**4. Molecular and cellular mechanisms of CAVD**

#### **4.1. Overview of CAVD progression**

The mature aortic valves are comprised of three ECM layers critical for normal leaflet struc‐ ture and function [24, 44, 117]. Collagen predominates in the fibrosa layer, which is oriented on the opposite side of blood flow, whereas elastin is enriched in the ventricularis layer on the flow side of the valve. Between the fibrosa and ventricularis layers, is the proteoglycanrich spongiosa layer [24, 44, 117]. This trilaminar ECM arrangement is preserved among spe‐ cies, and lends both strength and elasticity to the aortic valves [24]. In CAVD, the aortic valve becomes thickened and displays extensive ECM remodeling and mineralization [118-121]. Abnormal thickening (aortic valve sclerosis) and calcification of the aortic valve lead to stiffening of the valve leaflets and can reduce the effective valve opening (aortic valve stenosis), which can impede blood flow and lead to clinical symptoms such as syn‐ cope and angina [119, 122, 123]. Histologically, human explanted diseased aortic valves have extensive ECM remodeling and elastic fiber fragmentation with evidence of both macro‐ scopic calcific nodule formation as well as microscopic mineral deposits [119].

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‐

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

The mature aortic valves are comprised of three ECM layers critical for normal leaflet struc‐ ture and function [24, 44, 117]. Collagen predominates in the fibrosa layer, which is oriented on the opposite side of blood flow, whereas elastin is enriched in the ventricularis layer on the flow side of the valve. Between the fibrosa and ventricularis layers, is the proteoglycanrich spongiosa layer [24, 44, 117]. This trilaminar ECM arrangement is preserved among spe‐ cies, and lends both strength and elasticity to the aortic valves [24]. In CAVD, the aortic valve becomes thickened and displays extensive ECM remodeling and mineralization [118-121]. Abnormal thickening (aortic valve sclerosis) and calcification of the aortic valve lead to stiffening of the valve leaflets and can reduce the effective valve opening (aortic valve stenosis), which can impede blood flow and lead to clinical symptoms such as syn‐ cope and angina [119, 122, 123]. Histologically, human explanted diseased aortic valves have

ship exists in CAVD, but this has not yet been demonstrated.

70 Calcific Aortic Valve Disease

been demonstrated for osteoblast differentiation and bone mineralization.

**4. Molecular and cellular mechanisms of CAVD**

**4.1. Overview of CAVD progression**

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 the progression of CAVD pathogenesis.

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 (reviewed in [131]).

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, hypertension, and kidney disease, likely affect the pathology and underlying cause of CAVD [64, 139-142].

phatase (ALP), and Osx in disease [5, 13, 106, 118, 137, 152]. At a molecular level, BMP2 sig‐ naling is a key inducer of VIC calcification, which is thought to act through p-SMAD1/5/8 and phospho-ERK1/2 signaling to stimulate increases in both Runx2 and OPN expression [138]. Induction of VIC calcification by BMP2 stimulation is highly reminiscent of BMP sig‐ naling in bone development, suggesting that some parallels exist between osteogenic bone formation and VIC calcification [9]. Histological studies of explanted human valves further support a role for BMP signaling in valve calcification. Comparison of pediatric diseased valves, which do not acquire calcification, and adult calcified valves demonstrates that in‐ creased BMP signaling, evident in p-SMAD1/5/8 activation, is exclusive to adult valves with calcification, indicating that BMP signaling may contribute to valve calcification in human disease [13]. Additionally, TGFβ1 is also a potent inducer of osteogenic-like differentiation of VICs in cell culture, as it stimulates VIC activation and calcification, increases ALP activi‐ ty, and increases expression of ECM remodeling enzymes [126, 136, 137]. Negative regula‐ tors of valve calcification have been demonstrated through in vivo studies. One negative regulator of valve calcification is Notch signaling. Animals haploinsufficient for Notch sig‐ naling develop aortic valve calcification with increased BMP signaling and increased expres‐ sion of Runx2 in the valve leaflets [65, 153]. Studies in isolated aortic VICs further demonstrate that Notch signaling plays an important role in suppressing valve calcification as treatment of VICs with Notch inhibitors induces BMP signaling and subsequent increases in osteogenic gene expression [65, 153]. Another negative regulator of valve calcification is Sox9, which potentially acts through induction of proteoglycan expression, similar to what has been observed in developing cartilage [40, 49]. Conditional heterozygous Sox9 mutant mice develop valve calcification along with increased valve thickness and expression of the osteogenic genes *Runx2*, *osteonectin*, *OPN*, and *OPG* [40, 49]. Based on these studies, it is ap‐ parent that many factors involved in endochondral bone formation are active in the process

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 73

CAVD has been linked to chronic kidney disease in human patients and animal models [140, 154-157]. A prominent pathological feature of kidney disease is the inability to regulate cal‐ cium and phosphate metabolism [158]. Increased blood phosphate levels (hyperphosphate‐ mia) are highly associated with aortic valve sclerosis and valve calcification in humans [140]. Klotho-null mice are a model of accelerated aging that includes development of kidney fail‐ ure and hyperphosphatemia, along with cardiovascular disease [159-162]. Klotho-null mice exhibit extensive valve annulus calcification with increased expression of osteogenic genes, but minimal CD68 positive macrophage infiltration [73]. Thus, valve calcification in the klo‐ tho-null animals parallels bone formation, where increases in crucial osteogenic genes, such as *Col10a1, Runx2, OPN*, and *BSP*, are observed [73]. These observations suggest that in‐ creased blood phosphate levels could be one stimulus for inducing advanced aortic valve calcification with osteogenic gene expression, but this has not been definitively demonstrat‐

Atherogenic lipid deposition and inflammation in the valves also has been linked to induc‐ tion of osteogenic gene expression and disease [163-165]. Rabbit and mouse models of CAVD, induced with hypercholesterolemic or high fat diets, have increased lipid deposition

of aortic valve calcification.

ed [73, 140].

#### **4.2. Activation of progenitor cell and osteogenesis-related molecular pathways in CAVD**

#### *4.2.1. Expression of valve and bone progenitor cell genes in CAVD*

The mesenchymal markers Twist1, Msx2, and Sox9 are expressed in adult calcific aortic valves in mesenchymal-like activated VICs [12, 13, 106, 143, 144]. As discussed, these genes are expressed in both valve and bone mesenchymal progenitor cells. A recent study has compared gene expression in pediatric versus adult aortic valve disease and shown that the mesenchymal markers Twist1, Msx2, and Sox9 are increased in both [13]. The observation that both pediatric and adult diseased valves have increased expression of the mesenchymal markers suggests that this expression is related to VIC activation and proliferation, which is common to both, and not related to valve calcification, which is found only in advanced adult disease [13]. In both valve and bone progenitors, Twist1, Msx2, and Sox9 induce pro‐ liferation and promote a mesenchymal phenotype, thus reactivation in diseased valves is suggestive of a similar role in valve pathogenesis [38, 40-42, 84, 88, 145]. Although it is pre‐ sumed that resident VICs re-activate these early mesenchymal markers, other possibilities exist. EMT as a mechanism for VIC activation has not been established in CAVD, however, recent studies report EMT-like events in adult valves. Increased cyclic strain and altered he‐ modynamics, both recognized features of CAVD, can induce EMT in isolated sheep valve endothelial cells [146, 147]. In addition, cultured valve endothelial cells stimulated with TGFβ adapt a mesenchymal-like phenotype and express markers of both endothelial and mesenchymal cells, suggesting that they can undergo EMT [148, 149]. Likewise, disruption of Notch signaling in adult mice induces aortic valve thickening with evidence of endocar‐ dial EMT, as indicated by endocardial cells with more pseudopodial projections, loose endo‐ cardial cell-cell junctions, and αSMA expression [150]. Additional sources of mesenchymallike cells have been suggested. For example, circulating bone marrow-derived hematopoietic stem cells have been shown to integrate into the valve interstitium, adapt fi‐ broblast-like characteristics, and surround regions of prominent valve calcification in hu‐ man end stage CAVD [33, 130, 151]. It is uncertain what role reactivation of the mesenchymal markers Twist1, Msx2, and Sox9 have in potential valve repair mechanisms or in the progression of CAVD. It is possible that adult VICs maintain a certain "mesenchymalplasticity" and are able to revert back to an early progenitor-like mesenchymal cell during disease. Alternatively, they may be indicators of newly derived VICs arising from EMT or circulating progenitor cell populations in response to disease conditions.

