation has been identified to support Osx expression and bone formation42. In a manner analogous to the negative regulation of BMP signaling, secreted Wnt binding proteins known as secreted frizzled related proteins (sFRPs) inhibit Wnt ligand binding and activation of LRP5/6-Fzd cell surface receptors43. Two classes of non-signaling “ligands” -- members of the Dkk family44 and SOST/sclerostin4547 – bind to the osteogenic LRPs and inhibit signaling (Figure 3). In addition, intracellular inhibitors of Wnt/β-catenin signaling such as ICAT (inhibitor of β-catenin and TCF/LEF) antagonize protein-protein interactions between β-catenin and nuclear transcription factor complexes bound to DNA48 (Figure 3). Thus, the sequential and parallel interactions between the BMP and Wnt signaling cascades control osteochondral mineralization 34, with intracellular and extracellular fine-tuning of signal duration and strength.
BMP Signaling in Vascular Development and Disease
When BMP2, BMP4, and BMP6, were detected in calcified areas of atherosclerotic lesions 6, 49, 50 it was therefore presumed that they enhanced vascular calcification - even more so when it became evident that vascular calcification is largely driven by osteogenesis in the vascular media51, 52. Indeed, a causal relationship between BMP activity and vascular calcification has now been established5355. However, BMP signaling is not only driving ectopic calcification, but is essential for cardiovascular development, with critical roles in the establishment of endothelial tubes during vasculogenesis, the recruitment and differentiation of vascular smooth muscle cell (VSMC) precursor cells, and vascular patterning56, 57. Mutations in the genes coding for the ALK1 receptor and endoglin, an ALK1 co-receptor, cause hemorrhagic hereditary telangiectasia 58, which is characterized by abnormal angiogenesis and arteriovenous malformations. Furthermore, mutations in the BMPR2 and ALK1 receptors have been linked to pulmonary arterial hypertension 59, 60, characterized by a dysregulation of vascular cell growth and differentiation.
BMP activity is important for the regulation of phenotypic plasticity, proliferation and differentiation in VSMC52. BMP2 in particular has an inhibitory effect on VSMC proliferation and differentiation, whereas BMP7 promotes the VSMC phenotype in a manner reminiscent of TGF-β 52. Furthermore, BMP inhibition -- potentially in later steps -- appears to be a key factor in maintaining VSMC differentiation. Loss of MGP, an inhibitor of BMP2 and BMP4 6163, causes extensive calcification of elastic and muscular arteries 64. The loss of MGP results in a significant increase in aortic BMP activity and leads to osteochondrogenic trans-differentiation of VSMC with subsequent mineralization 65, supporting the notion that regulation of BMP activity is essential for maintaining a normal media. The potential anti-calcific roles of other BMP antagonists, such as noggin, CV2, gremlin, chordin-like 1, and cartilage oligomeric proteins 2 27 are not yet known although gremlin is over-expressed and may have anti-osteogenic effects in the calcified media in uremia66.
In addition to the aorta, MGP is also expressed in organs such as the lungs where it plays a role in the formation of the pulmonary arterial tree by inhibiting BMP4 67. BMP4 acts as an angiogenic factor and induces expression of ALK168, which is pivotal in endothelial maturation and recruitment of VSMC precursors. ALK1 activation in turn induces MGP, which provides negative feedback on BMP468. The interplay between BMP2, BMP4, ALK1, and MGP may affect morphogenetic processes that in some cases are linked to vascular calcification. BMP2 has been proposed to be an activation morphogen for calcifying vascular cells, promoting formation of large nodules and mineralization in vitro69, 70. The cellular patterns resulting from these interactions can be predicted using a reaction-diffusion model for the two types of morphogens69. In vivo, at least three types of patterns or morphogenetic processes might be affected by the balance between BMP and MGP or other inhibitors. The first process is the formation of the layered media, which includes normal VSMC differentiation. A disturbance in this process is exemplified by the abnormal and highly calcified media in the MGP null mouse64, 65. The two other processes, vascular branching and arteriovenous malformation (AVM) formation, have probably less to do with calcification but may present problems if the BMPs are targeted in anti-calcific therapies. Vascular branching is disturbed in the MGP transgenic mice, which exhibit loss of side branching and stunted growth of pulmonary arteries67. AVMs, on the other hand, are characteristic of ALK1 deficiency71 and can be described as a short-circuited and disrupted network in the capillary bed. Because ALK1 is regulated by BMP4 and in turn regulates MGP, the balance between BMP4 and MGP is likely to affect the capillary network. As highlighted by Rajamannan et al, osteogenesis and angiogenesis are coupled processes during vascular calcification72. Thus, the BMPs are true to their name in shaping the vasculature itself as well as inducing ectopic calcification.
Regulation of Vascular BMPs and BMP Antagonists: The Activated Endothelium
An activated endothelium may be a significant source of BMP and a potential calcific stimulus. BMP expression is easily activated in the endothelium in response to a number of different pathogenic stimuli, many of which are well known to stimulate atherosclerosis and vascular calcification. Sorescu, Jo and colleagues were the first to demonstrate that BMP4 is induced in aortic endothelial cells (ECs) by oscillatory shear stress, reactive oxygen species (ROS), and inflammatory cytokines 53, 73, 74. Subsequently, Csiszar, Ungvari, and colleagues showed that BMP2 is similarly induced by inflammatory cytokines and ROS and functions as an inflammatory mediator75, 76. Both BMP2 and BMP4 are also induced by high glucose levels, whereas BMP4 is more stimulated by high fat diets than BMP253, 55. Even though BMP2 and BMP4 are highly homologous on a protein level, initial results point to a difference in function when secreted from glucose-treated ECs. BMP4 promotes angiogenesis and adipogenic differentiation whereas BMP2 promotes mineralization53, 77, thus suggesting that BMP2 has a more direct link to calcification and osteogenic differentiation.
Interestingly, BMP inhibitors are induced in ECs in response to similar stimuli as BMP2 and BMP4, or by an increase in vascular BMP activity. In vitro, follistatin, noggin, and MGP and CV2 are induced by oscillatory shear stress78 or high glucose levels 53. Most of these inhibitors are also induced in the aortic wall of fat-fed apoE null mice or diabetic mice and rats. 53, 55. Enhanced BMP activity causes induction of some inhibitors such as MGP and CV2, but not others such as noggin or chordin55, pointing to a finely tuned mechanisms for regulation of BMP activity. Interestingly, HDL, a vasculo-protective factor, promotes higher expression of MGP in ECs and aortas of apoAI transgenic mice79, thereby pushing the balance towards BMP inhibition. In addition, CV2 is induced by statins in ECs80, and also promotes BMP inhibition. Thus, the balance between endothelial BMPs and their inhibitors may determine the health of the endothelium, and an efficient limitation of endothelial BMP activity may confer protection against atherosclerosis. This also emphasizes the consideration of “BMP activity” or “BMP tone” as a context-specific working concept arising from the expression of specific BMP ligands, receptors or antagonists.
It should also be kept in mind that the adventitial microvasculature is also exposed to oxidative stress and inflammatory mediators similar to the aortic endothelium and likely responds with higher BMP2/4 secretion. In addition, microvascular pericytes may contribute multipotent cells to the calcific process 8183. Thus, a calcific process could also be triggered from capillaries in the periphery of the vessel.
Vascular BMP activity is stimulated in cells other than ECs. BMP2 production is upregulated in SMC treated by high phosphate in the form of nanocrystals84, and in pericytes and mesangial fibroblasts by high glucose85. In addition, medial expression of BMPs, BMP receptors and inhibitors is increased in diabetic mice 8688. A subset of myofibroblasts also expresses BMP2 in response to diabetes and hyperlipidemia 51, even though myofibroblasts have been reported to down-regulate BMP6 as a potential step in vascular remodeling89.
Role of BMPs in Triggering the Calcification of Atherosclerotic Lesions
Endothelial BMP2 and BMP4 cause an induction of endothelial adhesion molecules in the ECs, in several instances using non-canonical signaling pathways 73, 75, 76. This provides a direct link between BMP activity and the initial stages of atherosclerosis, which sets the stage for atherosclerotic lesion development and ultimately lesion calcification. Once lesion formation gets under way, there may be local reactivation of developmental conditions where enhanced BMP activity could trigger osteogenesis in susceptible cells. Indeed, MGP, noggin and chordin are down-regulated in de-differentiated VSMCs54 which would make them more susceptible to BMP action. A number of BMP antagonists are later detected at increased levels in calcified vessels, and may in that context help limit the progression of the ectopic calcification.
As already discussed, the endothelium may have a role in triggering vascular calcification, and several populations of multipotent cells that respond to BMPs have been identified in the vasculature. These include the calcifying vascular cells (CVC) isolated from the aortic media 6, 90, myofibroblasts present in the adventitia of diabetic and hyperlipidemic LDLR−/− mice 51, and microvascular pericytes well known to have osteogenic potential81 (Figure 1). Circulating stem cells may also contribute to osteogenic changes in the artery wall 91, and VSMC “trans-differentiation” certainly contributes to the vascular endochondral ossification mechanisms in MGP−/− mice as elegantly demonstrated by Giachelli and colleagues65. However, the relative contribution of the respective cell population to local osteogenesis and vascular calcification in different arteriosclerotic disease states remains to be established.
