**2. The cellular and molecular regulation of valve development**

#### **2.1. Overview of valve development**

Valve development begins with the formation of endocardial cushions in the atrioventricu‐ lar (AV) canal and outflow tract (OFT) of the primitive heart tube, which occurs at embryon‐ ic day (E)9-10 in mice, E3 in chickens, and E31-35 in humans [8]. The first evidence of endocardial cushion formation is the separation of the endocardium and overlying myocar‐ dium in the AV canal by expansion of the cardiac jelly through increased expression of hya‐ luronan (Figure 1) [15]. These swellings are invested with mesenchymal cells that arise from endothelial-to-mesenchymal transformation (EMT) of the endocardium [16]. Similar swel‐ ling and induction of EMT occur approximately a day later in the cardiac OFT cushions that will contribute to the semilunar valves [17]. Endocardial EMT is induced by signaling mole‐ cules, including bone morphogenetic proteins (BMPs), emanating from the adjacent myocar‐ dium in the AVC and OFT [8, 18-20]. Once established, the endocardial cushions expand through increased extracellular matrix (ECM) production and cell proliferation of mesen‐ chymal and endothelial cells. The AV cushions subsequently fuse to separate right and left cardiac channels. In addition, lateral cushions are induced in the AV sulcus that will give rise to the mural leaflets of the mitral and tricuspid valves [21]. Neural crest cells (NCCs) migrate into the cushions of the cardiac OFT, contributing to the septum between the aortic and pulmonic roots and also to the morphogenesis of individual semilunar valve leaflets [21, 22]. At this point, distinct primordia of individual valve leaflets become apparent and proliferation of valve interstitial cells (VICs) is reduced [23]. Valve morphogenesis occurs with elongation and thinning of the valve primordia, in addition to ECM remodeling and stratification. In general, the development of the AV and semilunar (SL) valves is similar, but there are some differences in the sources of cells and structure of the resulting leaflets [8, 10, 11, 24]. In mature SL and AV valves, the ECM is stratified into collagen-rich fibrosa, pro‐ teoglycan-rich spongiosa, and elastin-rich (atrialis-ventricularis) layers oriented relative to blood flow [24].

#### **2.2. Embryonic origins of valve cell lineages**

Additional research in animal models and human patient specimens is necessary to deter‐ mine the detrimental molecular regulatory pathways that promote CAVD progression and also beneficial pathways that potentially inhibit CAVD. In the future, manipulation of these pathways could be exploited therapeutically in the treatment of patients with CAVD or with

**Figure 1. Molecular pathways active during endocardial cushion development and valve stratification are reacti‐ vated in CAVD.** (A) Early stages of OFT cushion development are marked by ECM deposition, EMT, and neural crest cell in‐ filtration. Factors necessary for EMT and mesenchymal cell function are expressed. (B) During late embryonic development and early postnatal development, the aortic valve becomes stratified and possesses three ECM layers. Factors necessary for ECM remodeling are active at this stage. (C) In CAVD, the ECM remodels and the valve becomes thickened. Calcification (black nodules) is typically observed in the collagen-rich fibrosa layer. Many factors expressed during OFT cushion develop‐ ment and valve stratification are reactivated during disease. Furthermore, osteogenic factors involved in bone develop‐ ment are also observed in CAVD. Please see text for details and references. OFT = outflow tract, EMT = epithelial-to-

Valve development begins with the formation of endocardial cushions in the atrioventricu‐ lar (AV) canal and outflow tract (OFT) of the primitive heart tube, which occurs at embryon‐ ic day (E)9-10 in mice, E3 in chickens, and E31-35 in humans [8]. The first evidence of endocardial cushion formation is the separation of the endocardium and overlying myocar‐ dium in the AV canal by expansion of the cardiac jelly through increased expression of hya‐

mesenchymal transition, ECM = extracellular matrix, CAVD = calcific aortic valve disease.

**2.1. Overview of valve development**

**2. The cellular and molecular regulation of valve development**

aortic valve sclerosis that precedes calcification.

60 Calcific Aortic Valve Disease

The primary embryonic source of adult semilunar valve interstitial cells is the endothelialderived cells of the endocardial cushions, that arise as a result of EMT as determined by Tie2-Cre lineage tracing in mice [23, 25]. Since the cardiac OFT is derived from the secon‐ dary heart field (SHF), semilunar VICs derived from OFT endocardium also are SHF-de‐ rived [20, 26]. NCC-derived cells are present in adult mouse semilunar valve leaflets as demonstrated by cell lineage tracing with Wnt1-Cre [27]. These cells are predominant throughout the aortic and pulmonic valve leaflets, but are enriched in the leaflets adjacent to the aorticopulmonary septum, which also is derived from NCCs [21, 28]. NCCs are required for semilunar valve morphogenesis and remodeling, likely by providing signals necessary for cell lineage differentiation and leaflet maturation [29, 30]. Another potential source of VICs is the epicardium, which contributes cells to the parietal leaflets of AV valves [31]. However, epicardial-derived cells (EPDCs) have not been not reported to contribute to the semilunar valves, based on Wt1-Cre fatemapping studies [31, 32]. Recent studies have re‐ ported that bone marrow-derived stem cells (BMSCs) are present in the developing and ma‐ ture semilunar valves [33, 34]. Additional work is necessary to determine if these cells have lineages and functions distinct from the predominant endocardial cushion-derived or neural crest-derived VICs. It is possible that valve cell lineages derived from different developmen‐ tal sources have distinct functions in normal and diseased aortic valves, but this has not yet been demonstrated. The sources of increased proliferative cells in diseased valves are rela‐ tively unknown, but could be any of these embryonic sources or, alternatively, an infiltrat‐ ing cell type.

#### **2.3. Transcription factors involved in valve development**

Several transcription factors have been implicated in various processes of endocardial cush‐ ion formation and EMT (reviewed in [8, 35]). Notch pathway function in EMT is dependent on the transcription factor RBPJ, which activates expression of the bHLH transcription factor *snail1* (Snai1) in endothelial cells [36]. Snai1 represses *ve-cadherin* gene expression, and loss of Snai1 in endothelial cells inhibits endocardial cushion formation [36, 37]. The mesenchy‐ mal valve progenitor cells of the endocardial cushions express several transcription factors characteristic of a variety of embryonic mesenchymal progenitor populations. These factors include, Twist1, Msx1/2, Tbx20, and Sox9 [18, 38-41]. Gain and loss of function studies have demonstrated critical roles for Tbx20, Twist1, and Sox9 in endocardial cushion mesenchy‐ mal cell proliferation [38-40]. Twist1 promotes *tbx20* expression directly and also regulates several genes associated with cell proliferation and migration [38, 42]. After endocardial cushion fusion and formation of valve primordia, mesenchymal genes, notably *twist1* [43], are down-regulated and cell proliferation is decreased [23, 24, 44]. In normal adult valves, there is little to no cell proliferation [24, 44], and expression of valve developmental tran‐ scription factors including Twist1, Sox9, and Msx2 is not detectable [13]. However, all of these factors are predominantly expressed in adult human CAVD (see below).