#### *4.2.2. Osteogenic factors in CAVD*

Molecular mechanisms of endochondral ossification and cartilaginous nodule formation are active in CAVD [7, 13]. Studies in human explanted diseased aortic valve tissues have dem‐ onstrated increased expression of the osteogenic factors BMP2, TGFβ1, Runx2, osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), bone sialoprotein (BSP), alkaline phos‐ phatase (ALP), and Osx in disease [5, 13, 106, 118, 137, 152]. At a molecular level, BMP2 sig‐ naling is a key inducer of VIC calcification, which is thought to act through p-SMAD1/5/8 and phospho-ERK1/2 signaling to stimulate increases in both Runx2 and OPN expression [138]. Induction of VIC calcification by BMP2 stimulation is highly reminiscent of BMP sig‐ naling in bone development, suggesting that some parallels exist between osteogenic bone formation and VIC calcification [9]. Histological studies of explanted human valves further support a role for BMP signaling in valve calcification. Comparison of pediatric diseased valves, which do not acquire calcification, and adult calcified valves demonstrates that in‐ creased BMP signaling, evident in p-SMAD1/5/8 activation, is exclusive to adult valves with calcification, indicating that BMP signaling may contribute to valve calcification in human disease [13]. Additionally, TGFβ1 is also a potent inducer of osteogenic-like differentiation of VICs in cell culture, as it stimulates VIC activation and calcification, increases ALP activi‐ ty, and increases expression of ECM remodeling enzymes [126, 136, 137]. Negative regula‐ tors of valve calcification have been demonstrated through in vivo studies. One negative regulator of valve calcification is Notch signaling. Animals haploinsufficient for Notch sig‐ naling develop aortic valve calcification with increased BMP signaling and increased expres‐ sion of Runx2 in the valve leaflets [65, 153]. Studies in isolated aortic VICs further demonstrate that Notch signaling plays an important role in suppressing valve calcification as treatment of VICs with Notch inhibitors induces BMP signaling and subsequent increases in osteogenic gene expression [65, 153]. Another negative regulator of valve calcification is Sox9, which potentially acts through induction of proteoglycan expression, similar to what has been observed in developing cartilage [40, 49]. Conditional heterozygous Sox9 mutant mice develop valve calcification along with increased valve thickness and expression of the osteogenic genes *Runx2*, *osteonectin*, *OPN*, and *OPG* [40, 49]. Based on these studies, it is ap‐ parent that many factors involved in endochondral bone formation are active in the process of aortic valve calcification.

hypertension, and kidney disease, likely affect the pathology and underlying cause of

**4.2. Activation of progenitor cell and osteogenesis-related molecular pathways in CAVD**

The mesenchymal markers Twist1, Msx2, and Sox9 are expressed in adult calcific aortic valves in mesenchymal-like activated VICs [12, 13, 106, 143, 144]. As discussed, these genes are expressed in both valve and bone mesenchymal progenitor cells. A recent study has compared gene expression in pediatric versus adult aortic valve disease and shown that the mesenchymal markers Twist1, Msx2, and Sox9 are increased in both [13]. The observation that both pediatric and adult diseased valves have increased expression of the mesenchymal markers suggests that this expression is related to VIC activation and proliferation, which is common to both, and not related to valve calcification, which is found only in advanced adult disease [13]. In both valve and bone progenitors, Twist1, Msx2, and Sox9 induce pro‐ liferation and promote a mesenchymal phenotype, thus reactivation in diseased valves is suggestive of a similar role in valve pathogenesis [38, 40-42, 84, 88, 145]. Although it is pre‐ sumed that resident VICs re-activate these early mesenchymal markers, other possibilities exist. EMT as a mechanism for VIC activation has not been established in CAVD, however, recent studies report EMT-like events in adult valves. Increased cyclic strain and altered he‐ modynamics, both recognized features of CAVD, can induce EMT in isolated sheep valve endothelial cells [146, 147]. In addition, cultured valve endothelial cells stimulated with TGFβ adapt a mesenchymal-like phenotype and express markers of both endothelial and mesenchymal cells, suggesting that they can undergo EMT [148, 149]. Likewise, disruption of Notch signaling in adult mice induces aortic valve thickening with evidence of endocar‐ dial EMT, as indicated by endocardial cells with more pseudopodial projections, loose endo‐ cardial cell-cell junctions, and αSMA expression [150]. Additional sources of mesenchymallike cells have been suggested. For example, circulating bone marrow-derived hematopoietic stem cells have been shown to integrate into the valve interstitium, adapt fi‐ broblast-like characteristics, and surround regions of prominent valve calcification in hu‐ man end stage CAVD [33, 130, 151]. It is uncertain what role reactivation of the mesenchymal markers Twist1, Msx2, and Sox9 have in potential valve repair mechanisms or in the progression of CAVD. It is possible that adult VICs maintain a certain "mesenchymalplasticity" and are able to revert back to an early progenitor-like mesenchymal cell during disease. Alternatively, they may be indicators of newly derived VICs arising from EMT or

*4.2.1. Expression of valve and bone progenitor cell genes in CAVD*

circulating progenitor cell populations in response to disease conditions.

Molecular mechanisms of endochondral ossification and cartilaginous nodule formation are active in CAVD [7, 13]. Studies in human explanted diseased aortic valve tissues have dem‐ onstrated increased expression of the osteogenic factors BMP2, TGFβ1, Runx2, osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), bone sialoprotein (BSP), alkaline phos‐

*4.2.2. Osteogenic factors in CAVD*

CAVD [64, 139-142].

72 Calcific Aortic Valve Disease

CAVD has been linked to chronic kidney disease in human patients and animal models [140, 154-157]. A prominent pathological feature of kidney disease is the inability to regulate cal‐ cium and phosphate metabolism [158]. Increased blood phosphate levels (hyperphosphate‐ mia) are highly associated with aortic valve sclerosis and valve calcification in humans [140]. Klotho-null mice are a model of accelerated aging that includes development of kidney fail‐ ure and hyperphosphatemia, along with cardiovascular disease [159-162]. Klotho-null mice exhibit extensive valve annulus calcification with increased expression of osteogenic genes, but minimal CD68 positive macrophage infiltration [73]. Thus, valve calcification in the klo‐ tho-null animals parallels bone formation, where increases in crucial osteogenic genes, such as *Col10a1, Runx2, OPN*, and *BSP*, are observed [73]. These observations suggest that in‐ creased blood phosphate levels could be one stimulus for inducing advanced aortic valve calcification with osteogenic gene expression, but this has not been definitively demonstrat‐ ed [73, 140].

Atherogenic lipid deposition and inflammation in the valves also has been linked to induc‐ tion of osteogenic gene expression and disease [163-165]. Rabbit and mouse models of CAVD, induced with hypercholesterolemic or high fat diets, have increased lipid deposition and macrophage infiltration associated with induction of osteogenic markers such as ALP, OCN, OPN, Runx2, and Osx [165-167]. Although osteogenic gene expression is induced in these models, this type of valve calcification closely mimics vascular calcification observed in atherosclerosis, rather than endochondral bone formation, due to the presence of exten‐ sive immune cell infiltration [165, 166]. In support, human aortic VICs stimulated with proinflammatory mimetics not only induce the expression of inflammatory cytokines, but also induce the expression of osteogenic factors, such as BMP2 and Runx2, again suggesting that this process may be similar to what is occurring in atherosclerotic disease [164, 168]. Based on this evidence, multiple physiologic factors likely contribute to osteogenic gene induction in calcified diseased aortic valves.

Whereas both BMP and TGFβ signaling have been found to induce VIC calcification, Notch signaling has been implicated as a negative regulator of valve calcification. Familial studies demonstrated that Notch1 haploinsufficiency is associated with CAVD and aortic stenosis (AS) [64]. During development, Notch1 is expressed in the endothelial cells lining the aortic valve cusps and is also observed at lower levels in the VICs, and this expression pattern is maintained into adulthood [64, 175]. Histological analysis of human explanted aortic valves demonstrates that activated Notch1 intracellular domain (NICD) expression is dramatically reduced in VICs directly adjacent to regions of aortic valve calcification [175]. This observa‐ tion is consistent with a mechanism whereby Notch signaling inhibits valve calcification and downregulation of Notch expression promotes valve calcification [175]. The idea that Notch signaling functions as a negative regulator of calcification was originally defined in endo‐ chondral bone formation, where downstream effectors of Notch signaling, Hes1 and Hey1, repress Runx2 transcriptional function, leading to expansion of hypertrophic cartilage and impaired osteoblast differentiation [115]. Notch1 heterozygous or RBPJ heterozygous mice develop CAVD, as evidenced by increased aortic valve calcification, and also display signifi‐ cant increases in BMP/pSMAD1/5/8 signaling and Runx2 expression in the aortic valves [65, 153]. Likewise, deletion of RBPJ in adult mice results in increased aortic valve thickness with evidence of VIC proliferation and potentially, endothelial EMT [150]. Together these in vivo studies support the idea that Notch signaling represses BMP expression, thereby indirectly repressing other osteogenic factors [65, 153]. Cell culture studies indicate that Notch inhibi‐ tion promotes calcification of VICs by repressing chondrogenic genes, including *Sox9*, and inducing expression of the osteogenic genes *OPN, osteonectin, Runx2, ALP*, and *BMP2* [65, 153, 175]. Specifically, Notch signaling in the aortic valves is thought to induce expression of Sox9, which is a negative regulator of calcification, and to repress the expression of both Runx2 and BMP2, which are known to stimulate osteogenic differentiation [64, 153, 175]. These studies suggest that, in the absence of a negative regulator of calcification, the resident VICs possess an intrinsic calcification mechanism, which becomes activated and subse‐ quently induces valve calcification. Combined, the evidence suggests that Notch signaling is a negative regulator of VIC osteogenic differentiation, and that the absence or dysregulation

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 75

Wnt/β-catenin signaling is important for osteoblast maturation during embryonic develop‐ ment and contributes to mineralized bone formation (reviewed in [80]). A number of studies have also shown activation of Wnt/β-catenin signaling in aortic valve calcification. Canoni‐ cal Wnt signaling acts through the frizzled receptors and the Wnt co-receptors Lrp5 and Lrp6, resulting in β-catenin nuclear localization and TCF/LEF1 activation [176]. Human ex‐ planted calcific AoVs have increased expression of Lrp5, β-catenin, and Wnt3a ligand as compared to control valves [143]. Increased Wnt signaling in diseased aortic valves also has been observed in multiple animal models of CAVD. Pigs and rabbits maintained on an athe‐ rogenic diet develop aortic valve disease and display increased expression levels of β-cate‐ nin and Lrp5 receptor [173, 177]. Likewise, in a subset of endothelial nitric oxide synthase (eNOS) deficient mice that develop BAV, expression of Wnt3a ligand and Lrp5 receptor is increased when the animals are fed a high cholesterol diet [178]. Cell culture studies also support the idea that Wnt/β-catenin signaling is important for VIC myofibroblast activation,

of Notch signaling can induce valvular calcification.