The anti-calcific role of MGP may depend on normal EC differentiation. Recently, MGP transgenic mice were crossed with ApoE−/− mice, a model of atherosclerosis and lesion calcification, to study the effect of BMP inhibition on lesions. The increase in MGP resulted in diminished SMAD1/5/8 signaling, inflammation, lesion formation and calcification after fat-feeding 55. The majority of the MGP was found in proximity to the endothelium, supporting the importance of the endothelium in preventing lesions and lesion calcification. MGP null mouse on the same ApoE−/− background had enhanced SMAD1/5/8 signaling 92, and extensive medial calcification as previously reported 64, 65. However, the MGP null mice lacked lesion formation and the expected induction of endothelial adhesion molecules and inflammatory markers, suggesting a truly abnormal endothelium. Interestingly, ALK2 was induced in the MGP-deficient aortas, and endothelial differentiation has recently been linked to ALK2 activity. Constitutively active ALK2 causes endothelial-to-mesenchymal transition and acquisition of a stem-cell-like phenotype in ECs, which may be triggered to undergo osteogenic differentiation91. In patients with fibrodysplasia ossificans progressiva, characterized by heterotopic calcification and caused by mutations in the ALK2 gene91, chondrocytes and osteoblasts expressed EC markers. The implication for vascular calcification is that the endothelium might be an additional cell source of osteoblastic cells via endothelial – mesenchymal transitioning (EMT) 93 -- dependent upon Smads and β-catenin94. Strong ALK2 activation by exogenous factors might therefore interfere with endothelial differentiation and protective function. Capillary ingrowth in atherosclerotic lesions may also promote calcification analogous to normal bone mineralization 77. This may be an indirect way for BMP4, via its angiogenic properties, to enhance the calcification process.
BMPs in Diabetic Medial Calcification & Chronic Kidney Disease
Calcification is extremely common in diabetic vasculopathy, often in the form of medial calcification (also referred to as Mönckeberg’s media sclerosis or elastocalcinosis). The media calcification occurs along the elastic lamellae95 and frequently co-exists with atherosclerotic lesion calcification. The first link between BMP activity and diabetic vasculopathy was found in adventitial myofibroblasts in the aorta of fat-fed diabetic LDLR−/− mice 51. BMP2 was instrumental in augmenting the BMP-2/Msx2-Wnt pathway leading to an osteogenic phenotype in a subset of the myofibroblasts. It was later discovered that diabetes in mice and rats activated BMP signaling throughout the vascular wall 53. Interestingly, the location of BMP2 and BMP4 differed in diabetic aortas in that BMP-4 was found in the endothelium and BMP2 all the way through the vascular wall 53. The high BMP4 was associated with high MGP, ALK1, ALK2 and MGP, which were all regulated as a group in ECs. When a MGP transgene was introduced to limit the BMP activity, MGP expression increased predominantly in the endothelium, even though BMP activity and calcification diminished in the entire media53, suggesting crosstalk between the endothelium and the media.
Diabetes is a common cause of chronic kidney disease (CKD), another powerful stimulator of vascular calcification96. The calcification in CKD has been primarily attributed to increased phosphate levels, which strongly promotes SMC mineralization in vitro and in vivo97. High phosphate levels stimulate secretion of BMP2 in SMC84 and BMP2 in turn upregulates the type III sodium-dependent phosphate co-transporter PiT1/SLC20A1, which mediates high phosphate-induced mineralization98100. Treatment with BMP7 counteracts vascular calcification in CKD, in part due to reduced phosphate levels and direct effects on SMC differentiation101. Therefore, it is important to understand all aspects of vascular BMP signaling in order to correctly target BMPs for anti-calcific effects.
BMPs, MGP, and Calcific Uremic Arteriolopathy
A particularly severe and rapidly deadly form of vascular calcification – calcific uremic arteriolopathy (CUA, a.k.a. calciphylaxis) -- arises in a subset of patients with CKD treated with warfarin for anticoagulation102104, and rarely in patients with severe chronic liver disease. Widespread calcium phosphate deposition in pulmonary, intestinal, and dermal soft tissues -- with predilection for the lower extremity, thighs and gluteal regions, and the abdominal pannus -- is characteristic for CUA. Histopathologically, arteriolar (< 0.6 mm diameter) medial calcification occurs -with concomitant fibroproliferative intimal vessel occlusion, dermal fat infarction, and skin necrosis. Immunohistochemistry demonstrates the expression of BMP4 in the periarteriolar adventitia of dermal CUA105. Warfarin inhibits the MGP gamma-carboxylation that is necessary for inhibition of BMP2/4 signaling62 as well as fetuin-dependent matrix vesicle clearance106, 107. Thus, it is highly probable that the warfarin-induced compromise in dermal arteriole MGP function contributes to the pathobiology of CUA. However, the precise reasons for the histoanatomic disease predilection remain to be explained – and other gamma-carboxylated proteins such as Gas6108 may also contribute to disease via Axl tyrosine kinase regulation of VSMC osteochondrocytic differentiation.
Calcific Aortic Valve Disease: Consequences and Clinical Considerations
Calcific aortic valve disease (CAVD) afflicts ca. 2% of our population over the age of 60109. Progressive fibrosis and valve calcium accumulation impairs leaflet compliance and coaptation, impeding outflow, increasing myocardial workload, and permitting variable regurgitation. Once symptoms occur – e.g., chest pain, syncope, congestive heart failure – progressive aortic valve stenosis conveys a two-year mortality that approaches 50% in the absence of surgical intervention110. Using echocardiographic criteria, Rosenhek and colleagues demonstrated that the presence of aortic valve calcium in patients with asymptomatic mild or moderate aortic stenosis was the single most significant predictor of clinical progression111, 112.
Numerous epidemiologic studies identified risk factors for CAVD development, which are similar to those of vascular atherosclerosis, including smoking, male gender, body mass index, hypertension, elevated lipid and inflammatory markers, bicuspid aortic valve, type II diabetes mellitus (T2DM) and/or metabolic syndrome and renal failure113. Although aortic stenosis may occur in individuals with otherwise anatomically normal tricuspid aortic valves, congenital valve abnormalities markedly increase the risk. Importantly, nearly half of older individuals with aortic stenosis also have a bicuspid aortic valve (BAV), an aortic valve that develops with two functional leaflets instead of the normal three109, 114, 115. BAV occurs in about 2–3% of the population and is the most common congenital cardiac malformation. Although the causes for the development of BAV are unclear, genetic factors have been identified in some cases. BAV disease tends to progress more rapidly for reasons that are poorly understood. Genetic mutations associated with BAV that cause cellular dysfunction may also predispose an individual to other congenital heart defects or to dilation and dissection of the ascending aorta114.
Detailed clinical histopathology reveals valve inflammation as a common theme whether CAVD arises with BAV, hypertension, dyslipidemia, or any of the other risk settings highlighted above. Furthermore, the pathological lesions of calcified aortic valves demonstrate the complexities and heterogeneity of soft tissue mineralization. Calcium accrual in areas of fibrosis, necrosis, atherosis/lipid accumulation, hypertrophic cartilage and ectopic bone can all be observed116, 117. Otto and colleagues described the fibro-fatty expansion and inflammation of the lamina fibrosa, displaced and/or split elastic lamina, and intracellular and extracellular lipid accumulation with stippled calcification in the earliest of valve lesions118. With progression, woven bone formation – complete with marrow elements – and nodular amorphous calcium phosphate accumulates, the latter presumably via epitaxial mineral deposition on virtually acellular concretions. Nevertheless, the molecular “fingerprints” of active osteochondrogenic mineralization are present in all calcifying human aortic valve specimens -- even if overt ossification (ectopic bone replete with marrow) is not observed116, 119121. Features of both membranous and endochondral osteogenic programs are elaborated by calcifying valve interstitial cells (VICs)121, with osteogenic potential highly dependent upon the stiffness of the extracellular matrix environment122. Additional osteoprogenitors may derive from chondrocytes originating at the base of the semilunar valve leaflet insertions123 and from valve ECs via EMT (see below). The frequent (~15%) ectopic bone formation and virtually omnipresent elaboration of osteochondrocytic programs in CAVD prompted evaluation for the potential contributions of Wnt/β-catenin signaling9.
Wnt/β-Catenin Signaling and the Biology of Calcific Aortic Stenosis
The first evidence that activation of Wnt/β-catenin signaling participated in the calcification of human aortic valves came from quantitative western blot analysis comparing calcifying human tricuspid aortic valves vs. normal aortic valves and mitral valves undergoing myxomatous degeneration. Accumulation of β-catenin, the prototypic mediator and marker of canonical Wnt signaling necessary for osteoblast formation23, was upregulated 3.5- to 4-fold in calcifying tricuspid aortic valves as compared to normal aortic valves or myxomatous mitral valves119. Expression of the osteochondrogenic transcription factor Runx2/Cbfa1 was also increased along with the chondrocytic transcription factor Sox9 and the bone-specific matrix proteins bone sialoprotein and osteocalcin. Additionally, expression of the Wnt co-receptor LRP5 and the ligand Wnt3 were identified as being increased in calcified aortic valves as assessed by immunohistochemistry119. Subsequent studies by Miller, Heistad and colleagues confirmed the upregulation of Runx2 and Msx2 in calcifying valves, with overlapping yet distinct patters of expression. Analyses of preclinical models have provided mechanistic insights into the role of Wnt/β-catenin signaling in diabetes- and dyslipidemia- induced arteriosclerotic calcification121. Transgenic augmentation of aortic valve and vascular Msx2 expression upregulates multiple arterial Wnt ligand expression and increases in arterial calcium deposition. Implementing the TOPGAL mouse that possesses a LacZ-based reporter for endogenous activation of β-catenin dependent-transcription, concomitant upregulation of Wnt/β-catenin was observed in calcifying aortic valves as well as arterial tunica media124. Consistent with this, the capacity of VICs to undergo myofibroblastic conversion ex vivo is also dependent upon β-catenin signaling that synergizes with TGFβ40. The specific Wnt ligands and LRP receptors participating in valve calcification have yet to be characterized, although LRP5 and Wnt3 are potential candidates based upon evaluation of calcifying human valve specimens.