BMP signaling, which promotes mesenchymal cell invasion [36, 51]. Likewise, vascular en‐ dothelial growth factor (VEGF) signaling promotes endocardial cushion endothelial cell pro‐ liferation and EMT [47, 52]. Furthermore, targeted mutagenesis of β-catenin has implicated Wnt/β-catenin signaling in endocardial cushion EMT and mesenchymal proliferation [53, 54]. Thus multiple pathways are involved in endocardial cushion EMT and mesenchymal cell proliferation. However, the intersection and specific cellular functions of these pathways

**A. Signaling pathways**

Notch EMT Inhibit OB differentiation represses calcification TGFβ EMT bone homeostasis promotes VIC calcification

BMP EMT, PG expression promotes OB specification active in CAVD Wnt/β-catenin EMT, fibrosa expression promotes OB differentiation active in CAVD

Twist1 ECC proliferation, migration represses differentiation active in CAVD Msx2 EMT, proliferation present in progenitors, OB active in CAVD

Runx2 not present promotes OB differentiation active in CAVD Osterix not present promotes OB differentiation reported in CAVD

**Table 1.** Signaling pathways and transcription factors involved in valvulogenesis, osteogenesis, and CAVDa, b

Please see text for details and references. bAbbreviations used: CAVD=calcific aortic valve dis‐ ease; ECM=extracellular matrix; EMT=endothelial to mesenchymal transition; OB=osteoblast;

Many of the signaling pathways important for endocardial cushion formation also have lat‐ er functions in valve lineage diversification, remodeling, and stratification. However, these functions have been difficult to elucidate due to limitations of available genetic tools and

endothelial proliferation, ECM remodeling

OC=osteoclast; PG=proteoglycan; VIC=valve interstitial cell.

RANKL ECM remodeling OC differentiation promotes VIC calcification **B. Transcription factors**

EMT, endothelial proliferation

promotes tenascin expression

Sox9 proliferation, PG expression

Role in valvulogenesis Role in osteogenesis Role in CAVD

OB proliferation, differentiation

Role in valvulogenesis Role in osteogenesis Role in CAVD

progenitor proliferation, cartilage differentiation

promotes OC differentiation promotes OB differentiation

angiogenesis angiogenesis

blocks VIC calcification

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 63

active in CAVD inhibits calcification

reported in CAVD

have not been fully determined.

VEGF

FGF

NFATc1

a

Additional regulatory pathways control heart valve ECM remodeling and compartmentali‐ zation. Loss of NFATc1 results in defective remodeling of the AV and SL valves in mice, with embryonic lethality by E14.5 [45, 46]. EMT occurs with loss of NFATc1, but valve pri‐ mordia fail to remodel and mature ECM molecules are not expressed in null mice or in cul‐ tured VICs with inhibition of receptor activator of nuclear factor κ-B ligand (RANKL) or calcineurin signaling upstream of NFATc1 activation [45, 47]. In addition to being required for endocardial cushion mesenchymal proliferation, Sox9 also promotes cartilage-like ECM gene expression in valve progenitor cells [48]. In late stage mouse embryos, loss of Sox9 in remodeling valves results in reduced proteoglycan expression, and Sox9 haploinsufficiency in adults leads to valve calcification [40, 49]. Conversely, the bHLH transcription factor Scleraxis, critical for tendon development, promotes expression of elastic/tendon-like matrix genes in cultured valve progenitor cells [48]. Loss of Scleraxis in mice is not lethal, but heart valve defects similar to myxomatous valve disease occur in these animals [50]. Little is known of the transcriptional regulatory mechanisms that control the development of the valve fibrosa layer, which is most critically involved in CAVD.

#### **2.4. Signaling pathways in valve development**

Several essential embryonic signaling pathways have been implicated in endocardial cush‐ ion formation and EMT (Table 1) (reviewed in [8]). Transforming growth factor (TGF)β sig‐ naling was the first pathway implicated in endocardial cushion formation and is required for EMT in chicken and mouse embryonic systems (reviewed in [16]). BMP signaling from the myocardium is required in endothelial cells for the initiation of EMT in the AV canal, and BMP2 and 4 are the predominant ligands involved in endocardial cushion development [18-20]. Notch signaling also is required for EMT as described above. Moreover, Notch sig‐ naling is required for expression of TGFβ ligands and receptors, in addition to activating BMP signaling, which promotes mesenchymal cell invasion [36, 51]. Likewise, vascular en‐ dothelial growth factor (VEGF) signaling promotes endocardial cushion endothelial cell pro‐ liferation and EMT [47, 52]. Furthermore, targeted mutagenesis of β-catenin has implicated Wnt/β-catenin signaling in endocardial cushion EMT and mesenchymal proliferation [53, 54]. Thus multiple pathways are involved in endocardial cushion EMT and mesenchymal cell proliferation. However, the intersection and specific cellular functions of these pathways have not been fully determined.

**2.3. Transcription factors involved in valve development**

62 Calcific Aortic Valve Disease

Several transcription factors have been implicated in various processes of endocardial cush‐ ion formation and EMT (reviewed in [8, 35]). Notch pathway function in EMT is dependent on the transcription factor RBPJ, which activates expression of the bHLH transcription factor *snail1* (Snai1) in endothelial cells [36]. Snai1 represses *ve-cadherin* gene expression, and loss of Snai1 in endothelial cells inhibits endocardial cushion formation [36, 37]. The mesenchy‐ mal valve progenitor cells of the endocardial cushions express several transcription factors characteristic of a variety of embryonic mesenchymal progenitor populations. These factors include, Twist1, Msx1/2, Tbx20, and Sox9 [18, 38-41]. Gain and loss of function studies have demonstrated critical roles for Tbx20, Twist1, and Sox9 in endocardial cushion mesenchy‐ mal cell proliferation [38-40]. Twist1 promotes *tbx20* expression directly and also regulates several genes associated with cell proliferation and migration [38, 42]. After endocardial cushion fusion and formation of valve primordia, mesenchymal genes, notably *twist1* [43], are down-regulated and cell proliferation is decreased [23, 24, 44]. In normal adult valves, there is little to no cell proliferation [24, 44], and expression of valve developmental tran‐ scription factors including Twist1, Sox9, and Msx2 is not detectable [13]. However, all of

these factors are predominantly expressed in adult human CAVD (see below).

valve fibrosa layer, which is most critically involved in CAVD.