#### *4.2.3. Valvulogenic- and osteogenic-related signaling pathways in CAVD*

As in both heart valve and endochondral bone development, BMP, TGFβ, Notch, and Wnt signaling have been implicated in the progression of CAVD (Figure 2; Table 1). Increased BMP ligand expression, particularly BMP2 and BMP4, has been demonstrated histologically in human explanted calcific aortic valves surrounding and throughout regions of valvular calcification [118, 129]. Furthermore, active BMP signaling, as indicated by pSMAD1/5/8 ex‐ pression, is present in both human explanted diseased aortic valves and animal models of CAVD [13, 169, 170]. Comparison of pediatric diseased valves void of calcification to heavily calcified adult diseased valves demonstrates extensive ECM remodeling and evidence of VIC activation in both; however, increased pSMAD1/5/8 signaling is exclusive to calcified valves [13]. The observation that pSMAD1/5/8 expression is found only in adult calcified valves is suggestive of a critical role for BMP signaling as an initiating osteogenic factor in CAVD [13]. Furthermore, increased pSMAD1/5/8 expression reportedly localizes to the fi‐ brosa layer of human calcific aortic valves, which is the primary sight of aortic valve calcifi‐ cation [169]. Cell culture studies support this and show that BMP2 stimulation promotes osteogenic-like aortic valve calcification in human aortic VICs by inducing the expression of the osteogenic factors Runx2, OPN, and ALP [138, 171]. Based on this evidence, active BMP signaling may be a potential therapeutic target to treat CAVD, however it has not yet been tested.

TGFβ signaling induces α SMA expression and myofibroblast differentiation of porcine aortic VICs, suggesting that TGFβ promotes VIC activation, potentially in response to physi‐ cal strain [125, 172]. Furthermore, TGFβ signaling may also have a role in aortic valve calcifi‐ cation, as human explanted calcific aortic valves have increased levels of TGFβ1 expression and ovine aortic VICs in culture calcify in response to TGFβ1 induction [136, 137]. TGFβ sig‐ naling has also been linked to both Wnt/β-catenin and FGF signaling pathways in CAVD [173, 174]. Specifically, FGF signals have been shown to induce MAPK signaling, which in‐ hibits aortic VIC αSMA expression and myofibroblast response to TGFβ [174]. In addition, TGFβ stimulation of aortic VICs induces nuclear localization and activation of β-catenin, which promotes VIC myofibroblast differentiation [173]. Although the role of TGFβ in CAVD is not well established in vivo, there is accumulating evidence for a role in VIC acti‐ vation and calcification from studies in cell culture systems.

Whereas both BMP and TGFβ signaling have been found to induce VIC calcification, Notch signaling has been implicated as a negative regulator of valve calcification. Familial studies demonstrated that Notch1 haploinsufficiency is associated with CAVD and aortic stenosis (AS) [64]. During development, Notch1 is expressed in the endothelial cells lining the aortic valve cusps and is also observed at lower levels in the VICs, and this expression pattern is maintained into adulthood [64, 175]. Histological analysis of human explanted aortic valves demonstrates that activated Notch1 intracellular domain (NICD) expression is dramatically reduced in VICs directly adjacent to regions of aortic valve calcification [175]. This observa‐ tion is consistent with a mechanism whereby Notch signaling inhibits valve calcification and downregulation of Notch expression promotes valve calcification [175]. The idea that Notch signaling functions as a negative regulator of calcification was originally defined in endo‐ chondral bone formation, where downstream effectors of Notch signaling, Hes1 and Hey1, repress Runx2 transcriptional function, leading to expansion of hypertrophic cartilage and impaired osteoblast differentiation [115]. Notch1 heterozygous or RBPJ heterozygous mice develop CAVD, as evidenced by increased aortic valve calcification, and also display signifi‐ cant increases in BMP/pSMAD1/5/8 signaling and Runx2 expression in the aortic valves [65, 153]. Likewise, deletion of RBPJ in adult mice results in increased aortic valve thickness with evidence of VIC proliferation and potentially, endothelial EMT [150]. Together these in vivo studies support the idea that Notch signaling represses BMP expression, thereby indirectly repressing other osteogenic factors [65, 153]. Cell culture studies indicate that Notch inhibi‐ tion promotes calcification of VICs by repressing chondrogenic genes, including *Sox9*, and inducing expression of the osteogenic genes *OPN, osteonectin, Runx2, ALP*, and *BMP2* [65, 153, 175]. Specifically, Notch signaling in the aortic valves is thought to induce expression of Sox9, which is a negative regulator of calcification, and to repress the expression of both Runx2 and BMP2, which are known to stimulate osteogenic differentiation [64, 153, 175]. These studies suggest that, in the absence of a negative regulator of calcification, the resident VICs possess an intrinsic calcification mechanism, which becomes activated and subse‐ quently induces valve calcification. Combined, the evidence suggests that Notch signaling is a negative regulator of VIC osteogenic differentiation, and that the absence or dysregulation of Notch signaling can induce valvular calcification.

and macrophage infiltration associated with induction of osteogenic markers such as ALP, OCN, OPN, Runx2, and Osx [165-167]. Although osteogenic gene expression is induced in these models, this type of valve calcification closely mimics vascular calcification observed in atherosclerosis, rather than endochondral bone formation, due to the presence of exten‐ sive immune cell infiltration [165, 166]. In support, human aortic VICs stimulated with proinflammatory mimetics not only induce the expression of inflammatory cytokines, but also induce the expression of osteogenic factors, such as BMP2 and Runx2, again suggesting that this process may be similar to what is occurring in atherosclerotic disease [164, 168]. Based on this evidence, multiple physiologic factors likely contribute to osteogenic gene induction

As in both heart valve and endochondral bone development, BMP, TGFβ, Notch, and Wnt signaling have been implicated in the progression of CAVD (Figure 2; Table 1). Increased BMP ligand expression, particularly BMP2 and BMP4, has been demonstrated histologically in human explanted calcific aortic valves surrounding and throughout regions of valvular calcification [118, 129]. Furthermore, active BMP signaling, as indicated by pSMAD1/5/8 ex‐ pression, is present in both human explanted diseased aortic valves and animal models of CAVD [13, 169, 170]. Comparison of pediatric diseased valves void of calcification to heavily calcified adult diseased valves demonstrates extensive ECM remodeling and evidence of VIC activation in both; however, increased pSMAD1/5/8 signaling is exclusive to calcified valves [13]. The observation that pSMAD1/5/8 expression is found only in adult calcified valves is suggestive of a critical role for BMP signaling as an initiating osteogenic factor in CAVD [13]. Furthermore, increased pSMAD1/5/8 expression reportedly localizes to the fi‐ brosa layer of human calcific aortic valves, which is the primary sight of aortic valve calcifi‐ cation [169]. Cell culture studies support this and show that BMP2 stimulation promotes osteogenic-like aortic valve calcification in human aortic VICs by inducing the expression of the osteogenic factors Runx2, OPN, and ALP [138, 171]. Based on this evidence, active BMP signaling may be a potential therapeutic target to treat CAVD, however it has not yet been

TGFβ signaling induces α SMA expression and myofibroblast differentiation of porcine aortic VICs, suggesting that TGFβ promotes VIC activation, potentially in response to physi‐ cal strain [125, 172]. Furthermore, TGFβ signaling may also have a role in aortic valve calcifi‐ cation, as human explanted calcific aortic valves have increased levels of TGFβ1 expression and ovine aortic VICs in culture calcify in response to TGFβ1 induction [136, 137]. TGFβ sig‐ naling has also been linked to both Wnt/β-catenin and FGF signaling pathways in CAVD [173, 174]. Specifically, FGF signals have been shown to induce MAPK signaling, which in‐ hibits aortic VIC αSMA expression and myofibroblast response to TGFβ [174]. In addition, TGFβ stimulation of aortic VICs induces nuclear localization and activation of β-catenin, which promotes VIC myofibroblast differentiation [173]. Although the role of TGFβ in CAVD is not well established in vivo, there is accumulating evidence for a role in VIC acti‐

in calcified diseased aortic valves.