At this, point, the important role of the endothelium –including both the aortic and ventricular surfaces of valve endothelium -- – should be re-emphasized. The biology of the aortic valve is regulated by valve ECs adjacent to valve interstitial myofibroblast cells125. These ECs maintain the health of the valve and mediate valve disease in the presence of cardiovascular risk factors and/or genetic signals. In the skeleton, angiogenesis and osteogenesis are tightly coupled processes77. In keeping with this relationship, Rajamannan et al have provided evidence of osteogenic-angiogenic “coupling” in ossifying human rheumatic valves analyzed following removal for valve replacement surgery72. The full spectrum of paracrine signals participating in valve cell-cell interaction – and the signals that trigger activation and differentiation of VICs along the osteogenic lineage -- have not been fully established. However, BMP-Wnt signaling in endothelial integration of valve matrix metabolism and remodeling will continue to emerge as being important. As mentioned above, the inflamed endothelium elaborates BMP2 and BMP4. Triggers include hemodynamic shear stress, abnormal endothelial nitric oxide synthase function, inflammatory cytokines and growth factors, and the intracellular metabolic environment arising from dyslipidemia, hyperglycemia, and uremia. Moreover, as D’Amore and colleagues first noted, endothelial cells express LRP5 and LRP6, in addition to multiple Wnt ligands126, 127 – and platelet –derived LRP5/6 antagonist induces an inflammatory EC phenotype128. Intriguingly, valve endothelial cells have the capacity to undergo mesenchymal transitioning (EnMT or EMT),129 a process whereby cadherin-mediated cell-cell interactions are re-organized, endothelial differentiation programs are down-regulated, and myofibroblastic gene regulatory programs are activated. Upon acquiring the myofibroblast phenotype, valve ECs post-EMT have the capacity to undergo osteogenic differentiation93 – resembling in this fashion the emerging role of the circulating endothelial progenitor cell (ePC) as a vascular osteoprogenitor91, 130. During development, members of the TGFβ superfamily drive and coordinate valve EMT in concert with Msx gene family members and β-catenin dependent signals94, 131. Finally, ECs of the valve ventricular surface elaborate gene regulatory programs distinct from those of the aortic surface132. The molecular regulators control post-natal valve EC fates and phenotypes have yet to be fully characterized. Of note, both Smad2 and β-catenin signaling have been implicated in the epithelial-mesenchymal transitioning that contributes to myofibroblast load in idiopathic pulmonary fibrosis133. Lessons learned from these studies may help guide productive experimental approaches to valve sclerosis that occurs either in the presence or absence of calcium accrual.
Wnt/β-catenin Signaling In Diabetic Arteriosclerosis and Vascular Fibrosis
When fed high fat diets (HFD) characteristic of westernized societies, male LDLR-deficient mice develop diet-induced obesity, dyslipidemia, and hyper-insulinemic diabetes characteristic of T2DM and the metabolic syndrome134, 135. Recent studies from MESA identify that these metabolic syndrome parameters convey risk for aortic and arterial calcification136. Consistent with this, male LDLR−/− mice on HFD develop progressively severe arteriosclerosis and fibrosis, with medial and atherosclerotic vascular mineralization134, 135, 137, 138. Early on, aortic upregulation of BMP2 and Msx2 is observed with subsequent Runx2 expression. Msx2-expressing adventitial myofibroblasts elaborate osteogenic Wnt3a, Wnt7a, and Wnt7b expression with concomitant paracrine activation of type I collagen and osterix gene expression, alkaline phosphatase activity, and osteogenic mineralization124, 137, 139, 140. Treatment with Dkk1, an inhibitor of LRP4/5/6 specifically down-regulated by Msx2, inhibits osteogenic mineralization by aortic myofibroblasts and other mesenchymal cell types124, 141.
As first predicted by Demer, Tintut, and colleagues142, inflammatory cytokine and oxidative stress cues appear to play an important role in the initiation of diabetic arteriosclerosis55, 137, 143. The prototypic inflammatory cytokine TNF, largely derived from adipose tissue macrophages, enhances oxylipid-dependent mineralization of calcifying vascular cells and adventitial myofibroblasts on rigid matrices such as tissue culture plastic. In addition to upregulating BMP2 production in endothelial cells, TNF directly activates arteriosclerotic Msx2 and Wnt/β-catenin signaling, the latter localized again to the tunica media with the TOPGAL reporter mouse137. Pharmacologic inhibition of endogenous oxidative stress signals generated by mitochondrial and Nox – family flavoenzymes inhibits TNF activation of myofibroblast Msx2 expression138. Recent data by others have confirmed that oxylipids such as oxLDL promote aortic VSMC mineralization in part via upregulation of osteogenic Msx2 signaling144146, although the Wnt/Dkk and β-catenin responses were not specifically evaluated. Moreover, oxLDL upregulates BMP2 expression in vascular myofibroblasts via tolloid receptors TLR2 and TLR4147. Thus, in the vasculature osteogenic BMP-Wnt signaling is entrained to innate immunity and inflammatory redox cues that initiate and propagate tissue fibrosis with osteogenic differentiation of local progenitors. Moreover, ROS signaling enhances Runx2-directed trans-activation that drives and reinforces the osteochondrogenic phenotype of trans-differentiating VSMCs148 (Figure 1). It is tempting to speculate that the “diabesity” associated inflammatory redox signals that drive BMP-Wnt signaling may promote macrovascular EMT -- and thus further contribute to fibrosis, calcification, and mural thickening in diabetic arteriosclerosis. Novel strategies that combine inhibition of inflammatory cytokine signaling with enhanced egress of inflammatory oxylipids149 may serve to inhibit arteriosclerotic disease initiation and progression via vascular down-regulation of these osteogenic morphogens.
Selective modulation of vascular BMP activity is a promising target for treatment or prevention of arterial calcification. Decreased BMP activity resulting from excess MGP limits calcification 53, 55 and BMP type I receptor inhibition by small molecules has been successfully used to limit heterotopic calcification in mice with fibrodysplasia ossificans progressiva 150. However, there are still many questions that need clarification in the targeting and design of BMP inhibitors. For example, what cells would be the best targets, endothelial, smooth muscle or adventitial cells? What would the best molecular targets, ligands, receptors or inhibitors? Similar strategies can be envisioned that would target the Wnt signaling cascade. However, since post-natal bone homeostasis and skeletal integrity is dependent upon BMP-Wnt signaling, can strategies be identified that inhibit osteogenic vascular BMP-Wnt actions without mechanism-based toxicity in the skeleton? Similar issues arise when one considers many other mechanisms for modulating osteogenic morphogens in vascular disease. For example, Notch1 sensitizes VSMC to pro-osteogenic BMP2 signaling151 including the upregulation of alkaline phosphatase via Msx2-dependent actions 152 while inhibiting bone marrow osteoprogenitor differentiation153. This suggests that antagonism of Notch1 signaling might safely help ameliorate pathogenic BMP signaling in VSMC-mediated arterial calcification. Yet, Notch1 also inhibits osteochondrogenic signaling in calcifying aortic valve cells 154, 155. Moreover, decreases in Notch1 tone predispose to calcinotic bicuspid aortic valve formation155157 and potential vascular tumor formation with hemorrhage158. Given the emerging difference in valvular vs. vascular Notch1 actions, it remains to be determined whether modulation of the Notch1 signaling pathways offer a significantly wide therapeutic window for pharmacologic intervention.
Alternatively, strategies can be envisioned that aggressively target the pivotal signals upstream of vascular BMP-Wnt activation to prevent disease initiation and slow arteriosclerotic progression. As discussed, inflammation, an abnormal oxidative stress environment, oxylipids stimulate the vascular endothelium to upregulate the secretion of BMPs important in osteogenic cell signaling 53, 75, 76, 138. These same signals down-regulate important mineralization defense mechanisms, including the degradation of inorganic pyrophosphate by alkaline phosphatase135, 159 and fetuin160. Furthermore, mechanical stretch plays a permissive role, allowing vascular osteoprogenitors to transition to a calcifying phenotype122. Thus, as contributors to pathobiology each of this processes are potential targets for pharmacologic intervention. HMG CoA Reductase agents, angiotensin converting enzyme (ACE) inhibitors, and angiotensin receptor blockers (ARBs), provide one approach. Indeed, in the Japanese Aortic Stenosis Study JASS, the use of ARBs was associated with reduced risk of disease progression in CAVD161. Interestingly, in preclinical models pulsatile hPTH(1–34) administration simultaneously reduces vascular 139and skeletal 162, 163oxidative stress signals – while simultaneously reducing vascular calcification and enhancing bone mass accrual140. Thus, novel anti-inflammatory ApoA1 mimetics such as D-4F164, 165 may help promote the egress of inflammatory oxylipids generated in the setting of diabetes and dyslipidemia in ways that simultaneously improve vascular and bone health149. However, in any one individual, certain pathophysiological stimuli (hyperphosphatemia, hyperlipidemia, hypertension, hyperglycemia) may be more critically important for intervention than others1 – and may differ with stage of disease. This may account for the disparate responses observed with respect to the impact of statin therapy upon CAVD166. With declining renal function, hyperphosphatemia plays an increasingly important role in vascular calcification167. Finally, once a significant amount of vascular calcium has accrued – or true ectopic ossification has occurred – a “point of no return” may be reached at which medical intervention by any means is no longer possible. Thus, careful patient characterization and selection will be required when testing known or novel therapeutic interventions to prevent or retard vascular calcification.