**2.4. Signaling pathways in valve development**

Additional regulatory pathways control heart valve ECM remodeling and compartmentali‐ zation. Loss of NFATc1 results in defective remodeling of the AV and SL valves in mice, with embryonic lethality by E14.5 [45, 46]. EMT occurs with loss of NFATc1, but valve pri‐ mordia fail to remodel and mature ECM molecules are not expressed in null mice or in cul‐ tured VICs with inhibition of receptor activator of nuclear factor κ-B ligand (RANKL) or calcineurin signaling upstream of NFATc1 activation [45, 47]. In addition to being required for endocardial cushion mesenchymal proliferation, Sox9 also promotes cartilage-like ECM gene expression in valve progenitor cells [48]. In late stage mouse embryos, loss of Sox9 in remodeling valves results in reduced proteoglycan expression, and Sox9 haploinsufficiency in adults leads to valve calcification [40, 49]. Conversely, the bHLH transcription factor Scleraxis, critical for tendon development, promotes expression of elastic/tendon-like matrix genes in cultured valve progenitor cells [48]. Loss of Scleraxis in mice is not lethal, but heart valve defects similar to myxomatous valve disease occur in these animals [50]. Little is known of the transcriptional regulatory mechanisms that control the development of the

Several essential embryonic signaling pathways have been implicated in endocardial cush‐ ion formation and EMT (Table 1) (reviewed in [8]). Transforming growth factor (TGF)β sig‐ naling was the first pathway implicated in endocardial cushion formation and is required for EMT in chicken and mouse embryonic systems (reviewed in [16]). BMP signaling from the myocardium is required in endothelial cells for the initiation of EMT in the AV canal, and BMP2 and 4 are the predominant ligands involved in endocardial cushion development [18-20]. Notch signaling also is required for EMT as described above. Moreover, Notch sig‐ naling is required for expression of TGFβ ligands and receptors, in addition to activating


a Please see text for details and references. bAbbreviations used: CAVD=calcific aortic valve dis‐ ease; ECM=extracellular matrix; EMT=endothelial to mesenchymal transition; OB=osteoblast; OC=osteoclast; PG=proteoglycan; VIC=valve interstitial cell.

**Table 1.** Signaling pathways and transcription factors involved in valvulogenesis, osteogenesis, and CAVDa, b

Many of the signaling pathways important for endocardial cushion formation also have lat‐ er functions in valve lineage diversification, remodeling, and stratification. However, these functions have been difficult to elucidate due to limitations of available genetic tools and critical requirements for these same regulatory pathways in endocardial cushion formation. BMP signaling, as indicated by phosphorylation of the intermediate signaling molecules Smad1/5/8, is active throughout endocardial cushion mesenchymal cells, is associated with mesenchymal cell proliferation [55], and also is active later in valve cell lineage diversifica‐ tion [48]. *BMP Receptor II* mutations and conditional mutagenesis results in thickening of semilunar valve leaflets at late fetal stages [56, 57]. Loss of inhibitory Smad6 leads to in‐ creased BMP signaling, in addition to thickening of valve leaflets and CAVD in adult ani‐ mals [58]. Studies in explanted avian valve progenitors have revealed antagonistic regulatory roles for BMP and fibroblast growth factor (FGF) signaling in promoting diversi‐ fied ECM gene expression, conserved with mechanisms that control cartilage and tendon lineage development [11, 48, 59]. Wnt pathway activation is evident throughout the remod‐ eling AV and semilunar valve primordia, as indicated in TopGal reporter mice [60]. Multi‐ ple Wnt ligands are expressed during valvulogenesis, but the function of Wnt signaling in heart valve remodeling has not yet been determined [60]. Thus, additional in vivo studies are necessary to determine the specific functions and intersecting regulatory mechanisms of these critical signaling pathways in valve leaflet development and also to determine specific contributions to valve degeneration and disease.

are likely to be additional factors necessary for congenital malformation of the aortic valve leaflets [65]. Loss of the zinc finger transcription factor GATA5 in mice [66] and mutations in human *GATA5* [67] are associated with BAV with incomplete penetrance. Likewise eNOS haploinsufficiency also leads to BAV, albeit with incomplete penetrance [68]. The mecha‐ nisms by which these genetic lesions lead to BAV in some individuals and not others are not known. However, based on the expression and function of Notch1, GATA5, and eNOS in endothelial cells, it is likely that these cells contribute to development of BAV in these mod‐ els. The link between BAV and CAVD could be due to similar regulatory mechanisms in de‐ velopment and disease or could, alternatively, result from induction of calcification in a hemodynamically compromised congenitally malformed aortic valve (see other chapters for

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 65

**2.5. Extracellular matrix composition and stratification of the developing valves**

The mature valve leaflets are composed of stratified ECM with layered compartments of fi‐ brillar collagen, proteoglycan, and elastin (Figure 1) (reviewed in [10, 69]). During heart valve remodeling, there is little proliferation of VICs, but the cells are highly synthetic and produce multiple ECM proteins of the mature leaflets [24, 44]. The distinct layers of matrix are integral to heart valve function and confer specific biomechanical properties to the valve leaflets [69]. The regulatory mechanisms for ECM remodeling and stratification are not well defined but are relevant to heart valve disease mechanisms. Periostin is required for colla‐ gen remodeling, and loss of periostin in mice leads to adult valve malformations and cardiac dysfunction [70, 71]. Likewise, mutations in *Collagen 1a2* or elastin haploinsufficiency also result in aortic valve dysmorphogenesis and adult disease [72, 73]. Gene expression of *CtsK*, a matrix remodeling enzyme expressed during heart valve elongation, is regulated by the RANKL/NFATc1 regulatory pathway [47, 61]. Additional ECM remodeling enzymes, in‐ cluding matrix metalloproteinase (MMP)13, a collagenase, and Adam-TS5 and 9 proteogly‐ can proteases, also are expressed during late valve morphogenesis and have been implicated in ECM maturation and organization [39, 74, 75]. Several ECM molecules required for nor‐ mal valve structure/function also are expressed during osteogenesis, and valve progenitors have gene expression profiles similar to bone progenitors [43]. *Osteopontin*, *osteonectin*, and *periostin* gene expression and collagen fiber deposition are increased during heart valve re‐ modeling [24, 43, 60]. However, the regulatory mechanisms for expression of these genes in valve development are not well defined. These proteins also are induced and mislocalized in pediatric and adult heart valve disease [13, 24, 70], but the pathways leading to their dysre‐

Many osteogenic regulatory interactions identified in developing bone also are active in CAVD (Table 1). The regulatory hierarchies and ECM composition of the developing valves,

a more complete discussion of BAV and CAVD).

gulation have not yet been fully characterized.

**3.1. Overview of skeletal development**

**3. Molecular mechanisms of osteogenesis**

The later stages of heart valve development are characterized by leaflet elongation, ECM re‐ modeling, and stratification, all of which are critical for mature valve structure and function [24]. Limited information is available on the regulation of these processes, but several regula‐ tory pathways have been implicated in late valve remodeling and morphogenesis. Strikingly these same pathways have been implicated in adult CAVD (see below). RANKL, expressed by valve endothelial cells, promotes ECM remodeling and Cathepsin K (Ctsk) expression by NFATc1 in a mechanism partially conserved with osteoclast differentiation and function [11, 47, 61]. The signaling mechanisms that control stratification and ECM organization of the valve leaflets are relatively unknown. Notch signaling is localized on the ventricularis surface of the remodeling aortic valve in mice [62], and Wnt/β-catenin signaling is active throughout aortic valve primordia at late gestation and in a subpopulation of VICs after birth [60]. Like‐ wise, Wnt signaling promotes expression of fibrosa genes *periostin* and *matrix gla-protein* (*mgp)* in cultured chicken embryo aortic VICs, but a role in valve stratification or lineage diversifica‐ tion has not yet been established in vivo [60]. Additional studies are necessary to demon‐ strate the specific functions and potential biomechanical stimulation of these pathways in an in vivo context. Since, both Notch and Wnt signaling pathways are required for initial stages of endocardial cushion formation, it has been difficult to establish their roles in the later stages of valvulogenesis in vivo using available conditional targeting approaches.