74 Calcific Aortic Valve Disease

tested.

*4.2.3. Valvulogenic- and osteogenic-related signaling pathways in CAVD*

vation and calcification from studies in cell culture systems.

Wnt/β-catenin signaling is important for osteoblast maturation during embryonic develop‐ ment and contributes to mineralized bone formation (reviewed in [80]). A number of studies have also shown activation of Wnt/β-catenin signaling in aortic valve calcification. Canoni‐ cal Wnt signaling acts through the frizzled receptors and the Wnt co-receptors Lrp5 and Lrp6, resulting in β-catenin nuclear localization and TCF/LEF1 activation [176]. Human ex‐ planted calcific AoVs have increased expression of Lrp5, β-catenin, and Wnt3a ligand as compared to control valves [143]. Increased Wnt signaling in diseased aortic valves also has been observed in multiple animal models of CAVD. Pigs and rabbits maintained on an athe‐ rogenic diet develop aortic valve disease and display increased expression levels of β-cate‐ nin and Lrp5 receptor [173, 177]. Likewise, in a subset of endothelial nitric oxide synthase (eNOS) deficient mice that develop BAV, expression of Wnt3a ligand and Lrp5 receptor is increased when the animals are fed a high cholesterol diet [178]. Cell culture studies also support the idea that Wnt/β-catenin signaling is important for VIC myofibroblast activation, proliferation, and chondrogenic gene induction. Studies in porcine aortic VICs show that Wnt3a treatment induces significant VIC proliferation and myofibroblast activation [173, 179]. Furthermore, Wnt3a treatment of embryonic chicken aortic VICs results in increased expression of *periostin* and *mgp*, but does not induce the expression of osteogenic-related genes, suggesting that Wnt3a signaling is not sufficient for VIC osteogenic differentiation [60]. However in adult valves, Wnt signaling can promote the VIC calcification response, as loss of Wnt signaling through the Lrp5 receptor in ApoE knockout mice results in decreased aortic valve calcification [180]. Together these studies demonstrate that Wnt signaling likely contributes to VIC activation, proliferation, and calcification in CAVD.

calcification, whereas RANKL expression is higher in stenotic calcified valves [103, 193]. This study concluded that OPG may be protective against valve calcification, whereas ele‐ vated RANKL expression may promote valve calcification by promoting upregulation of matrix remodeling enzymes [103, 193]. Furthermore, treatment of human aortic VICs with RANKL results in increased MMP1 and MMP2 activity with increased VIC proliferation, concomitant with increased calcification and osteogenic gene expression [103, 191]. In addi‐ tion, NFATc1 expression is increased in human explanted aortic valve leaflets with CAVD [106]. Together, these studies are consistent with signaling events during bone development, namely RANKL activation of NFATc1, stimulating matrix remodeling enzymes, and pro‐

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 77

A number of other signaling pathways are likely involved in ECM changes that occur dur‐ ing CAVD. In particular, TGFβ1 stimulation of cultured VICs stimulates myofibroblast dif‐ ferentiation, leading to increased levels of αSMA stress fibers in the VICs [125, 172]. It has been suggested that these myofibroblasts then exert a contractile force on the surrounding valve ECM and stimulate rearrangement of the matrix, particularly in fibronectin fibers [125]. Furthermore, TGFβ1 stimulation also induces increased type I collagen production and expression of the matrix remodeling enzymes MMP9 and MMP2 in cultured aortic VICs [136, 172]. These studies indicate that TGFβ1 signaling may be a key factor in ECM-related changes during CAVD pathogenesis. Moreover, Wnt signaling may work in concert with TGFβ1 to induce changes in ECM during CAVD [173]. TGFβ1 stimulation promotes nuclear localization and activation of β-catenin in cultured VICs, and, when combined, Wnt and TGFβ1 signaling dramatically increases myofibroblast activation [173]. In contrast to TGFβ1 and Wnt signaling, FGF signaling may work to inhibit ECM remodeling during valve dis‐ ease. FGF signaling has been shown to block TGFβ1 induced myofibroblast differentiation and αSMA expression in porcine aortic VICs through activation of phospho-ERK1/2 signal‐ ing [174]. In addition, FGF signaling inhibits myofibroblast contraction of a collagen matrix, supporting the idea that FGF signaling blocks TGFβ1 stimulation of matrix-related changes [174]. Many parallels exist between signaling factors involved in ECM changes in develop‐ ment and disease. In particular, RANKL, TGFβ1, Wnt, and FGF signaling have demonstrat‐ ed roles in ECM production and regulation in both heart valve and endochondral bone formation [8, 76]. The shared signaling pathways in these tissues, both in development and disease, suggest that developmental pathways may be reactivated in CAVD to induce ma‐

Currently, aortic valve replacement surgery is the only effective treatment option for CAVD [122]. There have been numerous studies, which are summarized below, testing the effec‐ tiveness of different pharmacotherapies on preventing the progression of AS. Unfortunately, studies on statin therapies, inhibitors of the renin-angiotensin-aldosterone system, and os‐ teoporosis treatments have not been proven to be effective at preventing the symptoms or the progression of CAVD/AS. Following the summary of these studies, additional treatment

moting calcification [192].

trix changes characteristic of the disease.

**5. Therapeutic mechanisms in CAVD**

#### **4.3. Matrix remodeling in CAVD**

Diseased aortic valves are characterized by changes in the ECM; in particular, disorganized collagen bundles and extensive elastic fiber fragmentation are observed [181, 182]. Insight into the role of elastin fiber disorganization in the pathogenesis of CAVD has been provided through studies of elastin haploinsufficient mice, which display elastin fiber fragmentation, abnormal ECM remodeling, and increased valve stiffness, suggesting that elastin homeosta‐ sis is important for maintaining valve function [72, 183]. Collagen synthesis and remodeling are dramatically increased in CAVD, however, overall collagen content in the valve is ac‐ tually decreased, suggesting that there is extensive collagen proteolysis during disease [184-186]. In contrast to collagens, expression of proteoglycans, including decorin, biglycan, versican, and hyaluronan, is increased particularly in regions of the diseased valve adjacent to calcific nodules [187]. These changes in ECM composition during CAVD can be compared to matrix remodeling events that occur during valve development and also in bone forma‐ tion. The decreased collagen content and increased proteoglycan matrix found in CAVD is similar to the primitive ECM characteristic of early valve development [188]. Furthermore, parallels can also be drawn between matrix remodeling in CAVD and bone development. Specifically, matrix remodeling in the immature bone is essential for providing a scaffold upon which the calcified matrix is deposited, and subsequent ECM degradation is essential for expansion of the calcified regions of newly forming bone [189]. The parallels between matrix remodeling in bone development and the disease process of CAVD suggest that valve matrix remodeling may contribute to valvular calcification.

Matrix degradation and remodeling in valvulogenesis, osteogenesis, and CAVD occurs con‐ comitant with increased activity of MMPs and cathepsins, along with increased RANKL sig‐ naling. A number of studies have shown significant increases in expression of multiple MMPs, including MMP1, MMP3, MMP7, MMP9, and MMP12, with increased cathepsins B, K, and S in human calcific diseased aortic valves, suggesting that extensive ECM remodel‐ ing is a key feature of disease [124, 163, 181, 182, 184, 190]. In bone, RANKL signals through the RANK receptor, which can be inhibited via binding to the soluble receptor OPG, and promotes the expression of proteolytic enzymes, such as MMPs and cathepsin K, through activation of NFATc1 [191, 192]. A similar mechanism has been identified in heart valve re‐ modeling [47, 61]. Comparison of sclerotic diseased aortic valves and advanced stenotic aortic valves determined that OPG levels are significantly higher in sclerotic valves without calcification, whereas RANKL expression is higher in stenotic calcified valves [103, 193]. This study concluded that OPG may be protective against valve calcification, whereas ele‐ vated RANKL expression may promote valve calcification by promoting upregulation of matrix remodeling enzymes [103, 193]. Furthermore, treatment of human aortic VICs with RANKL results in increased MMP1 and MMP2 activity with increased VIC proliferation, concomitant with increased calcification and osteogenic gene expression [103, 191]. In addi‐ tion, NFATc1 expression is increased in human explanted aortic valve leaflets with CAVD [106]. Together, these studies are consistent with signaling events during bone development, namely RANKL activation of NFATc1, stimulating matrix remodeling enzymes, and pro‐ moting calcification [192].