Acknowledgments
Sources of Funding
K.I.B. is supported by NIH grants HL081397 and HL030568. N.M.R. is supported by NIH grant HL085591. D.A.T. is supported by NIH grants HL069229, HL081138, HL088651, and by the Barnes-Jewish Hospital Foundation.
Non-standard Abbreviations and Acronyms
ACEangiotensin converting enzyme
AERapical ectodermal ridge
ALKactivin like kinase
ALPalkaline phosphatase (a.k.a. TNAP, akp2)
ApoApolipoprotein
ARBangiotensin receptor blocker
ATFactivating transcription factor
BAVbiscuspid aortic valve
BMPbone morphogenetic protein
BMPRBMP receptor
BSPbone sialoprotein
CAVDcalcific aortic valve disease
Cbfa1core-binding factor alpha subunit 1
CK1casein kinase 1
CKDchronic kidney disease
Colcollagen gene
Col1A1type I collagen gene for α-1 chain
Col1A2type I collagen gene for α-2 chain
COMP2cartilage oligomeric matrix protein 2
CV2crossveinless 2 (a.k.a. BMP-binding endothelial cell precursor-derived regulator)
CVCcalcifying vascular cell
Dkkdickkopf homolog
Dvldishevelled
ECendothelial cell
EMTendothelial-mesenchymal transitioning
ePCendothelial progenitor cell
FGFfibroblast growth factor
Fzdfrizzled homolog
G-protein alpha subunit
GlcGlucose
GPCRG protein coupled receptor
GRKG-protein coupled receptor kinase
GSKglycogen synthase kinase
HIFhypoxia induced factor
HFDdiabetogenic high fat western diet
HMG CoAhydroxymethylglutaryl coenzyme A
ICATinhibitor of β-catenin and TCF
IhhIndian Hedgehog
I-Smadinhibitory Smad
JASSJapanese Aortic Stenosis Study
LacZβ-galactosidase
LDLRlow density lipoprotein receptor
LEFlymphoid enhancer binding factor
LRPLDLR related protein
MAPKmitogen activated protein kinase
MESAMultiethnic Study of Atherosclerosis
MGPmatrix gamma-carboxylated protein
MMPmatrix metalloproteinase
Msxmuscle segment homeobox protein homolog
NFATnuclear factor of activated T cells
OCOsteocalcin
OPNOsteopontin
Osxosterix
oxLDLoxidized LDL
PiT1sodium phosphate transporter a.k.a. SLC20A1
PKAprotein kinase A
PKCprotein kinase C
PLCphospholipase C
PO4phosphorylated Ser/Thr residue
PTHparathyroid hormone
PTHrPPTH-related protein
PTH1RPTH/PTHrP receptor
ROR2receptor tyrosine kinase-like orphan receptor 2
ROSreactive oxygen specifies
R-Smadreceptor associated Smad
Runx2Runt-related transcription factor 2 (a.k.a. Cbfa1)
sFRPsecreted frizzled-related protein
Shhsonic hedgehog
Smadmothers against decapentaplegia homolog
SOSTsclerostin
SoxSry-related HMG- box gene
T2DMtype II diabetes mellitus
TGFtransforming growth factor
TCFT cell transcription factor
TNFtumor necrosis factor alpha
TLRtolloid like receptor
TOPGALTCF/LEF optimal promoter-galactosidase reporter mouse
VEGFvascular endothelial growth factor
VSMCvascular smooth muscle cell
Wntwingless-type MMTV integration site family member
ZPAzone of polarizing activity

Footnotes
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Disclosures
None.
1. Demer LL, Tintut Y. Vascular calcification: pathobiology of a multifaceted disease. Circulation. 2008;117(22):2938–2948. [PubMed]
2. Tanimura A, McGregor DH, Anderson HC. Calcification in atherosclerosis. I. Human studies. J Exp Pathol. 1986;2(4):261–273. [PubMed]
3. Tanimura A, McGregor DH, Anderson HC. Matrix vesicles in atherosclerotic calcification. Proc Soc Exp Biol Med. 1983;172(2):173–177. [PubMed]
4. Urist MR, Strates BS. Bone formation in implants of partially and wholly demineralized bone matrix. Including observations on acetone-fixed intra and extracellular proteins. Clin Orthop Relat Res. 1970;71:271–278. [PubMed]
5. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA. Novel regulators of bone formation: molecular clones and activities. Science. 1988;242(4885):1528–1534. [PubMed]
6. Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest. 1993;91(4):1800–1809. [PMC free article] [PubMed]
7. Wan M, Cao X. BMP signaling in skeletal development. Biochem Biophys Res Commun. 2005;328(3):651–657. [PubMed]
8. Tsuji K, Bandyopadhyay A, Harfe BD, Cox K, Kakar S, Gerstenfeld L, Einhorn T, Tabin CJ, Rosen V. BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing. Nat Genet. 2006;38(12):1424–1429. [PubMed]
9. Johnson ML, Rajamannan N. Diseases of Wnt signaling. Rev Endocr Metab Disord. 2006;7(1–2):41–49. [PubMed]
10. Towler DA, Shao JS, Cheng SL, Pingsterhaus JM, Loewy AP. Osteogenic regulation of vascular calcification. Ann N Y Acad Sci. 2006;1068:327–333. [PubMed]
11. Karsenty G, Kronenberg HM, Settembre C. Genetic control of bone formation. Annu Rev Cell Dev Biol. 2009;25:629–648. [PubMed]
12. Cohen MM., Jr Merging the old skeletal biology with the new. I. Intramembranous ossification, endochondral ossification, ectopic bone, secondary cartilage, and pathologic considerations. J Craniofac Genet Dev Biol. 2000;20(2):84–93. [PubMed]
13. Cohen MM., Jr The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A. 2006;140(23):2646–2706. [PubMed]
14. Capdevila J, Izpisua Belmonte JC. Patterning mechanisms controlling vertebrate limb development. Annu Rev Cell Dev Biol. 2001;17:87–132. [PubMed]
15. Yang Y. Growth and patterning in the limb: signaling gradients make the decision. Sci Signal. 2009;2(53):pe3. [PubMed]
16. Benazet JD, Zeller R. Vertebrate limb development: moving from classical morphogen gradients to an integrated 4-dimensional patterning system. Cold Spring Harb Perspect Biol. 2009;1(4):a001339. [PMC free article] [PubMed]
17. Adamska M, MacDonald BT, Sarmast ZH, Oliver ER, Meisler MH. En1 and Wnt7a interact with Dkk1 during limb development in the mouse. Dev Biol. 2004;272(1):134–144. [PubMed]
18. Kronenberg HM. Developmental regulation of the growth plate. Nature. 2003;423(6937):332–336. [PubMed]
19. Canalis E. Update in new anabolic therapies for osteoporosis. J Clin Endocrinol Metab. 2010;95(4):1496–1504. [PubMed]
20. de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S, Huang W. Transcriptional mechanisms of chondrocyte differentiation. Matrix Biol. 2000;19(5):389–394. [PubMed]
21. Smits P, Li P, Mandel J, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B, Lefebvre V. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell. 2001;1(2):277–290. [PubMed]
22. Araldi E, Schipani E. Hypoxia, HIFs and bone development. Bone. 2010;47(2):190–196. [PMC free article] [PubMed]
23. Hu H, Hilton MJ, Tu X, Yu K, Ornitz DM, Long F. Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development. 2005;132(1):49–60. [PubMed]
24. Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, Maeda T, Takano Y, Uchiyama M, Heaney S, Peters H, Tang Z, Maxson R, Maas R. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet. 2000;24(4):391–395. [PubMed]
25. Koga T, Matsui Y, Asagiri M, Kodama T, de Crombrugghe B, Nakashima K, Takayanagi H. NFAT and Osterix cooperatively regulate bone formation. Nat Med. 2005;11(8):880–885. [PubMed]
26. Urist MR, Strates BS. The classic: Bone morphogenetic protein. Clin Orthop Relat Res. 2009;467(12):3051–3062. [PMC free article] [PubMed]
27. Umulis D, O'Connor MB, Blair SS. The extracellular regulation of bone morphogenetic protein signaling. Development. 2009;136(22):3715–3728. [PubMed]
28. Chang H, Brown CW, Matzuk MM. Genetic analysis of the mammalian transforming growth factor-beta superfamily. Endocr Rev. 2002;23(6):787–823. [PubMed]
29. Du Y, Wang Y, Wang L, Liu B, Tian Q, Liu CJ, Zhang T, Xu Q, Zhu Y, Ake O, Qi Y, Tang C, Kong W, Wang X. Cartilage oligomeric matrix protein inhibits vascular smooth muscle calcification by interacting with bone morphogenetic protein-2. Circ Res. 2011;108(8):917–928. [PubMed]
30. David L, Feige JJ, Bailly S. Emerging role of bone morphogenetic proteins in angiogenesis. Cytokine Growth Factor Rev. 2009;20(3):203–212. [PubMed]
31. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 2003;113(6):685–700. [PubMed]
32. Miyazono K, Maeda S, Imamura T. BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine Growth Factor Rev. 2005;16(3):251–263. [PubMed]
33. Rawadi G, Vayssiere B, Dunn F, Baron R, Roman-Roman S. BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res. 2003;18(10):1842–1853. [PubMed]