Bicuspid aortic valve is arguably the most common congenital heart malformation with an incidence of 1-2% in the US adult population [63]. BAV often does not often manifest in valve dysfunction in early life, but malformed aortic valves are predisposed to calcification. Strikingly, the majority of stenotic aortic valves that are replaced in adults are congenitally malformed [4]. However, the molecular and cellular mechanisms of BAV are not well de‐ fined. In humans, mutations in *NOTCH1* are associated with BAV, but the mechanisms by which valve leaflet number is regulated by Notch signaling have not yet been identified [64]. Likewise, Notch1 haploinsufficiency in mice leads to BAV at very low penetrance and there are likely to be additional factors necessary for congenital malformation of the aortic valve leaflets [65]. Loss of the zinc finger transcription factor GATA5 in mice [66] and mutations in human *GATA5* [67] are associated with BAV with incomplete penetrance. Likewise eNOS haploinsufficiency also leads to BAV, albeit with incomplete penetrance [68]. The mecha‐ nisms by which these genetic lesions lead to BAV in some individuals and not others are not known. However, based on the expression and function of Notch1, GATA5, and eNOS in endothelial cells, it is likely that these cells contribute to development of BAV in these mod‐ els. The link between BAV and CAVD could be due to similar regulatory mechanisms in de‐ velopment and disease or could, alternatively, result from induction of calcification in a hemodynamically compromised congenitally malformed aortic valve (see other chapters for a more complete discussion of BAV and CAVD).

#### **2.5. Extracellular matrix composition and stratification of the developing valves**

The mature valve leaflets are composed of stratified ECM with layered compartments of fi‐ brillar collagen, proteoglycan, and elastin (Figure 1) (reviewed in [10, 69]). During heart valve remodeling, there is little proliferation of VICs, but the cells are highly synthetic and produce multiple ECM proteins of the mature leaflets [24, 44]. The distinct layers of matrix are integral to heart valve function and confer specific biomechanical properties to the valve leaflets [69]. The regulatory mechanisms for ECM remodeling and stratification are not well defined but are relevant to heart valve disease mechanisms. Periostin is required for colla‐ gen remodeling, and loss of periostin in mice leads to adult valve malformations and cardiac dysfunction [70, 71]. Likewise, mutations in *Collagen 1a2* or elastin haploinsufficiency also result in aortic valve dysmorphogenesis and adult disease [72, 73]. Gene expression of *CtsK*, a matrix remodeling enzyme expressed during heart valve elongation, is regulated by the RANKL/NFATc1 regulatory pathway [47, 61]. Additional ECM remodeling enzymes, in‐ cluding matrix metalloproteinase (MMP)13, a collagenase, and Adam-TS5 and 9 proteogly‐ can proteases, also are expressed during late valve morphogenesis and have been implicated in ECM maturation and organization [39, 74, 75]. Several ECM molecules required for nor‐ mal valve structure/function also are expressed during osteogenesis, and valve progenitors have gene expression profiles similar to bone progenitors [43]. *Osteopontin*, *osteonectin*, and *periostin* gene expression and collagen fiber deposition are increased during heart valve re‐ modeling [24, 43, 60]. However, the regulatory mechanisms for expression of these genes in valve development are not well defined. These proteins also are induced and mislocalized in pediatric and adult heart valve disease [13, 24, 70], but the pathways leading to their dysre‐ gulation have not yet been fully characterized.

#### **3. Molecular mechanisms of osteogenesis**

#### **3.1. Overview of skeletal development**

critical requirements for these same regulatory pathways in endocardial cushion formation. BMP signaling, as indicated by phosphorylation of the intermediate signaling molecules Smad1/5/8, is active throughout endocardial cushion mesenchymal cells, is associated with mesenchymal cell proliferation [55], and also is active later in valve cell lineage diversifica‐ tion [48]. *BMP Receptor II* mutations and conditional mutagenesis results in thickening of semilunar valve leaflets at late fetal stages [56, 57]. Loss of inhibitory Smad6 leads to in‐ creased BMP signaling, in addition to thickening of valve leaflets and CAVD in adult ani‐ mals [58]. Studies in explanted avian valve progenitors have revealed antagonistic regulatory roles for BMP and fibroblast growth factor (FGF) signaling in promoting diversi‐ fied ECM gene expression, conserved with mechanisms that control cartilage and tendon lineage development [11, 48, 59]. Wnt pathway activation is evident throughout the remod‐ eling AV and semilunar valve primordia, as indicated in TopGal reporter mice [60]. Multi‐ ple Wnt ligands are expressed during valvulogenesis, but the function of Wnt signaling in heart valve remodeling has not yet been determined [60]. Thus, additional in vivo studies are necessary to determine the specific functions and intersecting regulatory mechanisms of these critical signaling pathways in valve leaflet development and also to determine specific

The later stages of heart valve development are characterized by leaflet elongation, ECM re‐ modeling, and stratification, all of which are critical for mature valve structure and function [24]. Limited information is available on the regulation of these processes, but several regula‐ tory pathways have been implicated in late valve remodeling and morphogenesis. Strikingly these same pathways have been implicated in adult CAVD (see below). RANKL, expressed by valve endothelial cells, promotes ECM remodeling and Cathepsin K (Ctsk) expression by NFATc1 in a mechanism partially conserved with osteoclast differentiation and function [11, 47, 61]. The signaling mechanisms that control stratification and ECM organization of the valve leaflets are relatively unknown. Notch signaling is localized on the ventricularis surface of the remodeling aortic valve in mice [62], and Wnt/β-catenin signaling is active throughout aortic valve primordia at late gestation and in a subpopulation of VICs after birth [60]. Like‐ wise, Wnt signaling promotes expression of fibrosa genes *periostin* and *matrix gla-protein* (*mgp)* in cultured chicken embryo aortic VICs, but a role in valve stratification or lineage diversifica‐ tion has not yet been established in vivo [60]. Additional studies are necessary to demon‐ strate the specific functions and potential biomechanical stimulation of these pathways in an in vivo context. Since, both Notch and Wnt signaling pathways are required for initial stages of endocardial cushion formation, it has been difficult to establish their roles in the later stages

of valvulogenesis in vivo using available conditional targeting approaches.

Bicuspid aortic valve is arguably the most common congenital heart malformation with an incidence of 1-2% in the US adult population [63]. BAV often does not often manifest in valve dysfunction in early life, but malformed aortic valves are predisposed to calcification. Strikingly, the majority of stenotic aortic valves that are replaced in adults are congenitally malformed [4]. However, the molecular and cellular mechanisms of BAV are not well de‐ fined. In humans, mutations in *NOTCH1* are associated with BAV, but the mechanisms by which valve leaflet number is regulated by Notch signaling have not yet been identified [64]. Likewise, Notch1 haploinsufficiency in mice leads to BAV at very low penetrance and there

contributions to valve degeneration and disease.