A number of other signaling pathways are likely involved in ECM changes that occur dur‐ ing CAVD. In particular, TGFβ1 stimulation of cultured VICs stimulates myofibroblast dif‐ ferentiation, leading to increased levels of αSMA stress fibers in the VICs [125, 172]. It has been suggested that these myofibroblasts then exert a contractile force on the surrounding valve ECM and stimulate rearrangement of the matrix, particularly in fibronectin fibers [125]. Furthermore, TGFβ1 stimulation also induces increased type I collagen production and expression of the matrix remodeling enzymes MMP9 and MMP2 in cultured aortic VICs [136, 172]. These studies indicate that TGFβ1 signaling may be a key factor in ECM-related changes during CAVD pathogenesis. Moreover, Wnt signaling may work in concert with TGFβ1 to induce changes in ECM during CAVD [173]. TGFβ1 stimulation promotes nuclear localization and activation of β-catenin in cultured VICs, and, when combined, Wnt and TGFβ1 signaling dramatically increases myofibroblast activation [173]. In contrast to TGFβ1 and Wnt signaling, FGF signaling may work to inhibit ECM remodeling during valve dis‐ ease. FGF signaling has been shown to block TGFβ1 induced myofibroblast differentiation and αSMA expression in porcine aortic VICs through activation of phospho-ERK1/2 signal‐ ing [174]. In addition, FGF signaling inhibits myofibroblast contraction of a collagen matrix, supporting the idea that FGF signaling blocks TGFβ1 stimulation of matrix-related changes [174]. Many parallels exist between signaling factors involved in ECM changes in develop‐ ment and disease. In particular, RANKL, TGFβ1, Wnt, and FGF signaling have demonstrat‐ ed roles in ECM production and regulation in both heart valve and endochondral bone formation [8, 76]. The shared signaling pathways in these tissues, both in development and disease, suggest that developmental pathways may be reactivated in CAVD to induce ma‐ trix changes characteristic of the disease.

### **5. Therapeutic mechanisms in CAVD**

proliferation, and chondrogenic gene induction. Studies in porcine aortic VICs show that Wnt3a treatment induces significant VIC proliferation and myofibroblast activation [173, 179]. Furthermore, Wnt3a treatment of embryonic chicken aortic VICs results in increased expression of *periostin* and *mgp*, but does not induce the expression of osteogenic-related genes, suggesting that Wnt3a signaling is not sufficient for VIC osteogenic differentiation [60]. However in adult valves, Wnt signaling can promote the VIC calcification response, as loss of Wnt signaling through the Lrp5 receptor in ApoE knockout mice results in decreased aortic valve calcification [180]. Together these studies demonstrate that Wnt signaling likely

Diseased aortic valves are characterized by changes in the ECM; in particular, disorganized collagen bundles and extensive elastic fiber fragmentation are observed [181, 182]. Insight into the role of elastin fiber disorganization in the pathogenesis of CAVD has been provided through studies of elastin haploinsufficient mice, which display elastin fiber fragmentation, abnormal ECM remodeling, and increased valve stiffness, suggesting that elastin homeosta‐ sis is important for maintaining valve function [72, 183]. Collagen synthesis and remodeling are dramatically increased in CAVD, however, overall collagen content in the valve is ac‐ tually decreased, suggesting that there is extensive collagen proteolysis during disease [184-186]. In contrast to collagens, expression of proteoglycans, including decorin, biglycan, versican, and hyaluronan, is increased particularly in regions of the diseased valve adjacent to calcific nodules [187]. These changes in ECM composition during CAVD can be compared to matrix remodeling events that occur during valve development and also in bone forma‐ tion. The decreased collagen content and increased proteoglycan matrix found in CAVD is similar to the primitive ECM characteristic of early valve development [188]. Furthermore, parallels can also be drawn between matrix remodeling in CAVD and bone development. Specifically, matrix remodeling in the immature bone is essential for providing a scaffold upon which the calcified matrix is deposited, and subsequent ECM degradation is essential for expansion of the calcified regions of newly forming bone [189]. The parallels between matrix remodeling in bone development and the disease process of CAVD suggest that

Matrix degradation and remodeling in valvulogenesis, osteogenesis, and CAVD occurs con‐ comitant with increased activity of MMPs and cathepsins, along with increased RANKL sig‐ naling. A number of studies have shown significant increases in expression of multiple MMPs, including MMP1, MMP3, MMP7, MMP9, and MMP12, with increased cathepsins B, K, and S in human calcific diseased aortic valves, suggesting that extensive ECM remodel‐ ing is a key feature of disease [124, 163, 181, 182, 184, 190]. In bone, RANKL signals through the RANK receptor, which can be inhibited via binding to the soluble receptor OPG, and promotes the expression of proteolytic enzymes, such as MMPs and cathepsin K, through activation of NFATc1 [191, 192]. A similar mechanism has been identified in heart valve re‐ modeling [47, 61]. Comparison of sclerotic diseased aortic valves and advanced stenotic aortic valves determined that OPG levels are significantly higher in sclerotic valves without

contributes to VIC activation, proliferation, and calcification in CAVD.

valve matrix remodeling may contribute to valvular calcification.

**4.3. Matrix remodeling in CAVD**

76 Calcific Aortic Valve Disease

Currently, aortic valve replacement surgery is the only effective treatment option for CAVD [122]. There have been numerous studies, which are summarized below, testing the effec‐ tiveness of different pharmacotherapies on preventing the progression of AS. Unfortunately, studies on statin therapies, inhibitors of the renin-angiotensin-aldosterone system, and os‐ teoporosis treatments have not been proven to be effective at preventing the symptoms or the progression of CAVD/AS. Following the summary of these studies, additional treatment options, related to the expression of developmental and osteogenic-related genes in CAVD, are discussed.

tion: Measuring Effects of Rosuvastatin), found that statin therapy was not effective at treat‐ ing the progression of AS [199-201]. In these trials, three different statin therapies were investigated in patients with mild to moderate AS and it was determined that statin therapy did not alter the progression of CAVD/AS nor prevent outcomes such as the necessity to un‐ dergo aortic valve replacement surgery [199-201]. In response to the negative outcomes of these large clinical trials, the use of statin therapy was next investigated in patients with the earliest form of CAVD/AS, aortic valve sclerosis, to determine if statin use could prevent, rather than reverse, AS [202]. In this report, statin therapy was significantly associated with a decreased development of AS and a decreased need for aortic valve replacement surgery, suggesting that statin therapy may be an effective treatment if started at the earliest stages of the disease, prior to any indication of valve calcification [202]. Based on these studies, it can be concluded that in humans, statin therapy is ineffective at preventing the progression of AS and reversing aortic valve calcification. However, statin therapy may be useful at pre‐

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 79

venting the onset of AS in patients with the earliest stages of aortic valve thickening.

Another potential therapy to prevent the progression of CAVD/AS is the use of the anti-hy‐ pertensive angiotensin-converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB). Currently, ACEIs and ARBs are prescribed to treat hypertension, and func‐ tion by acting on the renin-angiotensin-aldosterone system to ultimately inhibit the vasocon‐ strictor effects of angiotensin II [203]. Previous reports have identified the overlapping expression of angiotensin-converting enzyme (ACE) and angiotensin II in calcified human aortic valves surrounding regions of valvular calcification [133]. It has been hypothesized that ACE inhibition may prevent the progression of CAVD by reducing ACE activity in the diseased valve leaflet [204-206]. A study conducted in ApoE knockout mice with induced chronic renal failure concluded that animals treated with the ACEI enalapril had significant‐ ly reduced levels of pathologic aortic valve leaflet thickening and valve fibrosis than un‐ treated animals [206]. Similarly, in a rabbit model of CAVD in which the animals were fed a high vitamin D diet, treatment with the ACEI ramipril significantly reduced the progression to AS, improved valve endothelial cell integrity, and reduced aortic valve calcification [205]. It is uncertain how ACE and angiotensin receptor (AR) inhibition would directly affect mo‐ lecular changes in the valve leaflets. However, in a study of rabbits fed a high cholesterol diet, treatment with the ARB olmesartan decreased the number of α SMA positive myofibro‐ blasts and reduced expression of the osteogenic markers *Runx2* and OPN, compared to un‐ treated control animals [204]. These animal studies suggest that ACE and/or AR inhibition may reduce pathologic changes in aortic valve disease by limiting valve fibrosis, reducing

**5.2. Angiotensin converting enzyme inhibitors/Angiotensin receptor blockers**

myofibroblast activation, and decreasing osteogenic gene expression.