34. Baron R, Rawadi G. Wnt signaling and the regulation of bone mass. Curr Osteoporos Rep. 2007;5(2):73–80. [PubMed]
35. Mikels AJ, Nusse R. Wnts as ligands: processing, secretion and reception. Oncogene. 2006;25(57):7461–7468. [PubMed]
36. van Amerongen R, Nusse R. Towards an integrated view of Wnt signaling in development. Development. 2009;136(19):3205–3214. [PubMed]
37. Minami Y, Oishi I, Endo M, Nishita M. Ror-family receptor tyrosine kinases in noncanonical Wnt signaling: their implications in developmental morphogenesis and human diseases. Dev Dyn. 2010;239(1):1–15. [PubMed]
38. Shitashige M, Hirohashi S, Yamada T. Wnt signaling inside the nucleus. Cancer Sci. 2008;99(4):631–637. [PubMed]
39. Shafer SL, Towler DA. Transcriptional regulation of SM22alpha by Wnt3a: convergence with TGFbeta(1)/Smad signaling at a novel regulatory element. J Mol Cell Cardiol. 2009;46(5):621–635. [PMC free article] [PubMed]
40. Chen JH, Chen WL, Sider KL, Yip CY, Simmons CA. {beta}-Catenin Mediates Mechanically Regulated, Transforming Growth Factor-{beta}1-Induced Myofibroblast Differentiation of Aortic Valve Interstitial Cells. Arterioscler Thromb Vasc Biol. 2011;31(3):590–597. [PubMed]
41. Niehrs C, Shen J. Regulation of Lrp6 phosphorylation. Cell Mol Life Sci. 2010;67(15):2551–2562. [PubMed]
42. Tu X, Joeng KS, Nakayama KI, Nakayama K, Rajagopal J, Carroll TJ, McMahon AP, Long F. Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation. Dev Cell. 2007;12(1):113–127. [PMC free article] [PubMed]
43. Bergmann MW. WNT signaling in adult cardiac hypertrophy and remodeling: lessons learned from cardiac development. Circ Res. 2010;107(10):1198–1208. [PubMed]
44. Pinzone JJ, Hall BM, Thudi NK, Vonau M, Qiang YW, Rosol TJ, Shaughnessy JD., Jr The role of Dickkopf-1 in bone development, homeostasis, and disease. Blood. 2009;113(3):517–525. [PubMed]
45. Collette NM, Genetos DC, Murugesh D, Harland RM, Loots GG. Genetic evidence that SOST inhibits WNT signaling in the limb. Dev Biol. 2010;342(2):169–179. [PMC free article] [PubMed]
46. Semenov M, Tamai K, He X. SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. J Biol Chem. 2005;280(29):26770–26775. [PubMed]
47. Li X, Zhang Y, Kang H, Liu W, Liu P, Zhang J, Harris SE, Wu D. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J Biol Chem. 2005;280(20):19883–19887. [PubMed]
48. Stow JL. ICAT is a multipotent inhibitor of beta-catenin. Focus on "role for ICAT in beta-catenin-dependent nuclear signaling and cadherin functions". Am J Physiol Cell Physiol. 2004;286(4):C745–C746. [PubMed]
49. Dhore CR, Cleutjens JP, Lutgens E, Cleutjens KB, Geusens PP, Kitslaar PJ, Tordoir JH, Spronk HM, Vermeer C, Daemen MJ. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2001;21(12):1998–2003. [PubMed]
50. Schluesener HJ, Meyermann R. Immunolocalization of BMP-6, a novel TGF-beta-related cytokine, in normal and atherosclerotic smooth muscle cells. Atherosclerosis. 1995;113(2):153–156. [PubMed]
51. Shao JS, Cai J, Towler DA. Molecular mechanisms of vascular calcification: lessons learned from the aorta. Arterioscler. Thromb. Vasc. Biol. 2006;26(7):1423–1430. [PubMed]
52. Hruska KA, Mathew S, Saab G. Bone morphogenetic proteins in vascular calcification. Circ Res. 2005;97(2):105–114. [PubMed]
53. Bostrom KI, Jumabay M, Matveyenko A, Nicholas SB, Yao Y. Activation of vascular bone morphogenetic protein signaling in diabetes mellitus. Circ Res. 2011;108(4):446–457. [PMC free article] [PubMed]
54. Nakagawa Y, Ikeda K, Akakabe Y, Koide M, Uraoka M, Yutaka KT, Kurimoto-Nakano R, Takahashi T, Matoba S, Yamada H, Okigaki M, Matsubara H. Paracrine osteogenic signals via bone morphogenetic protein-2 accelerate the atherosclerotic intimal calcification in vivo. Arterioscler Thromb Vasc Biol. 2010;30(10):1908–1915. [PubMed]
55. Yao Y, Bennett BJ, Wang X, Rosenfeld ME, Giachelli C, Lusis AJ, Bostrom KI. Inhibition of bone morphogenetic proteins protects against atherosclerosis and vascular calcification. Circ Res. 2010;107(4):485–494. [PMC free article] [PubMed]
56. David L, Feige JJ, Bailly S. Emerging role of bone morphogenetic proteins in angiogenesis. Cytokine Growth Factor Rev. 2009;20(3):203–212. [PubMed]
57. Moreno-Miralles I, Schisler JC, Patterson C. New insights into bone morphogenetic protein signaling: focus on angiogenesis. Curr Opin Hematol. 2009;16(3):195–201. [PMC free article] [PubMed]
58. Govani FS, Shovlin CL. Hereditary haemorrhagic telangiectasia: a clinical and scientific review. Eur J Hum Genet. 2009;17(7):860–871. [PMC free article] [PubMed]
59. Long L, Crosby A, Yang X, Southwood M, Upton PD, Kim DK, Morrell NW. Altered bone morphogenetic protein and transforming growth factor-beta signaling in rat models of pulmonary hypertension: potential for activin receptor-like kinase-5 inhibition in prevention and progression of disease. Circulation. 2009;119(4):566–576. [PubMed]
60. Yang J, Davies RJ, Southwood M, Long L, Yang X, Sobolewski A, Upton PD, Trembath RC, Morrell NW. Mutations in bone morphogenetic protein type II receptor cause dysregulation of Id gene expression in pulmonary artery smooth muscle cells: implications for familial pulmonary arterial hypertension. Circ Res. 2008;102(10):1212–1221. [PubMed]
61. Zebboudj AF, Imura M, Bostrom K. Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2. J Biol Chem. 2002;277(6):4388–4394. [PubMed]
62. Yao Y, Shahbazian A, Bostrom KI. Proline and gamma-carboxylated glutamate residues in matrix Gla protein are critical for binding of bone morphogenetic protein-4. Circ Res. 2008;102(9):1065–1074. [PubMed]
63. Yao Y, Zebboudj AF, Shao E, Perez M, Bostrom K. Regulation of bone morphogenetic protein-4 by matrix GLA protein in vascular endothelial cells involves activin-like kinase receptor 1. J Biol Chem. 2006;281(45):33921–33930. [PubMed]
64. Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature. 1997;386(6620):78–81. [PubMed]
65. Speer MY, Yang HY, Brabb T, Leaf E, Look A, Lin WL, Frutkin A, Dichek D, Giachelli CM. Smooth muscle cells give rise to osteochondrogenic precursors and chondrocytes in calcifying arteries. Circ Res. 2009;104(6):733–741. [PMC free article] [PubMed]
66. Jara A, Chacon C, Burgos ME, Droguett A, Valdivieso A, Ortiz M, Troncoso P, Mezzano S. Expression of gremlin, a bone morphogenetic protein antagonist, is associated with vascular calcification in uraemia. Nephrol Dial Transplant. 2009;24(4):1121–1129. [PubMed]
67. Yao Y, Nowak S, Yochelis A, Garfinkel A, Bostrom KI. Matrix GLA protein, an inhibitory morphogen in pulmonary vascular development. J Biol Chem. 2007;282(41):30131–30142. [PubMed]
68. Shao ES, Lin L, Yao Y, Bostrom KI. Expression of vascular endothelial growth factor is coordinately regulated by the activin-like kinase receptors 1 and 5 in endothelial cells. Blood. 2009;114(10):2197–2206. [PubMed]
69. Garfinkel A, Tintut Y, Petrasek D, Bostrom K, Demer LL. Pattern formation by vascular mesenchymal cells. Proc Natl Acad Sci U S A. 2004;101(25):9247–9250. [PubMed]
70. Zebboudj AF, Shin V, Bostrom K. Matrix GLA protein and BMP-2 regulate osteoinduction in calcifying vascular cells. J Cell Biochem. 2003;90(4):756–765. [PubMed]
71. Govani FS, Shovlin CL. Fine mapping of the hereditary haemorrhagic telangiectasia (HHT)3 locus on chromosome 5 excludes VE-Cadherin-2, Sprouty4 and other interval genes. J Angiogenes Res. 2010;2:15. [PMC free article] [PubMed]
72. Rajamannan NM, Nealis TB, Subramaniam M, Pandya S, Stock SR, Ignatiev CI, Sebo TJ, Rosengart TK, Edwards WD, McCarthy PM, Bonow RO, Spelsberg TC. Calcified rheumatic valve neoangiogenesis is associated with vascular endothelial growth factor expression and osteoblast-like bone formation. Circulation. 2005;111(24):3296–3301. [PubMed]
73. Sorescu GP, Song H, Tressel SL, Hwang J, Dikalov S, Smith DA, Boyd NL, Platt MO, Lassegue B, Griendling KK, Jo H. Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress induces monocyte adhesion by stimulating reactive oxygen species production from a nox1-based NADPH oxidase. Circ. Res. 2004;95(8):773–779. [PubMed]