64 Calcific Aortic Valve Disease

Many osteogenic regulatory interactions identified in developing bone also are active in CAVD (Table 1). The regulatory hierarchies and ECM composition of the developing valves, most notably the collagen rich fibrosa layer, are similar to those observed in osteoblast pre‐ cursor cells [43]. Both the bone substratum and valve ECM are composed primarily of fibril‐ lar collagen. Thus, it is not surprising that there are extensive similarities in their composition and developmental regulation. Normally, heart valves do not progress to min‐ eralization, but striking similarities have been identified between osteogenic pathways that regulate bone mineralization and CAVD mechanisms [7]. Thus the molecular understanding of normal development of bone has clear implications for pathogenic mechanisms of con‐ nective tissue mineralization, including CAVD.

formation [83]. Bone mineralization occurs with the deposition of calcium phosphate and hydroxyapatite by osteocytes and is dependent on Runx2 and Osx function [80]. Bone ho‐ meostasis is maintained throughout life by the osteogenic activity of osteocytes and bone re‐

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 67

**Figure 2.** Hierarchies of signaling pathways and transcription factors regulate the differentiation of chondrogenic and osteogenic progenitor cells during skeletal development. Early osteochondrogenic progenitor cells express BMPs, Twist1, Msx1/2, and Sox9. Wnt/β-catenin signaling promotes pre-osteoblast differentiation while inhibiting chondro‐ cyte differentiation. In contrast, Notch signaling promotes cartilage differentiation and inhibits osteoblast differentia‐ tion. BMP signaling is further required for osteocyte differentiation in the final stages of bone maturation. Sox5, 6, and 9 are transcription factors crucial for maintaining the chondrogenic lineage, whereas, Runx2, Osx, and ATF4 are tran‐ scription factors necessary for osteoblast and osteocyte differentiation and maturation. Many of these factors are also expressed during calcific aortic valve disease and have been implicated in pathologic calcification. Please see text for details and references. Activating factors are shown in green, inhibitory factors are shown in red, and signaling path‐

Twist1 is expressed early in the osteochondroprogenitor lineage and inhibits terminal differ‐ entiation of cartilage and bone [84]. In preosteoblasts, Twist1 binds to Runx2 and inhibits its transcriptional activation of bone differentiation genes including *osteocalcin* [84]. Similarly, Twist1 can inhibit cartilage differentiation by binding to Sox9 and preventing activation of cartilage-specific gene expression [85]. Mutations in human *TWIST1* cause Saethre-Chotzen syndrome, characterized by premature bone differentiation evident in premature fusion of

**3.2. Transcriptional regulation of osteoblast lineage development and bone**

sorption activity of osteoclasts [80].

ways are indicated in blue.

**differentiation**

The osteogenic precursors of the developing axial skeleton and long bones of the limbs are derived primarily from paraxial mesoderm of the developing somites and also lateral plate mesoderm, the main source of cardiac precursor cells [76, 77]. Additional progenitors of the craniofacial skeleton are derived from cranial neural crest [78]. Most axial skeletal elements develop by endochondral bone formation that occurs through a cartilage intermediate [76, 77]. Alternatively, the craniofacial bones of the skull form through membranous ossification in which condensed osteogenic progenitors differentiate directly into bone and do not go through a cartilage intermediate [76]. The osteochondroprogenitors present in the axial and appendicular skeletal elements develop into both bone and cartilage lineages [77, 79, 80]. Ex‐ tensive research over the past several years has defined transcriptional regulatory mecha‐ nisms and signaling events that control the development of cartilage and bone (Figure 2) [79, 80].

Mature cartilage is composed predominantly of chondroitin sulfate proteoglycans that pro‐ vides cushioning and flexibility to cartilaginous structures [77, 81]. In addition, the proteo‐ glycan-rich ECM is angiostatic and mature cartilage is avascular [81]. Interestingly, the predominant proteoglycan composition and lack of vasculature also are features of the ma‐ ture aortic valve leaflet spongiosa layer [82]. Likewise, the cartilage ECM inhibits minerali‐ zation, and a similar role has been hypothesized for the proteoglycan-rich matrix of the aortic valve [49]. During normal axial bone development, osteoblasts from the laterally placed periostium differentiate into trabecular bone, and secondary ossification centers at the ends of the bone displace the growth plate hypertrophic cartilage [79, 80]. During bone differentiation, hypertrophic cartilage cells must die for mineralization to occur in a process of endochondral ossification, which could be related to dystrophic mechanisms of CAVD [79, 80].

Bone cell lineage maturation goes through multiple stages defined by molecular regulatory mechanisms that also are active in valve development and disease processes [80]. Osteo‐ chondroprogenitor cells express several mesenchymal transcription factors, including Twist1, Msx2, and Sox9, that also are predominant in valve progenitor cells and diseased aortic valves [79]. Immature pre-osteoblasts express high levels of fibrillar type 1 collagen, in addition to periostin, osteonectin, and osteopontin, similar to normal differentiated VICs [43, 60]. Differentiated osteoblasts are not yet mineralized but express the transcription fac‐ tor Runx2, in addition to osteocalcin and bone sialoprotein involved in bone mineralization and also in valve calcification, [7, 80]. Later stage osteoblasts and osteocytes express the tran‐ scription factor Osterix (Osx), which is regulated by Runx2 and is required for mature bone formation [83]. Bone mineralization occurs with the deposition of calcium phosphate and hydroxyapatite by osteocytes and is dependent on Runx2 and Osx function [80]. Bone ho‐ meostasis is maintained throughout life by the osteogenic activity of osteocytes and bone re‐ sorption activity of osteoclasts [80].

most notably the collagen rich fibrosa layer, are similar to those observed in osteoblast pre‐ cursor cells [43]. Both the bone substratum and valve ECM are composed primarily of fibril‐ lar collagen. Thus, it is not surprising that there are extensive similarities in their composition and developmental regulation. Normally, heart valves do not progress to min‐ eralization, but striking similarities have been identified between osteogenic pathways that regulate bone mineralization and CAVD mechanisms [7]. Thus the molecular understanding of normal development of bone has clear implications for pathogenic mechanisms of con‐

The osteogenic precursors of the developing axial skeleton and long bones of the limbs are derived primarily from paraxial mesoderm of the developing somites and also lateral plate mesoderm, the main source of cardiac precursor cells [76, 77]. Additional progenitors of the craniofacial skeleton are derived from cranial neural crest [78]. Most axial skeletal elements develop by endochondral bone formation that occurs through a cartilage intermediate [76, 77]. Alternatively, the craniofacial bones of the skull form through membranous ossification in which condensed osteogenic progenitors differentiate directly into bone and do not go through a cartilage intermediate [76]. The osteochondroprogenitors present in the axial and appendicular skeletal elements develop into both bone and cartilage lineages [77, 79, 80]. Ex‐ tensive research over the past several years has defined transcriptional regulatory mecha‐ nisms and signaling events that control the development of cartilage and bone (Figure 2)

Mature cartilage is composed predominantly of chondroitin sulfate proteoglycans that pro‐ vides cushioning and flexibility to cartilaginous structures [77, 81]. In addition, the proteo‐ glycan-rich ECM is angiostatic and mature cartilage is avascular [81]. Interestingly, the predominant proteoglycan composition and lack of vasculature also are features of the ma‐ ture aortic valve leaflet spongiosa layer [82]. Likewise, the cartilage ECM inhibits minerali‐ zation, and a similar role has been hypothesized for the proteoglycan-rich matrix of the aortic valve [49]. During normal axial bone development, osteoblasts from the laterally placed periostium differentiate into trabecular bone, and secondary ossification centers at the ends of the bone displace the growth plate hypertrophic cartilage [79, 80]. During bone differentiation, hypertrophic cartilage cells must die for mineralization to occur in a process of endochondral ossification, which could be related to dystrophic mechanisms of CAVD