Clinical studies testing the therapeutic benefits of ACEIs and ARBs in CAVD progression have had mixed results. In human explanted aortic valve tissues, ARB therapy is associated with reduced aortic valve remodeling and calcification [207]. As in animal studies, this his‐ tological analysis suggests that AR inhibition may limit aortic valve calcification [207]. In a small pilot clinical study (Symptomatic Cardiac Obstruction – Pilot Study of Enalapril in

#### **5.1. Statins**

Lipid deposition and the accumulation of apolipoproteins (Apo) in the aortic valve leaflets have long been associated with CAVD, and many studies have compared the progression of CAVD to atherosclerotic disease [119, 133, 134, 194]. Therefore, it has been hypothesized that cholesterol lowering therapy with statin drugs may be an effective treatment strategy to de‐ lay the progression of CAVD. A specialized mouse model called "Reversa" mice develop signs of CAVD when fed a high cholesterol diet, however, when serum cholesterol is low‐ ered via a genetic deletion of the microsomal triglyceride transfer protein (Mttp), reduced levels of aortic valve calcification, as well as decreased expression of the osteogenic markers pSMAD1/5/8, Msx2, Osx, β-catenin, and Runx2, are observed [167, 170]. Thus reducing plas‐ ma cholesterol may reduce CAVD pathogenesis, particularly in terms of reducing osteogen‐ ic gene expression in the diseased valves. Similarly, statin treatment of human or porcine aortic VICs cultured concomitantly with osteogenic media results in decreased expression of the osteogenic genes ALP, OCN, Lrp5, and OPN, and reduced calcific nodule formation [171, 177, 195]. However, when statin treatment of aortic VICs is initiated after osteogenic transformation or calcific nodule formation, it is ineffective at reducing calcification and ex‐ pression of osteogenic markers, indicating that statin therapy cannot reverse aortic valve cal‐ cification and osteogenic differentiation once it has occurred [195, 196]. Results from animal studies are equally contradictory. Rabbits fed a high cholesterol diet supplemented with atorvastatin have decreased aortic valve thickness, reduced VIC proliferation, and reduced expression of Lrp5, β -catenin, OPN, *Runx2*, and *ALP*, compared to those animals fed only a high cholesterol diet [165, 177]. Similarly, endothelial nitric oxide synthase (eNOS) deficient mice, displaying a BAV phenotype and fed a high cholesterol diet, have reduced *Lrp5* and *Wnt3a* expression as well as reduced aortic valve calcification when treated with statins, compared to animals fed only a high cholesterol diet [178]. In contrast, a long term study in rabbits fed a high cholesterol diet showed that atorvastatin therapy initiated after aortic valve disease is established is not effective at reducing the amount of aortic valve calcifica‐ tion present, although some improvements in other histological parameters were noted [197]. Based on both cell culture and animal studies, statin therapy may improve some measures of aortic valve calcification, specifically in terms of reducing osteogenic gene ex‐ pression, however, firm conclusions as to potential efficacy as a CAVD treatment cannot be drawn.

Clinicial studies investigating the use of statin therapy in patients with CAVD are also wide‐ ly contradictory. An early study investigating the use of statin therapy in patients with mod‐ erate to severe aortic stenosis (AS) reported that patients treated with statins had less hemodynamic progression of AS over a 2 year time period than patients who were not on statin therapy [198]. In contrast, three larger prospective clinical studies, SALTIRE (Scottish Aortic Stenosis and Lipid Lowering Trial, Impact on Regression), SEAS (Simvastatin and Ezetimibe in Aortic Stenosis), and ASTRONOMER (Aortic Stenosis Progression Observa‐ tion: Measuring Effects of Rosuvastatin), found that statin therapy was not effective at treat‐ ing the progression of AS [199-201]. In these trials, three different statin therapies were investigated in patients with mild to moderate AS and it was determined that statin therapy did not alter the progression of CAVD/AS nor prevent outcomes such as the necessity to un‐ dergo aortic valve replacement surgery [199-201]. In response to the negative outcomes of these large clinical trials, the use of statin therapy was next investigated in patients with the earliest form of CAVD/AS, aortic valve sclerosis, to determine if statin use could prevent, rather than reverse, AS [202]. In this report, statin therapy was significantly associated with a decreased development of AS and a decreased need for aortic valve replacement surgery, suggesting that statin therapy may be an effective treatment if started at the earliest stages of the disease, prior to any indication of valve calcification [202]. Based on these studies, it can be concluded that in humans, statin therapy is ineffective at preventing the progression of AS and reversing aortic valve calcification. However, statin therapy may be useful at pre‐ venting the onset of AS in patients with the earliest stages of aortic valve thickening.

#### **5.2. Angiotensin converting enzyme inhibitors/Angiotensin receptor blockers**

options, related to the expression of developmental and osteogenic-related genes in CAVD,

Lipid deposition and the accumulation of apolipoproteins (Apo) in the aortic valve leaflets have long been associated with CAVD, and many studies have compared the progression of CAVD to atherosclerotic disease [119, 133, 134, 194]. Therefore, it has been hypothesized that cholesterol lowering therapy with statin drugs may be an effective treatment strategy to de‐ lay the progression of CAVD. A specialized mouse model called "Reversa" mice develop signs of CAVD when fed a high cholesterol diet, however, when serum cholesterol is low‐ ered via a genetic deletion of the microsomal triglyceride transfer protein (Mttp), reduced levels of aortic valve calcification, as well as decreased expression of the osteogenic markers pSMAD1/5/8, Msx2, Osx, β-catenin, and Runx2, are observed [167, 170]. Thus reducing plas‐ ma cholesterol may reduce CAVD pathogenesis, particularly in terms of reducing osteogen‐ ic gene expression in the diseased valves. Similarly, statin treatment of human or porcine aortic VICs cultured concomitantly with osteogenic media results in decreased expression of the osteogenic genes ALP, OCN, Lrp5, and OPN, and reduced calcific nodule formation [171, 177, 195]. However, when statin treatment of aortic VICs is initiated after osteogenic transformation or calcific nodule formation, it is ineffective at reducing calcification and ex‐ pression of osteogenic markers, indicating that statin therapy cannot reverse aortic valve cal‐ cification and osteogenic differentiation once it has occurred [195, 196]. Results from animal studies are equally contradictory. Rabbits fed a high cholesterol diet supplemented with atorvastatin have decreased aortic valve thickness, reduced VIC proliferation, and reduced expression of Lrp5, β -catenin, OPN, *Runx2*, and *ALP*, compared to those animals fed only a high cholesterol diet [165, 177]. Similarly, endothelial nitric oxide synthase (eNOS) deficient mice, displaying a BAV phenotype and fed a high cholesterol diet, have reduced *Lrp5* and *Wnt3a* expression as well as reduced aortic valve calcification when treated with statins, compared to animals fed only a high cholesterol diet [178]. In contrast, a long term study in rabbits fed a high cholesterol diet showed that atorvastatin therapy initiated after aortic valve disease is established is not effective at reducing the amount of aortic valve calcifica‐ tion present, although some improvements in other histological parameters were noted [197]. Based on both cell culture and animal studies, statin therapy may improve some measures of aortic valve calcification, specifically in terms of reducing osteogenic gene ex‐ pression, however, firm conclusions as to potential efficacy as a CAVD treatment cannot be

Clinicial studies investigating the use of statin therapy in patients with CAVD are also wide‐ ly contradictory. An early study investigating the use of statin therapy in patients with mod‐ erate to severe aortic stenosis (AS) reported that patients treated with statins had less hemodynamic progression of AS over a 2 year time period than patients who were not on statin therapy [198]. In contrast, three larger prospective clinical studies, SALTIRE (Scottish Aortic Stenosis and Lipid Lowering Trial, Impact on Regression), SEAS (Simvastatin and Ezetimibe in Aortic Stenosis), and ASTRONOMER (Aortic Stenosis Progression Observa‐

are discussed.

78 Calcific Aortic Valve Disease

**5.1. Statins**

drawn.

Another potential therapy to prevent the progression of CAVD/AS is the use of the anti-hy‐ pertensive angiotensin-converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB). Currently, ACEIs and ARBs are prescribed to treat hypertension, and func‐ tion by acting on the renin-angiotensin-aldosterone system to ultimately inhibit the vasocon‐ strictor effects of angiotensin II [203]. Previous reports have identified the overlapping expression of angiotensin-converting enzyme (ACE) and angiotensin II in calcified human aortic valves surrounding regions of valvular calcification [133]. It has been hypothesized that ACE inhibition may prevent the progression of CAVD by reducing ACE activity in the diseased valve leaflet [204-206]. A study conducted in ApoE knockout mice with induced chronic renal failure concluded that animals treated with the ACEI enalapril had significant‐ ly reduced levels of pathologic aortic valve leaflet thickening and valve fibrosis than un‐ treated animals [206]. Similarly, in a rabbit model of CAVD in which the animals were fed a high vitamin D diet, treatment with the ACEI ramipril significantly reduced the progression to AS, improved valve endothelial cell integrity, and reduced aortic valve calcification [205]. It is uncertain how ACE and angiotensin receptor (AR) inhibition would directly affect mo‐ lecular changes in the valve leaflets. However, in a study of rabbits fed a high cholesterol diet, treatment with the ARB olmesartan decreased the number of α SMA positive myofibro‐ blasts and reduced expression of the osteogenic markers *Runx2* and OPN, compared to un‐ treated control animals [204]. These animal studies suggest that ACE and/or AR inhibition may reduce pathologic changes in aortic valve disease by limiting valve fibrosis, reducing myofibroblast activation, and decreasing osteogenic gene expression.