74. Sorescu GP, Sykes M, Weiss D, Platt MO, Saha A, Hwang J, Boyd N, Boo YC, Vega JD, Taylor WR, Jo H. Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response. J Biol Chem. 2003;278(33):31128–31135. [PubMed]
75. Csiszar A, Smith KE, Koller A, Kaley G, Edwards JG, Ungvari Z. Regulation of bone morphogenetic protein-2 expression in endothelial cells: role of nuclear factor-kappaB activation by tumor necrosis factor-alpha, H2O2, and high intravascular pressure. Circulation. 2005;111(18):2364–2372. [PubMed]
76. Csiszar A, Ahmad M, Smith KE, Labinskyy N, Gao Q, Kaley G, Edwards JG, Wolin MS, Ungvari Z. Bone morphogenetic protein-2 induces proinflammatory endothelial phenotype. Am J Pathol. 2006;168(2):629–638. [PubMed]
77. Towler DA. The osteogenic-angiogenic interface: novel insights into the biology of bone formation and fracture repair. Curr Osteoporos Rep. 2008;6(2):67–71. [PubMed]
78. Chang K, Weiss D, Suo J, Vega JD, Giddens D, Taylor WR, Jo H. Bone morphogenic protein antagonists are coexpressed with bone morphogenic protein 4 in endothelial cells exposed to unstable flow in vitro in mouse aortas and in human coronary arteries: role of bone morphogenic protein antagonists in inflammation and atherosclerosis. Circulation. 2007;116(11):1258–1266. [PubMed]
79. Yao Y, Shao ES, Jumabay M, Shahbazian A, Ji S, Bostrom KI. High-density lipoproteins affect endothelial BMP-signaling by modulating expression of the activin-like kinase receptor 1 and 2. Arterioscler Thromb Vasc Biol. 2008;28(12):2266–2274. [PMC free article] [PubMed]
80. Helbing T, Rothweiler R, Heinke J, Goetz L, Diehl P, Zirlik A, Patterson C, Bode C, Moser M. BMPER is upregulated by statins and modulates endothelial inflammation by intercellular adhesion molecule-1. Arterioscler Thromb Vasc Biol. 2010;30(3):554–560. [PMC free article] [PubMed]
81. Canfield AE, Doherty MJ, Wood AC, Farrington C, Ashton B, Begum N, Harvey B, Poole A, Grant ME, Boot-Handford RP. Role of pericytes in vascular calcification: a review. Z Kardiol. 2000;89 Suppl 2:20–27. [PubMed]
82. Collett GD, Canfield AE. Angiogenesis and pericytes in the initiation of ectopic calcification. Circ Res. 2005;96(9):930–938. [PubMed]
83. Doherty MJ, Ashton BA, Walsh S, Beresford JN, Grant ME, Canfield AE. Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res. 1998;13(5):828–838. [PubMed]
84. Sage AP, Lu J, Tintut Y, Demer LL. Hyperphosphatemia-induced nanocrystals upregulate the expression of bone morphogenetic protein-2 and osteopontin genes in mouse smooth muscle cells in vitro. Kidney Int. 2011;79(4):414–422. [PMC free article] [PubMed]
85. Chen NX, Duan D, O'Neill KD, Moe SM. High glucose increases the expression of Cbfa1 and BMP-2 and enhances the calcification of vascular smooth muscle cells. Nephrol Dial Transplant. 2006;21(12):3435–3442. [PubMed]
86. Nett PC, Ortmann J, Celeiro J, Haas E, Hofmann-Lehmann R, Tornillo L, Terraciano LM, Barton M. Transcriptional regulation of vascular bone morphogenetic protein by endothelin receptors in early autoimmune diabetes mellitus. Life Sci. 2006;78(19):2213–2218. [PubMed]
87. Bostrom KI, Jumabay M, Matveyenko A, Nicholas SB, Yao Y. Activation of Vascular Bone Morphogenetic Protein Signaling in Diabetes Mellitus. Circ. Res. 2010 [PMC free article] [PubMed]
88. Nett PC, Ortmann J, Celeiro J, Haas E, Hofmann-Lehmann R, Tornillo L, Terraciano LM, Barton M. Transcriptional regulation of vascular bone morphogenetic protein by endothelin receptors in early autoimmune diabetes mellitus. Life Sci. 2006;78(19):2213–2218. [PubMed]
89. Nguyen TQ, Chon H, van Nieuwenhoven FA, Braam B, Verhaar MC, Goldschmeding R. Myofibroblast progenitor cells are increased in number in patients with type 1 diabetes and express less bone morphogenetic protein 6: a novel clue to adverse tissue remodelling? Diabetologia. 2006;49(5):1039–1048. [PubMed]
90. Watson KE, Bostrom K, Ravindranath R, Lam T, Norton B, Demer LL. TGF-beta 1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest. 1994;93(5):2106–2113. [PMC free article] [PubMed]
91. Medici D, Shore EM, Lounev VY, Kaplan FS, Kalluri R, Olsen BR. Conversion of vascular endothelial cells into multipotent stem-like cells. Nat Med. 2010;16(12):1400–1406. [PMC free article] [PubMed]
92. Yao Y, Bennett BJ, Wang X, Rosenfeld ME, Giachelli C, Lusis AJ, Bostrom KI. Inhibition of bone morphogenetic proteins protects against atherosclerosis and vascular calcification. Circ. Res. 2010;107(4):485–494. [PMC free article] [PubMed]
93. Wylie-Sears J, Aikawa E, Levine RA, Yang JH, Bischoff J. Mitral valve endothelial cells with osteogenic differentiation potential. Arterioscler Thromb Vasc Biol. 2011;31(3):598–607. [PMC free article] [PubMed]
94. Liebner S, Cattelino A, Gallini R, Rudini N, Iurlaro M, Piccolo S, Dejana E. Beta-catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse. J Cell Biol. 2004;166(3):359–367. [PMC free article] [PubMed]
95. Shanahan CM, Cary NR, Salisbury JR, Proudfoot D, Weissberg PL, Edmonds ME. Medial localization of mineralization-regulating proteins in association with Monckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation. 1999;100(21):2168–2176. [PubMed]
96. Amann K. Media calcification and intima calcification are distinct entities in chronic kidney disease. Clin J Am Soc Nephrol. 2008;3(6):1599–1605. [PubMed]
97. Speer MY, Giachelli CM. Regulation of cardiovascular calcification. Cardiovasc Pathol. 2004;13(2):63–70. [PubMed]
98. Li X, Yang HY, Giachelli CM. BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells. Atherosclerosis. 2008;199(2):271–277. [PMC free article] [PubMed]
99. Li X, Giachelli CM. Sodium-dependent phosphate cotransporters and vascular calcification. Curr Opin Nephrol Hypertens. 2007;16(4):325–328. [PubMed]
100. Li X, Yang HY, Giachelli CM. Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification. Circ Res. 2006;98(7):905–912. [PubMed]
101. Maciel TT, Kempf H, Campos AH. Targeting bone morphogenetic protein signaling on renal and vascular diseases. Curr Opin Nephrol Hypertens. 2010;19(1):26–31. [PubMed]
102. Araya CE, Fennell RS, Neiberger RE, Dharnidharka VR. Sodium thiosulfate treatment for calcific uremic arteriolopathy in children and young adults. Clin J Am Soc Nephrol. 2006;1(6):1161–1166. [PubMed]
103. Sowers KM, Hayden MR. Calcific uremic arteriolopathy: pathophysiology, reactive oxygen species and therapeutic approaches. Oxid Med Cell Longev. 2010;3(2):109–121. [PMC free article] [PubMed]
104. Rifkin BS, Perazella MA. Calcific uremic arteriolopathy (calciphylaxis) Mayo Clin Proc. 2006;81(1):9. [PubMed]
105. Griethe W, Schmitt R, Jurgensen JS, Bachmann S, Eckardt KU, Schindler R. Bone morphogenic protein-4 expression in vascular lesions of calciphylaxis. J Nephrol. 2003;16(5):728–732. [PubMed]
106. Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol. 2004;15(11):2857–2867. [PubMed]
107. Reynolds JL, Skepper JN, McNair R, Kasama T, Gupta K, Weissberg PL, Jahnen-Dechent W, Shanahan CM. Multifunctional roles for serum protein fetuin-a in inhibition of human vascular smooth muscle cell calcification. J Am Soc Nephrol. 2005;16(10):2920–2930. [PubMed]
108. Collett G, Wood A, Alexander MY, Varnum BC, Boot-Handford RP, Ohanian V, Ohanian J, Fridell YW, Canfield AE. Receptor tyrosine kinase Axl modulates the osteogenic differentiation of pericytes. Circ Res. 2003;92(10):1123–1129. [PubMed]
109. Rajamannan NM, Bonow RO, Rahimtoola SH. Calcific aortic stenosis: an update. Nat Clin Pract Cardiovasc Med. 2007;4(5):254–262. [PubMed]
110. Otto CM. Valvular aortic stenosis: disease severity and timing of intervention. J Am Coll Cardiol. 2006;47(11):2141–2151. [PubMed]
111. Rosenhek R, Klaar U, Schemper M, Scholten C, Heger M, Gabriel H, Binder T, Maurer G, Baumgartner H. Mild and moderate aortic stenosis. Natural history and risk stratification by echocardiography. Eur Heart J. 2004;25(3):199–205. [PubMed]