Bone cell lineage maturation goes through multiple stages defined by molecular regulatory mechanisms that also are active in valve development and disease processes [80]. Osteo‐ chondroprogenitor cells express several mesenchymal transcription factors, including Twist1, Msx2, and Sox9, that also are predominant in valve progenitor cells and diseased aortic valves [79]. Immature pre-osteoblasts express high levels of fibrillar type 1 collagen, in addition to periostin, osteonectin, and osteopontin, similar to normal differentiated VICs [43, 60]. Differentiated osteoblasts are not yet mineralized but express the transcription fac‐ tor Runx2, in addition to osteocalcin and bone sialoprotein involved in bone mineralization and also in valve calcification, [7, 80]. Later stage osteoblasts and osteocytes express the tran‐ scription factor Osterix (Osx), which is regulated by Runx2 and is required for mature bone

nective tissue mineralization, including CAVD.

[79, 80].

66 Calcific Aortic Valve Disease

[79, 80].

**Figure 2.** Hierarchies of signaling pathways and transcription factors regulate the differentiation of chondrogenic and osteogenic progenitor cells during skeletal development. Early osteochondrogenic progenitor cells express BMPs, Twist1, Msx1/2, and Sox9. Wnt/β-catenin signaling promotes pre-osteoblast differentiation while inhibiting chondro‐ cyte differentiation. In contrast, Notch signaling promotes cartilage differentiation and inhibits osteoblast differentia‐ tion. BMP signaling is further required for osteocyte differentiation in the final stages of bone maturation. Sox5, 6, and 9 are transcription factors crucial for maintaining the chondrogenic lineage, whereas, Runx2, Osx, and ATF4 are tran‐ scription factors necessary for osteoblast and osteocyte differentiation and maturation. Many of these factors are also expressed during calcific aortic valve disease and have been implicated in pathologic calcification. Please see text for details and references. Activating factors are shown in green, inhibitory factors are shown in red, and signaling path‐ ways are indicated in blue.

#### **3.2. Transcriptional regulation of osteoblast lineage development and bone differentiation**

Twist1 is expressed early in the osteochondroprogenitor lineage and inhibits terminal differ‐ entiation of cartilage and bone [84]. In preosteoblasts, Twist1 binds to Runx2 and inhibits its transcriptional activation of bone differentiation genes including *osteocalcin* [84]. Similarly, Twist1 can inhibit cartilage differentiation by binding to Sox9 and preventing activation of cartilage-specific gene expression [85]. Mutations in human *TWIST1* cause Saethre-Chotzen syndrome, characterized by premature bone differentiation evident in premature fusion of cranial sutures of the skull [86]. Msx2 also is involved in early mesenchymal stages of osteo‐ chondroprogenitor development and is down regulated during osteoblast differentiation [79]. Persistent Msx2 expression in osteoblasts prevents differentiation and mineralization, while antisense mRNA-mediated loss of Msx2 accelerates these processes [87]. Thus Msx2 is expressed in osteoblast progenitor cells but has an inhibitory role in osteogenic differentia‐ tion. Together Twist1 and Msx2 act to maintain undifferentiated osteochondroprogenitors during development.

critical mediator of bone calcification and resorption [96]. A similar balance of OPG and RANKL signaling in CAVD has been proposed [103]. While NFATc1 is a critical regulator of heart valve remodeling during development and activates valvular *CtsK* expression [47, 61],

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 69

Additional transcription factors involved in bone differentiation are not generally found in CAVD, although there are conflicting reports. Most notable is Osx, which is required for ter‐ minal differentiation of osteoblasts and mineralization of bone [83]. Osx, promotes expres‐ sion of *collagen 1a1* and the matrix metalloproteinase *mmp13*, which also are upregulated in aortic valve disease [73, 101, 104, 105]. Studies based on antibody staining demonstrate Osx expression in Notch signaling-deficient calcified mouse valves [65] and human CAVD [106]. ATF4 is an additional transcription factor critical for bone differentiation, mineralization, and homeostasis that has not been found in developing or diseased valves [79]. Further studies are necessary to determine if *ATF4* or *Osx* gene expression is induced or if they con‐

Multiple signaling pathways control the stages of bone cell lineage determination, differen‐ tiation and maturation [80, 107]. These include BMP, Wnt, and Notch pathways, also active in developing and diseased heart valves, as well as FGF, hedgehog, insulin-like growth fac‐ tor (IGF), and retinoic acid (RA) pathways, not yet characterized in heart valve pathogenesis [80]. BMP, Wnt, and Notch pathways are required at multiple stages of osteogenesis and have distinct regulatory interactions that control transcription factor function and cell typespecific gene expression in cartilage and bone cell lineages (Figure 2). In addition, these pathways crosstalk with each other in synergistic and antagonistic regulatory interactions. Strikingly many of these same regulatory interactions occur in heart valve development and

Bone morphogenetic proteins were originally identified based on their ability to induce ec‐ topic bone formation [108]; however, in vivo functions in normal bone development are less clear [109]. In the developing limb buds, BMP signaling has a critical role in mesenchymal condensation, Sox9 activation, and cartilage lineage differentiation [89]. Thus BMP signaling is an important regulator of the earliest stages of skeletal development. Later in differentiat‐ ing osteoblasts, BMP signaling through Smad1/5/8 phosphorylation (pSmad1/5/8) promotes osteogenic differentiation and calcification [110]. Runx2 directly binds to activated Smads1 and 5 to cooperatively activate osteoblast gene expression in response to BMP signaling [109]. Conditional loss of BMP2 and BMP4 in the osteoblast lineage in mice inhibits late stage differentiation into Osx1-positive osteocytes, and BMP signaling is required for bone homeostasis after birth [80, 109]. Surprisingly, earlier stages of bone lineage development

Wnt/β-catenin signaling is required for osteoblast differentiation as demonstrated by loss of osteoblast differentiation with conditional loss of β-catenin in osteochondroprogenitor cells in mice [80]. In addition, loss of β-catenin in pre-osteoblasts leads to ectopic cartilage forma‐ tion, thus implicating Wnt signaling in osteogenic versus chondrogenic cell fate determina‐

its role in CAVD and adult valve homeostasis has not been determined.

tribute to valve mineralization in CAVD.

pathogenesis (Table 1) [8].

**3.3. Signaling pathways involved in bone development**

are apparently unaffected with conditional loss of these ligands.

Sox9 functions in the expansion of cartilage progenitors and promotes cartilage differentia‐ tion, while inhibiting bone differentiation [77, 80]. Sox9 is required for osteochondroproge‐ nitor lineage specification but is not expressed in differentiated osteoblasts [88]. At early stages of cartilage lineage development, Sox9 promotes cell proliferation and later is re‐ quired for cartilage lineage differentiation [88]. BMP signaling induces *Sox9* gene expression in cartilage progenitor cells [89], and Sox9 regulates expression of cartilage marker genes *Col2a1* and *aggrecan* [90, 91]. Sox9 transcriptional activity can be inhibited by binding to Twist1, thus inhibiting differentiation of early stage osteochondroprogenitor cells [85]. At later stages of cartilage maturation, Sox9 inhibits Runx2 transcriptional activity, thus pro‐ moting hypertrophic cartilage and inhibiting osteogenic differentiation [92]. Thus downre‐ gulation of Sox9 is required in osteoblasts for differentiation and mineralization of bone.