Clinical studies testing the therapeutic benefits of ACEIs and ARBs in CAVD progression have had mixed results. In human explanted aortic valve tissues, ARB therapy is associated with reduced aortic valve remodeling and calcification [207]. As in animal studies, this his‐ tological analysis suggests that AR inhibition may limit aortic valve calcification [207]. In a small pilot clinical study (Symptomatic Cardiac Obstruction – Pilot Study of Enalapril in Aortic Stenosis), use of the ACEI Enalapril was associated with improved clinical symptoms in patients with severe symptomatic AS [208]. The majority of studies investigating the use of ACEIs or ARBs in CAVD/AS with positive outcomes have been retrospective. In three dif‐ ferent retrospective studies, ACEI or ARB use in patients with mild to moderate AS was as‐ sociated with decreased mortality, decreased number of adverse cardiovascular events, slower progression of AS, and less accumulation of valvular calcification [209-211]. Addi‐ tionally, one prospective study followed a small random population of patients over a 4 year period and reported that the use of ACEIs or ARBs was significantly associated with reduced CAVD/AS disease progression [212]. Together, these studies provide evidence that ACEI and ARB may delay CAVD progression. Conversely, there have also been a number of studies that show no association between ACEI and ARB use and improved outcomes in CAVD progression. The JASS study (Japanese Aortic Stenosis Study) reported that ARB therapy in patients with moderate to severe AS had no beneficial outcomes in CAVD pro‐ gression, although patients with mild asymptomatic AS had some indication of reduced progression to AS [213]. Furthermore, a large study in patients with very mild asymptomat‐ ic AS found that patients on ACEI or ARB therapy had no improvement in the progression of AS compared against a control group [202]. Similarly, a small 2-year study observed no difference in the hemodynamic progression of AS with ACEI use versus non-use [198]. Based on both animal and clinical studies, it is unclear whether ACEI or ARB therapy is an effective treatment option to prevent the progression of CAVD/AS, however there are indi‐ cations that perhaps this therapy may limit valve calcification [204, 207]. A placebo control‐ led, blinded trial will be necessary to determine the effectiveness of these therapies in treating CAVD.

**5.4. Bisphosphonates**

prospective studies will be necessary.

**5.5. Nitric oxide bioavailability**

Bisphosphonates (BP) are a class of drugs that mimic inorganic pyrophosphate and prevent ectopic soft tissue calcification and inhibit bone resorption [216]. In adults, especially wom‐ en, BPs are commonly prescribed to treat excessive bone resorption associated with osteopo‐ rosis [216]. Human aortic VICs, grown on collagen gels in the presence of a specialized thiol bisphosphonate, have decreased ALP activity and reduced cellular aggregation, a step that precedes calcific nodule formation, as compared to cells grown on collagen alone [217]. This study suggests that bisphosphonates may inhibit VIC calcification in vitro and could serve as a potential therapeutic strategy to prevent aortic valve calcification [217]. Due to the abili‐ ty of BPs to prevent ectopic calcification in bone and the availability of patient populations currently using BPs, a number of studies have investigated the use of BPs in the inhibition of aortic valve calcification. Three small retrospective human studies compared measurements of AS progression over a 2-year period in patients with AS taking BPs versus those not tak‐ ing BPs [218-220]. The results of these studies suggest a modest reduction in the progression of AS in those patients taking BPs [218-220]. The large MESA study (Multi-Ethnic Study of Atherosclerosis) followed women taking BPs, compared to those not taking BPs, and their development of CAVD/AS over time [221]. The results of this study were mixed and showed that, in older women, BP therapy was associated with a slight benefit in terms of aortic valve calcification, whereas, younger women taking BPs had significantly more pro‐ gression of aortic valve calcification compared to women not taking BPs [221]. Most recent‐ ly, a large retrospective study investigated the progression of AS in women with mild to moderate AS over a 5-year period and compared the outcomes in patients on BP therapy versus those not taking BPs [222]. The evidence from this study shows that there was no change in survival, or in the number of aortic valve replacement surgeries, in women taking BPs compared to those not taking BPs, suggesting that BP therapy does not suppress the progression of CAVD/AS [222]. Thus far, the outcomes of the human studies investigating the use of bisphosphonate therapy in CAVD demonstrate that this therapy is ineffective at preventing or delaying the progression of CAVD/AS. To definitively determine whether or not BP therapy is effective at suppressing the progression of CAVD/AS, placebo-controlled

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 81

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO) from L-arginine, and eNOS expression has been identified in the endothelial cells lining the aortic valves [68]. eNOS deficiency has been linked to defective aortic valve development, as approximately 50% of eNOS deficient mice develop a bicuspid, rather than tricuspid, aortic valve [68]. eNOS deficient mice with a BAV phenotype fed a high cholesterol diet develop hemody‐ namic symptoms of AS and also display microscopic mineralization in the aortic valve leaf‐ lets, indicating that eNOS activity may be important for suppressing aortic valve calcification [178]. Nitric Oxide deficiency is also an indicator of endothelial cell dysfunction, and systemic endothelial cell dysfunction is prevalent in patients with aortic valve sclerosis/ stenosis [223-225]. The uncoupling, or dysfunction, of eNOS results in decreased NO pro‐

#### **5.3. Aldosterone-receptor antagonists**

Aldosterone is a component of the renin-angiotensin-aldosterone system that plays a key role in the kidney to regulate water and sodium reabsorption and effectively raise blood pressure [203]. Aldosterone-receptor antagonists (ARA) are commonly prescribed for their diuretic effects [203]. Recently, there have been two studies investigating the use of ARAs in the treatment of CAVD/AS. In an animal study, rabbits fed a high cholesterol diet develop aortic valve sclerosis, with thickening of the valve leaflets and microscopic calcific deposits, which was blocked by treatment with the ARA eplerenone [214]. In addition to reducing valve fibrosis and mineralization, evidence of macrophage infiltration was also reduced [214]. Conversely, in a small placebo-controlled human trial of patients with moderate to se‐ vere asymptomatic AS, there was no difference in the progression of AS in those patients receiving the ARA eplerenone versus placebo [215]. To our knowledge, no molecular evi‐ dence has been reported in studies on ARA therapy in CAVD and it is unknown whether ARA therapy affects myofibroblast activation or osteogenic gene induction. Additional clini‐ cal studies will be necessary to determine if ARA use can prevent AS progression if therapy is started in early disease stages.

#### **5.4. Bisphosphonates**

Aortic Stenosis), use of the ACEI Enalapril was associated with improved clinical symptoms in patients with severe symptomatic AS [208]. The majority of studies investigating the use of ACEIs or ARBs in CAVD/AS with positive outcomes have been retrospective. In three dif‐ ferent retrospective studies, ACEI or ARB use in patients with mild to moderate AS was as‐ sociated with decreased mortality, decreased number of adverse cardiovascular events, slower progression of AS, and less accumulation of valvular calcification [209-211]. Addi‐ tionally, one prospective study followed a small random population of patients over a 4 year period and reported that the use of ACEIs or ARBs was significantly associated with reduced CAVD/AS disease progression [212]. Together, these studies provide evidence that ACEI and ARB may delay CAVD progression. Conversely, there have also been a number of studies that show no association between ACEI and ARB use and improved outcomes in CAVD progression. The JASS study (Japanese Aortic Stenosis Study) reported that ARB therapy in patients with moderate to severe AS had no beneficial outcomes in CAVD pro‐ gression, although patients with mild asymptomatic AS had some indication of reduced progression to AS [213]. Furthermore, a large study in patients with very mild asymptomat‐ ic AS found that patients on ACEI or ARB therapy had no improvement in the progression of AS compared against a control group [202]. Similarly, a small 2-year study observed no difference in the hemodynamic progression of AS with ACEI use versus non-use [198]. Based on both animal and clinical studies, it is unclear whether ACEI or ARB therapy is an effective treatment option to prevent the progression of CAVD/AS, however there are indi‐ cations that perhaps this therapy may limit valve calcification [204, 207]. A placebo control‐ led, blinded trial will be necessary to determine the effectiveness of these therapies in

Aldosterone is a component of the renin-angiotensin-aldosterone system that plays a key role in the kidney to regulate water and sodium reabsorption and effectively raise blood pressure [203]. Aldosterone-receptor antagonists (ARA) are commonly prescribed for their diuretic effects [203]. Recently, there have been two studies investigating the use of ARAs in the treatment of CAVD/AS. In an animal study, rabbits fed a high cholesterol diet develop aortic valve sclerosis, with thickening of the valve leaflets and microscopic calcific deposits, which was blocked by treatment with the ARA eplerenone [214]. In addition to reducing valve fibrosis and mineralization, evidence of macrophage infiltration was also reduced [214]. Conversely, in a small placebo-controlled human trial of patients with moderate to se‐ vere asymptomatic AS, there was no difference in the progression of AS in those patients receiving the ARA eplerenone versus placebo [215]. To our knowledge, no molecular evi‐ dence has been reported in studies on ARA therapy in CAVD and it is unknown whether ARA therapy affects myofibroblast activation or osteogenic gene induction. Additional clini‐ cal studies will be necessary to determine if ARA use can prevent AS progression if therapy

treating CAVD.

80 Calcific Aortic Valve Disease

**5.3. Aldosterone-receptor antagonists**

is started in early disease stages.