112. Rosenhek R, Binder T, Porenta G, Lang I, Christ G, Schemper M, Maurer G, Baumgartner H. Predictors of outcome in severe, asymptomatic aortic stenosis. N Engl J Med. 2000;343(9):611–617. [PubMed]
113. O'Brien KD. Pathogenesis of calcific aortic valve disease: a disease process comes of age (and a good deal more) Arterioscler Thromb Vasc Biol. 2006;26(8):1721–1728. [PubMed]
114. Rajamannan NM. Bicuspid aortic valve disease: the role of oxidative stress in Lrp5 bone formation. Cardiovasc Pathol. 2011 [PMC free article] [PubMed]
115. Rajamannan NM, Gersh B, Bonow RO. Calcific aortic stenosis: from bench to the bedside--emerging clinical and cellular concepts. Heart. 2003;89(7):801–805. [PMC free article] [PubMed]
116. Rajamannan NM, Subramaniam M, Rickard D, Stock SR, Donovan J, Springett M, Orszulak T, Fullerton DA, Tajik AJ, Bonow RO, Spelsberg T. Human aortic valve calcification is associated with an osteoblast phenotype. Circulation. 2003;107(17):2181–2184. [PubMed]
117. Mohler ER, 3rd, Gannon F, Reynolds C, Zimmerman R, Keane MG, Kaplan FS. Bone formation and inflammation in cardiac valves. Circulation. 2001;103(11):1522–1528. [PubMed]
118. Otto CM, Kuusisto J, Reichenbach DD, Gown AM, O'Brien KD. Characterization of the early lesion of 'degenerative' valvular aortic stenosis. Histological and immunohistochemical studies. Circulation. 1994;90(2):844–853. [PubMed]
119. Caira FC, Stock SR, Gleason TG, McGee EC, Huang J, Bonow RO, Spelsberg TC, McCarthy PM, Rahimtoola SH, Rajamannan NM. Human degenerative valve disease is associated with up-regulation of low-density lipoprotein receptor-related protein 5 receptor-mediated bone formation. J Am Coll Cardiol. 2006;47(8):1707–1712. [PubMed]
120. Rajamannan NM, Subramaniam M, Springett M, Sebo TC, Niekrasz M, McConnell JP, Singh RJ, Stone NJ, Bonow RO, Spelsberg TC. Atorvastatin inhibits hypercholesterolemia-induced cellular proliferation and bone matrix production in the rabbit aortic valve. Circulation. 2002;105(22):2660–2665. [PubMed]
121. Miller JD, Chu Y, Brooks RM, Richenbacher WE, Pena-Silva R, Heistad DD. Dysregulation of antioxidant mechanisms contributes to increased oxidative stress in calcific aortic valvular stenosis in humans. J Am Coll Cardiol. 2008;52(10):843–850. [PMC free article] [PubMed]
122. Yip CY, Chen JH, Zhao R, Simmons CA. Calcification by valve interstitial cells is regulated by the stiffness of the extracellular matrix. Arterioscler Thromb Vasc Biol. 2009;29(6):936–942. [PubMed]
123. Chen JH, Yip CY, Sone ED, Simmons CA. Identification and characterization of aortic valve mesenchymal progenitor cells with robust osteogenic calcification potential. Am J Pathol. 2009;174(3):1109–1119. [PubMed]
124. Shao JS, Cheng SL, Pingsterhaus JM, Charlton-Kachigian N, Loewy AP, Towler DA. Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals. J Clin Invest. 2005;115(5):1210–1220. [PMC free article] [PubMed]
125. Rajamannan NM. Calcific aortic stenosis: lessons learned from experimental and clinical studies. Arterioscler Thromb Vasc Biol. 2009;29(2):162–168. [PMC free article] [PubMed]
126. Goodwin AM, Sullivan KM, D'Amore PA. Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling. Dev Dyn. 2006;235(11):3110–3120. [PubMed]
127. Goodwin AM, D'Amore PA. Wnt signaling in the vasculature. Angiogenesis. 2002;5(1–2):1–9. [PubMed]
128. Ueland T, Otterdal K, Lekva T, Halvorsen B, Gabrielsen A, Sandberg WJ, Paulsson-Berne G, Pedersen TM, Folkersen L, Gullestad L, Oie E, Hansson GK, Aukrust P. Dickkopf-1 enhances inflammatory interaction between platelets and endothelial cells and shows increased expression in atherosclerosis. Arterioscler Thromb Vasc Biol. 2009;29(8):1228–1234. [PubMed]
129. Paruchuri S, Yang JH, Aikawa E, Melero-Martin JM, Khan ZA, Loukogeorgakis S, Schoen FJ, Bischoff J. Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2. Circ Res. 2006;99(8):861–869. [PMC free article] [PubMed]
130. Gossl M, Modder UI, Atkinson EJ, Lerman A, Khosla S. Osteocalcin expression by circulating endothelial progenitor cells in patients with coronary atherosclerosis. J Am Coll Cardiol. 2008;52(16):1314–1325. [PMC free article] [PubMed]
131. Chen YH, Ishii M, Sucov HM, Maxson RE., Jr Msx1 and Msx2 are required for endothelial-mesenchymal transformation of the atrioventricular cushions and patterning of the atrioventricular myocardium. BMC Dev Biol. 2008;8:75. [PMC free article] [PubMed]
132. Simmons CA, Grant GR, Manduchi E, Davies PF. Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves. Circ Res. 2005;96(7):792–799. [PMC free article] [PubMed]
133. Kim KK, Wei Y, Szekeres C, Kugler MC, Wolters PJ, Hill ML, Frank JA, Brumwell AN, Wheeler SE, Kreidberg JA, Chapman HA. Epithelial cell alpha3beta1 integrin links beta-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis. J Clin Invest. 2009;119(1):213–224. [PMC free article] [PubMed]
134. Towler DA, Bidder M, Latifi T, Coleman T, Semenkovich CF. Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice. J Biol Chem. 1998;273(46):30427–30434. [PubMed]
135. Shao JS, Cheng SL, Sadhu J, Towler DA. Inflammation and the osteogenic regulation of vascular calcification: a review and perspective. Hypertension. 2010;55(3):579–592. [PMC free article] [PubMed]
136. Katz R, Wong ND, Kronmal R, Takasu J, Shavelle DM, Probstfield JL, Bertoni AG, Budoff MJ, O'Brien KD. Features of the metabolic syndrome and diabetes mellitus as predictors of aortic valve calcification in the Multi-Ethnic Study of Atherosclerosis. Circulation. 2006;113(17):2113–2119. [PubMed]
137. Al-Aly Z, Shao JS, Lai CF, Huang E, Cai J, Behrmann A, Cheng SL, Towler DA. Aortic Msx2-Wnt calcification cascade is regulated by TNF-alpha-dependent signals in diabetic Ldlr−/− mice. Arterioscler Thromb Vasc Biol. 2007;27(12):2589–2596. [PubMed]
138. Shao JS, Aly ZA, Lai CF, Cheng SL, Cai J, Huang E, Behrmann A, Towler DA. Vascular Bmp Msx2 Wnt signaling and oxidative stress in arterial calcification. Ann N Y Acad Sci. 2007;1117:40–50. [PubMed]
139. Cheng SL, Shao JS, Halstead LR, Distelhorst K, Sierra O, Towler DA. Activation of vascular smooth muscle parathyroid hormone receptor inhibits Wnt/beta-catenin signaling and aortic fibrosis in diabetic arteriosclerosis. Circ Res. 2010;107(2):271–282. [PMC free article] [PubMed]
140. Shao JS, Cheng SL, Charlton-Kachigian N, Loewy AP, Towler DA. Teriparatide (human parathyroid hormone (1–34)) inhibits osteogenic vascular calcification in diabetic low density lipoprotein receptor-deficient mice. J Biol Chem. 2003;278(50):50195–50202. [PubMed]
141. Cheng SL, Shao JS, Cai J, Sierra OL, Towler DA. Msx2 exerts bone anabolism via canonical Wnt signaling. J Biol Chem. 2008;283(29):20505–20522. [PubMed]
142. Tintut Y, Patel J, Parhami F, Demer LL. Tumor necrosis factor-alpha promotes in vitro calcification of vascular cells via the cAMP pathway. Circulation. 2000;102(21):2636–2642. [PubMed]
143. Yao Y, Watson AD, Ji S, Bostrom KI. Heat shock protein 70 enhances vascular bone morphogenetic protein-4 signaling by binding matrix Gla protein. Circ Res. 2009;105(6):575–584. [PMC free article] [PubMed]
144. Preusch MR, Rattazzi M, Albrecht C, Merle U, Tuckermann J, Schutz G, Blessing E, Zoppellaro G, Pauletto P, Krempien R, Rosenfeld ME, Katus HA, Bea F. Critical role of macrophages in glucocorticoid driven vascular calcification in a mouse-model of atherosclerosis. Arterioscler Thromb Vasc Biol. 2008;28(12):2158–2164. [PubMed]
145. Taylor J, Butcher M, Zeadin M, Politano A, Shaughnessy SG. Oxidized low-density lipoprotein promotes osteoblast differentiation in primary cultures of vascular smooth muscle cells by up-regulating Osterix expression in an Msx2-dependent manner. J Cell Biochem. 2011;112(2):581–588. [PubMed]
146. Yan J, Stringer SE, Hamilton A, Charlton-Menys V, Gotting C, Muller B, Aeschlimann D, Alexander MY. Decorin GAG Synthesis and TGF-{beta} Signaling Mediate Ox-LDL-Induced Mineralization of Human Vascular Smooth Muscle Cells. Arterioscler Thromb Vasc Biol. 2011;31(3):608–615. [PubMed]