Runx2, originally called Cbfa1, has been defined as a master regulatory gene in bone forma‐ tion [79, 93]. Gain and loss of function studies in mice demonstrate that Runx2 is both neces‐ sary and sufficient for osteoblast differentiation [93]. During bone development, Runx2 directly regulates *osteocalcin* gene expression [93]. Runx2 transcriptional function can be in‐ hibited by interaction with Twist1 and also by Hey1, downstream of Notch signaling [84, 94]. Mice lacking Runx2 lack mineralized bone, and haploinsufficiency of Runx2 results in reduced bone formation in mice and humans [80]. Induction of a dominant negative form of Runx2 in differentiated osteoblasts after birth also leads to reduced bone mineralization, demonstrating a role for Runx2 in bone homeostasis and mineralization throughout life [95]. Runx2 has not been implicated in normal heart valve development, and its expression in de‐ veloping valves has not been reported, consistent with the lack of calcification in normal valves. Likewise, in adult valves Runx2 is not normally expressed, but its expression is in‐ duced in CAVD in both humans and mice [13, 73]. The presence of Runx2 in diseased aortic valves and association with calcification is consistent with a role in mineralization, as has been established for bone cell lineages.

NFATc1 is a critical transcription factor in osteoclast differentiation and also has been impli‐ cated in osteoblast development [80, 96]. Osteoclasts, derived from a macrophage lineage, have bone resorptive activity and are necessary for bone homeostasis [96]. During osteoclast development, RANKL signaling induces activation of NFATc1, which promotes the tran‐ scription of bone matrix remodeling genes including C*tsK* and *mmp9* [97, 98]. RANKL activi‐ ty in bone is antagonized by the receptor decoy osteoprotegerin (OPG) that promotes bone calcification [99, 100]. In osteoblasts, NFATc1 promotes cell proliferation and also enhances differentiation by cooperating with Osx to promote *Col1a1* gene expression [101, 102]. Thus, the balance of RANKL and OPG signaling acting on NFATc1 transcriptional function is a critical mediator of bone calcification and resorption [96]. A similar balance of OPG and RANKL signaling in CAVD has been proposed [103]. While NFATc1 is a critical regulator of heart valve remodeling during development and activates valvular *CtsK* expression [47, 61], its role in CAVD and adult valve homeostasis has not been determined.

Additional transcription factors involved in bone differentiation are not generally found in CAVD, although there are conflicting reports. Most notable is Osx, which is required for ter‐ minal differentiation of osteoblasts and mineralization of bone [83]. Osx, promotes expres‐ sion of *collagen 1a1* and the matrix metalloproteinase *mmp13*, which also are upregulated in aortic valve disease [73, 101, 104, 105]. Studies based on antibody staining demonstrate Osx expression in Notch signaling-deficient calcified mouse valves [65] and human CAVD [106]. ATF4 is an additional transcription factor critical for bone differentiation, mineralization, and homeostasis that has not been found in developing or diseased valves [79]. Further studies are necessary to determine if *ATF4* or *Osx* gene expression is induced or if they con‐ tribute to valve mineralization in CAVD.

#### **3.3. Signaling pathways involved in bone development**

cranial sutures of the skull [86]. Msx2 also is involved in early mesenchymal stages of osteo‐ chondroprogenitor development and is down regulated during osteoblast differentiation [79]. Persistent Msx2 expression in osteoblasts prevents differentiation and mineralization, while antisense mRNA-mediated loss of Msx2 accelerates these processes [87]. Thus Msx2 is expressed in osteoblast progenitor cells but has an inhibitory role in osteogenic differentia‐ tion. Together Twist1 and Msx2 act to maintain undifferentiated osteochondroprogenitors

Sox9 functions in the expansion of cartilage progenitors and promotes cartilage differentia‐ tion, while inhibiting bone differentiation [77, 80]. Sox9 is required for osteochondroproge‐ nitor lineage specification but is not expressed in differentiated osteoblasts [88]. At early stages of cartilage lineage development, Sox9 promotes cell proliferation and later is re‐ quired for cartilage lineage differentiation [88]. BMP signaling induces *Sox9* gene expression in cartilage progenitor cells [89], and Sox9 regulates expression of cartilage marker genes *Col2a1* and *aggrecan* [90, 91]. Sox9 transcriptional activity can be inhibited by binding to Twist1, thus inhibiting differentiation of early stage osteochondroprogenitor cells [85]. At later stages of cartilage maturation, Sox9 inhibits Runx2 transcriptional activity, thus pro‐ moting hypertrophic cartilage and inhibiting osteogenic differentiation [92]. Thus downre‐ gulation of Sox9 is required in osteoblasts for differentiation and mineralization of bone.

Runx2, originally called Cbfa1, has been defined as a master regulatory gene in bone forma‐ tion [79, 93]. Gain and loss of function studies in mice demonstrate that Runx2 is both neces‐ sary and sufficient for osteoblast differentiation [93]. During bone development, Runx2 directly regulates *osteocalcin* gene expression [93]. Runx2 transcriptional function can be in‐ hibited by interaction with Twist1 and also by Hey1, downstream of Notch signaling [84, 94]. Mice lacking Runx2 lack mineralized bone, and haploinsufficiency of Runx2 results in reduced bone formation in mice and humans [80]. Induction of a dominant negative form of Runx2 in differentiated osteoblasts after birth also leads to reduced bone mineralization, demonstrating a role for Runx2 in bone homeostasis and mineralization throughout life [95]. Runx2 has not been implicated in normal heart valve development, and its expression in de‐ veloping valves has not been reported, consistent with the lack of calcification in normal valves. Likewise, in adult valves Runx2 is not normally expressed, but its expression is in‐ duced in CAVD in both humans and mice [13, 73]. The presence of Runx2 in diseased aortic valves and association with calcification is consistent with a role in mineralization, as has

NFATc1 is a critical transcription factor in osteoclast differentiation and also has been impli‐ cated in osteoblast development [80, 96]. Osteoclasts, derived from a macrophage lineage, have bone resorptive activity and are necessary for bone homeostasis [96]. During osteoclast development, RANKL signaling induces activation of NFATc1, which promotes the tran‐ scription of bone matrix remodeling genes including C*tsK* and *mmp9* [97, 98]. RANKL activi‐ ty in bone is antagonized by the receptor decoy osteoprotegerin (OPG) that promotes bone calcification [99, 100]. In osteoblasts, NFATc1 promotes cell proliferation and also enhances differentiation by cooperating with Osx to promote *Col1a1* gene expression [101, 102]. Thus, the balance of RANKL and OPG signaling acting on NFATc1 transcriptional function is a

during development.

68 Calcific Aortic Valve Disease

been established for bone cell lineages.

Multiple signaling pathways control the stages of bone cell lineage determination, differen‐ tiation and maturation [80, 107]. These include BMP, Wnt, and Notch pathways, also active in developing and diseased heart valves, as well as FGF, hedgehog, insulin-like growth fac‐ tor (IGF), and retinoic acid (RA) pathways, not yet characterized in heart valve pathogenesis [80]. BMP, Wnt, and Notch pathways are required at multiple stages of osteogenesis and have distinct regulatory interactions that control transcription factor function and cell typespecific gene expression in cartilage and bone cell lineages (Figure 2). In addition, these pathways crosstalk with each other in synergistic and antagonistic regulatory interactions. Strikingly many of these same regulatory interactions occur in heart valve development and pathogenesis (Table 1) [8].

Bone morphogenetic proteins were originally identified based on their ability to induce ec‐ topic bone formation [108]; however, in vivo functions in normal bone development are less clear [109]. In the developing limb buds, BMP signaling has a critical role in mesenchymal condensation, Sox9 activation, and cartilage lineage differentiation [89]. Thus BMP signaling is an important regulator of the earliest stages of skeletal development. Later in differentiat‐ ing osteoblasts, BMP signaling through Smad1/5/8 phosphorylation (pSmad1/5/8) promotes osteogenic differentiation and calcification [110]. Runx2 directly binds to activated Smads1 and 5 to cooperatively activate osteoblast gene expression in response to BMP signaling [109]. Conditional loss of BMP2 and BMP4 in the osteoblast lineage in mice inhibits late stage differentiation into Osx1-positive osteocytes, and BMP signaling is required for bone homeostasis after birth [80, 109]. Surprisingly, earlier stages of bone lineage development are apparently unaffected with conditional loss of these ligands.

Wnt/β-catenin signaling is required for osteoblast differentiation as demonstrated by loss of osteoblast differentiation with conditional loss of β-catenin in osteochondroprogenitor cells in mice [80]. In addition, loss of β-catenin in pre-osteoblasts leads to ectopic cartilage forma‐ tion, thus implicating Wnt signaling in osteogenic versus chondrogenic cell fate determina‐ tion. At a molecular level, Wnt/β-catenin signaling promotes osteoblast lineage differentiation, while inhibiting chondrogenesis, by activating Runx2, while inhibiting Sox9 [77]. In bone lineages, BMP and Wnt signaling act synergistically to promote calcification, although neither pathway alone is sufficient to induce a full osteogenic response [111]. Dur‐ ing the initial differentiation of bone progenitor cells, regulatory elements of *Runx2* and *Msx2* genes are bound by Smad1, downstream of BMP signaling, and also by Lef1, activated by Wnt signaling, for cooperative gene activation [112]. Postnatally, Wnt signaling through the Lrp5 receptor is required for bone accrual in mice and humans [80]. In developing bone, osteogenic differentiation and calcification are dependent on sequential activation of BMP, followed by Wnt/β-catenin, signaling [110]. It is possible that a similar regulatory relation‐ ship exists in CAVD, but this has not yet been demonstrated.

extensive ECM remodeling and elastic fiber fragmentation with evidence of both macro‐

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 71

Changes in the resident VICs are apparent in CAVD. Under normal conditions, aortic VICs are quiescent and non-proliferative [13, 24, 104, 124]. However, in disease, a subset of aortic VICs exhibits features of myofibroblast activation, which is characterized by expression of α-smooth muscle actin (αSMA), MMP13, non-muscle myosin heavy chain (SMemb), and markers of proliferation [13, 104, 119, 124, 125]. In vivo, the factors responsible for inducing myofibroblast activation are not well defined. However, in culture, TGFβ1 stimulation and mechanical strain are potent inducers of VIC myofibroblast activation [125, 126]. Activated VICs also exhibit characteristics of valve and bone precursor cells as they induce expression of the common mesenchymal markers Sox9, Twist1, and Msx2 [13]. Currently it is unknown where the mesenchymal-like cells come from and what role these proliferative cells play in

Valve calcification, apparent as hydroxyapatite deposits on the surface of or within the leaf‐ lets, is a prominent feature of CAVD [119, 127, 128]. Histologically, valve calcific nodules are primarily acellular [13, 129]. Although traditionally thought to be a completely passive dep‐ osition of mineral, in some cases, valve calcification is coincident with endochondral bonelike and cartilaginous-like nodules [129, 130]. Aortic valve calcification is observed primarily in the regions of the valves exposed to the greatest physical strain, specifically at the hinge region of the valve and along the line of leaflet coaptation [120]. Furthermore, calcification is predominantly found in the fibrosa layer of the diseased valve, which is similar to early bone matrix as it is contains primarily fibrillar collagen [44]. Expression of other bone matrix molecules, such as osteocalcin and osteopontin, are induced during disease [5]. Further‐ more, expression of osteogenic factors, such as Runx2, BMP2, and alkaline phosphatase, also is induced in VICs from calcified valves, suggesting that resident VICs may have the poten‐ tial to undergo osteogenic transdifferentiation and actively contribute to valve calcification

Extrinsic factors have been implicated in valve calcification. For example, lipid deposition and immune cell infiltration are common histopathological features of CAVD, and it has been proposed that aortic valve calcification occurs by mechanisms similar to arterial calci‐ fication in atherosclerosis [119, 132-135]. In addition, altered external physical forces elicit changes in resident VICs, which play an active role in pathological valve calcification [126]. In contrast to VIC response to immune cell infiltration and altered physical forces, cell intrinsic mechanisms may also contribute to valve calcification, as stimulation with factors such as BMP2 or TGFβ1 in cell culture studies can induce VIC calcification in the absence of inflammatory stimulation or altered physical forces [126, 136-138]. Together, these studies suggest that not only is valve calcification an active cell-regulated process, but that many factors likely contribute to progression of calcification during disease. It is also likely that not all CAVD is created equal. Genetic predisposition, the presence of a malformed aortic valve, and other disease comorbidities, such as coronary artery disease,

scopic calcific nodule formation as well as microscopic mineral deposits [119].

the progression of CAVD pathogenesis.

(reviewed in [131]).

Notch activation inhibits osteogenesis through suppression of the Wnt/β-catenin pathway and Runx2 transcription factor activity [94, 113, 114]. Loss of Notch1 or Notch2 function pro‐ motes osteoblast differentiation and leads to increased bone mass in mice [115]. Notch path‐ way activation inhibits the progression of osteoblast differentiation through direct binding of the activated Notch1 intracellular domain (N1ICD) to β-catenin, thereby counteracting Wnt-mediated induction of osteogenesis [113, 114]. In addition, the Notch target gene *Hey1* encodes a transcriptional repressor that binds and inhibits Runx2 transcriptional function [115]. Precise levels of Notch signaling are required for cell proliferation and chondrogenic differentiation, with defects in these processes occurring with increased or decreased Notch signaling in mice [116]. In early cartilage precursors, Notch signaling is required for cell pro‐ liferation, but increased Notch signaling inhibits terminal differentiation of chondrocytes and endochondral ossification [116]. Loss of Notch signaling has been implicated in CAVD [64], but it is not known if this occurs through inhibition of Wnt/β-catenin signaling, as has been demonstrated for osteoblast differentiation and bone mineralization.