147. Su X, Ao L, Shi Y, Johnson TR, Fullerton DA, Meng X. Oxidized low-density lipoprotein induces bone morphogenetic protein-2 in coronary artery endothelial cells via Toll-like receptors 2 and 4. J Biol Chem. 2011 [PubMed]
148. Byon CH, Javed A, Dai Q, Kappes JC, Clemens TL, Darley-Usmar VM, McDonald JM, Chen Y. Oxidative stress induces vascular calcification through modulation of the osteogenic transcription factor Runx2 by AKT signaling. J Biol Chem. 2008;283(22):15319–15327. [PubMed]
149. Sage AP, Lu J, Atti E, Tetradis S, Ascenzi MG, Adams DJ, Demer LL, Tintut Y. Hyperlipidemia induces resistance to PTH bone anabolism in mice via oxidized lipids. J Bone Miner Res. 2010 [PubMed]
150. Yu PB, Deng DY, Lai CS, Hong CC, Cuny GD, Bouxsein ML, Hong DW, McManus PM, Katagiri T, Sachidanandan C, Kamiya N, Fukuda T, Mishina Y, Peterson RT, Bloch KD. BMP type I receptor inhibition reduces heterotopic [corrected] ossification. Nat Med. 2008;14(12):1363–1369. [PMC free article] [PubMed]
151. Shimizu T, Tanaka T, Iso T, Matsui H, Ooyama Y, Kawai-Kowase K, Arai M, Kurabayashi M. Notch enhances bone morphogenetic protein2 (BMP2) responsiveness of Msx2 gene to induce osteogenic differentiation and mineralization of vascular smooth muscle cells. J Biol Chem. 2011 [PubMed]
152. Shimizu T, Tanaka T, Iso T, Doi H, Sato H, Kawai-Kowase K, Arai M, Kurabayashi M. Notch signaling induces osteogenic differentiation and mineralization of vascular smooth muscle cells: role of Msx2 gene induction via Notch-RBP-Jk signaling. Arterioscler Thromb Vasc Biol. 2009;29(7):1104–1111. [PubMed]
153. Hilton MJ, Tu X, Wu X, Bai S, Zhao H, Kobayashi T, Kronenberg HM, Teitelbaum SL, Ross FP, Kopan R, Long F. Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation. Nat Med. 2008;14(3):306–314. [PMC free article] [PubMed]
154. Nigam V, Srivastava D. Notch1 represses osteogenic pathways in aortic valve cells. J Mol Cell Cardiol. 2009;47(6):828–834. [PMC free article] [PubMed]
155. Garg V, Muth AN, Ransom JF, Schluterman MK, Barnes R, King IN, Grossfeld PD, Srivastava D. Mutations in NOTCH1 cause aortic valve disease. Nature. 2005;437(7056):270–274. [PubMed]
156. McKellar SH, Tester DJ, Yagubyan M, Majumdar R, Ackerman MJ, Sundt TM., 3rd Novel NOTCH1 mutations in patients with bicuspid aortic valve disease and thoracic aortic aneurysms. J Thorac Cardiovasc Surg. 2007;134(2):290–296. [PubMed]
157. Ellison JW, Yagubyan M, Majumdar R, Sarkar G, Bolander ME, Atkinson EJ, Sarano ME, Sundt TM. Evidence of genetic locus heterogeneity for familial bicuspid aortic valve. J Surg Res. 2007;142(1):28–31. [PubMed]
158. Liu Z, Turkoz A, Jackson EN, Corbo JC, Engelbach JA, Garbow JR, Piwnica-Worms DR, Kopan R. Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice. J Clin Invest. 2011;121(2):800–808. [PMC free article] [PubMed]
159. Johnson K, Polewski M, van Etten D, Terkeltaub R. Chondrogenesis mediated by PPi depletion promotes spontaneous aortic calcification in NPP1−/− mice. Arterioscler Thromb Vasc Biol. 2005;25(4):686–691. [PubMed]
160. Floege J, Ketteler M. Vascular calcification in patients with end-stage renal disease. Nephrol Dial Transplant. 2004;19 Suppl 5:V59–V66. [PubMed]
161. Yamamoto K, Yamamoto H, Yoshida K, Kisanuki A, Hirano Y, Ohte N, Akasaka T, Takeuchi M, Nakatani S, Ohtani T, Sozu T, Masuyama T. Prognostic factors for progression of early- and late-stage calcific aortic valve disease in Japanese: the Japanese Aortic Stenosis Study (JASS) Retrospective Analysis. Hypertens Res. 2010;33(3):269–274. [PubMed]
162. Jilka RL, Almeida M, Ambrogini E, Han L, Roberson PK, Weinstein RS, Manolagas SC. Decreased oxidative stress and greater bone anabolism in the aged, when compared to the young, murine skeleton with parathyroid hormone administration. Aging Cell. 2010;9(5):851–867. [PMC free article] [PubMed]
163. Weinstein RS, Jilka RL, Almeida M, Roberson PK, Manolagas SC. Intermittent parathyroid hormone administration counteracts the adverse effects of glucocorticoids on osteoblast and osteocyte viability, bone formation, and strength in mice. Endocrinology. 2010;151(6):2641–2649. [PubMed]
164. Navab M, Anantharamaiah GM, Fogelman AM. The effect of apolipoprotein mimetic peptides in inflammatory disorders other than atherosclerosis. Trends Cardiovasc Med. 2008;18(2):61–66. [PubMed]
165. Navab M, Anantharamaiah GM, Reddy ST, Hama S, Hough G, Grijalva VR, Yu N, Ansell BJ, Datta G, Garber DW, Fogelman AM. Apolipoprotein A-I mimetic peptides. Arterioscler Thromb Vasc Biol. 2005;25(7):1325–1331. [PubMed]
166. Rajamannan NM. Reassessment of statins to retard the progression of aortic stenosis. Curr Cardiol Rep. 2007;9(2):99–104. [PubMed]
167. Giachelli CM, Speer MY, Li X, Rajachar RM, Yang H. Regulation of vascular calcification: roles of phosphate and osteopontin. Circ Res. 2005;96(7):717–722. [PubMed]
168. Koay EJ, Athanasiou KA. Hypoxic chondrogenic differentiation of human embryonic stem cells enhances cartilage protein synthesis and biomechanical functionality. Osteoarthritis Cartilage. 2008;16(12):1450–1456. [PubMed]
169. Kanichai M, Ferguson D, Prendergast PJ, Campbell VA. Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: a role for AKT and hypoxia-inducible factor (HIF)-1alpha. J Cell Physiol. 2008;216(3):708–715. [PubMed]
170. Egli RJ, Bastian JD, Ganz R, Hofstetter W, Leunig M. Hypoxic expansion promotes the chondrogenic potential of articular chondrocytes. J Orthop Res. 2008;26(7):977–985. [PubMed]
171. Ross S, Hill CS. How the Smads regulate transcription. Int J Biochem Cell Biol. 2008;40(3):383–408. [PubMed]
172. Zhang YW, Yasui N, Ito K, Huang G, Fujii M, Hanai J, Nogami H, Ochi T, Miyazono K, Ito Y. A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia. Proc Natl Acad Sci U S A. 2000;97(19):10549–10554. [PubMed]
173. Lin SL, Li B, Rao S, Yeo EJ, Hudson TE, Nowlin BT, Pei H, Chen L, Zheng JJ, Carroll TJ, Pollard JW, McMahon AP, Lang RA, Duffield JS. Macrophage Wnt7b is critical for kidney repair and regeneration. Proc Natl Acad Sci U S A. 2010;107(9):4194–4199. [PubMed]
174. Lobov IB, Rao S, Carroll TJ, Vallance JE, Ito M, Ondr JK, Kurup S, Glass DA, Patel MS, Shu W, Morrisey EE, McMahon AP, Karsenty G, Lang RA. WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature. Nature. 2005;437(7057):417–421. [PubMed]
175. Baron R, Rawadi G, Roman-Roman S. Wnt signaling: a key regulator of bone mass. Curr Top Dev Biol. 2006;76:103–127. [PubMed]
176. Rooney B, O'Donovan H, Gaffney A, Browne M, Faherty N, Curran SP, Sadlier D, Godson C, Brazil DP, Crean J. CTGF/CCN2 activates canonical Wnt signalling in mesangial cells through LRP6: implications for the pathogenesis of diabetic nephropathy. FEBS Lett. 2011;585(3):531–538. [PubMed]
177. Nam JS, Turcotte TJ, Smith PF, Choi S, Yoon JK. Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate beta-catenin-dependent gene expression. J Biol Chem. 2006;281(19):13247–13257. [PubMed]
178. Binnerts ME, Kim KA, Bright JM, Patel SM, Tran K, Zhou M, Leung JM, Liu Y, Lomas WE, 3rd, Dixon M, Hazell SA, Wagle M, Nie WS, Tomasevic N, Williams J, Zhan X, Levy MD, Funk WD, Abo A. R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6. Proc Natl Acad Sci U S A. 2007;104(37):14700–14705. [PubMed]
179. Wei Q, Yokota C, Semenov MV, Doble B, Woodgett J, He X. R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and beta-catenin signaling. J Biol Chem. 2007;282(21):15903–15911. [PubMed]
180. Kim KA, Wagle M, Tran K, Zhan X, Dixon MA, Liu S, Gros D, Korver W, Yonkovich S, Tomasevic N, Binnerts M, Abo A. R-Spondin family members regulate the Wnt pathway by a common mechanism. Mol Biol Cell. 2008;19(6):2588–2596. [PMC free article] [PubMed]
181. Faverman L, Mikhaylova L, Malmquist J, Nurminskaya M. Extracellular transglutaminase 2 activates beta-catenin signaling in calcifying vascular smooth muscle cells. FEBS Lett. 2008;582(10):1552–1557. [PubMed]