Bisphosphonates (BP) are a class of drugs that mimic inorganic pyrophosphate and prevent ectopic soft tissue calcification and inhibit bone resorption [216]. In adults, especially wom‐ en, BPs are commonly prescribed to treat excessive bone resorption associated with osteopo‐ rosis [216]. Human aortic VICs, grown on collagen gels in the presence of a specialized thiol bisphosphonate, have decreased ALP activity and reduced cellular aggregation, a step that precedes calcific nodule formation, as compared to cells grown on collagen alone [217]. This study suggests that bisphosphonates may inhibit VIC calcification in vitro and could serve as a potential therapeutic strategy to prevent aortic valve calcification [217]. Due to the abili‐ ty of BPs to prevent ectopic calcification in bone and the availability of patient populations currently using BPs, a number of studies have investigated the use of BPs in the inhibition of aortic valve calcification. Three small retrospective human studies compared measurements of AS progression over a 2-year period in patients with AS taking BPs versus those not tak‐ ing BPs [218-220]. The results of these studies suggest a modest reduction in the progression of AS in those patients taking BPs [218-220]. The large MESA study (Multi-Ethnic Study of Atherosclerosis) followed women taking BPs, compared to those not taking BPs, and their development of CAVD/AS over time [221]. The results of this study were mixed and showed that, in older women, BP therapy was associated with a slight benefit in terms of aortic valve calcification, whereas, younger women taking BPs had significantly more pro‐ gression of aortic valve calcification compared to women not taking BPs [221]. Most recent‐ ly, a large retrospective study investigated the progression of AS in women with mild to moderate AS over a 5-year period and compared the outcomes in patients on BP therapy versus those not taking BPs [222]. The evidence from this study shows that there was no change in survival, or in the number of aortic valve replacement surgeries, in women taking BPs compared to those not taking BPs, suggesting that BP therapy does not suppress the progression of CAVD/AS [222]. Thus far, the outcomes of the human studies investigating the use of bisphosphonate therapy in CAVD demonstrate that this therapy is ineffective at preventing or delaying the progression of CAVD/AS. To definitively determine whether or not BP therapy is effective at suppressing the progression of CAVD/AS, placebo-controlled prospective studies will be necessary.

#### **5.5. Nitric oxide bioavailability**

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO) from L-arginine, and eNOS expression has been identified in the endothelial cells lining the aortic valves [68]. eNOS deficiency has been linked to defective aortic valve development, as approximately 50% of eNOS deficient mice develop a bicuspid, rather than tricuspid, aortic valve [68]. eNOS deficient mice with a BAV phenotype fed a high cholesterol diet develop hemody‐ namic symptoms of AS and also display microscopic mineralization in the aortic valve leaf‐ lets, indicating that eNOS activity may be important for suppressing aortic valve calcification [178]. Nitric Oxide deficiency is also an indicator of endothelial cell dysfunction, and systemic endothelial cell dysfunction is prevalent in patients with aortic valve sclerosis/ stenosis [223-225]. The uncoupling, or dysfunction, of eNOS results in decreased NO pro‐ duction and increased generation of reactive oxygen species (ROS) [223]. ROS activity is present in calcific lesions of human stenotic aortic valves, and it has been suggested that ROS activity may speed aortic valve calcification [144, 226]. In animal studies, rabbits fed a high cholesterol/high vitamin D diet develop aortic valve thickening, small deposits of valve calcification, and increased ROS activity in cells surrounding regions of valve calcification [226]. Furthermore, ROS activity was co-localized to clusters of cells expressing Runx2 and OPN, suggesting that ROS activity is associated with VICs displaying an osteogenic-like phenotype [226]. In VIC culture studies, TGFβ1 stimulation induces increased ROS activity, along with calcific nodule formation and ALP activity [227]. Increasing the availability of NO, via NO donors such as sodium nitroprusside, partially blocks both nodule formation and ALP activity, suggesting that NO levels are important for reducing ROS and inhibiting calcification in VICs [227]. There have been a number of small clinical studies investigating the levels of the NOS inhibitor, asymmetric dimethylarginine (ADMA), an indicator of en‐ dothelial cell dysfunction, in patients with moderate to severe AS [228-230]. In these studies, plasma levels of ADMA are significantly higher in patients with moderate to severe AS, compared to patients with mild AS or no disease, suggesting that NO production is disrupt‐ ed in CAVD/AS [228-230]. Combined, these studies suggest that increased ROS production is associated with aortic valve calcification and the induction of osteogenic gene expression. Thus, increasing the bioavailability of NO may be a potential therapeutic avenue to block ROS activity, and thereby disease progression, in CAVD.

to determine whether COX2 inhibition/NSAID use could reduce CAVD progression. How‐ ever, one caveat is that COX2 inhibitor therapy can be associated with some rare but signifi‐ cant adverse cardiovascular events such as myocardial infarction and stroke [252, 253]. Perhaps therapeutics designed toward downstream targets of PG signaling, such as p38 MAPK, could improve outcomes of CAVD patients without the cardiovascular side effects

Developmental Pathways in CAVD http://dx.doi.org/10.5772/54356 83

**5.7. Development of new therapeutic approaches based on valvulogenic and osteogenic**

As reviewed above, Notch, Wnt, and BMP signaling have been implicated in the progres‐ sion of CAVD. Pharmacotherapies designed to act as Wnt and BMP inhibitors, or Notch ag‐ onists, could be a potential avenue for new therapeutics to treat the progression of CAVD. BMP signaling is thought to be a specific indicator of aortic valve calcification as active BMP signaling is observed in adult diseased valves with prominent calcification and is not found in pediatric diseased valves void of calcification [13]. Furthermore, BMP2 signaling stimu‐ lates VIC calcific nodule formation and induces osteogenic gene expression [138, 171]. It is possible that therapies designed to inhibit BMP signaling will block osteogenic-like calcifica‐ tion in diseased aortic valves. Likewise, inhibition of the Wnt/β-catenin signaling pathway may also serve to reduce aortic valve calcification during disease, which is supported by evi‐ dence from animal studies in ApoE knockout mice. When fed an atherogenic diet, ApoE knockout mice reportedly develop aortic valve calcification, however, when the Wnt co-re‐ ceptor Lrp5 is genetically deleted in these mice, the amount of aortic valve calcification is significantly reduced [180]. Therefore, Wnt inhibition may be another potential therapeutic approach for treating CAVD. Lastly, strategies to maintain Notch signaling in the valves may be another potential way to inhibit calcification in CAVD. Notch inhibition of calcifica‐ tion and osteogenic gene expression has been demonstrated in aortic VICs in culture and re‐ duced Notch signaling in vivo leads to CAVD in mice [65, 153, 175]. Furthermore, Notch1 haploinsufficiency in humans is associated with CAVD, indicating that maintaining Notch signaling is important for valve homeostasis [64]. Thus, therapeutic strategies designed to affect one or more of these pathways may serve to prevent valve calcification in CAVD. A potential limitation of this approach is that BMP, Wnt, and Notch signaling pathways are involved in many homeostatic and disease processes. For example, Wnt signaling is in‐ creased in many types of cancer, and all three pathways are involved in bone homeostasis. Therefore the development of therapeutics based on these molecular mechanisms must take into account potential effects on multiple organ systems. Nevertheless, targeted approaches based on these pathways could represent a new therapeutic avenue in the development of

There are numerous examples of shared molecular pathways between valvulogenesis, os‐ teogenesis, and disease pathogenesis of CAVD. In valvulogenesis, signaling factors involved

of selective coxibs [254].

**molecular mechanisms.**

pharmacologic based approaches to CAVD.

**6. Conclusions and future directions**

#### **5.6. NSAIDs/COX2 inhibitors**

Previous reports have demonstrated that immune cell infiltration is common in CAVD [119, 132, 135]. Non-steroidal anti-inflammatory drugs (NSAIDS) are commonly used to treat pain and inflammation, and act by inhibiting the enzymes COX1 and/or COX2 [231-233]. These enzymes function by converting arachidonic acid to prostaglandins (PGs) [233, 234]. There has been one study conducted in human aortic VICs, demonstrating that stimulation of VICs with pro-inflammatory mimetics induces the expression of COX2 and the release of prostaglandins [235]. This study suggests that COX2 inhibition may be one way to treat the immune response associated with CAVD [235]. Interestingly, COX2 and PG signaling are al‐ so involved in bone formation as well as cellular responses to physical stress and strain, processes that also likely contribute to CAVD (reviewed in [131]). In bone, PG signaling has an anabolic effect, and PG treatment of osteoblast cultures results in increased expression of OCN, BMP2, Runx2, OPN, ALP, and BSP [236-244]. In osteoblast cultures, BMP2 stimulation induces COX2 expression through upregulation of Runx2, which binds to and activates the COX2 promoter [245, 246]. Downstream, PG signaling induces the expression of p38 MAPK through the activation of protein kinase A [243, 247]. Furthermore, fluid shear stress and other physical forces induce COX2 expression in osteoblast-like cells, suggesting that in‐ creased COX2 and PG signaling is a cellular response to altered mechanical forces [248, 249]. The combined results of these studies suggest that COX2/PG signaling may be an effective therapeutic target to treat CAVD progression, as COX2/PG signaling plays a role in inflam‐ mation, osteogenesis, and cellular response to physical strain, all of which are thought to be pathological mechanisms involved in CAVD [131, 243, 244, 248-251]. It would be interesting to determine whether COX2 inhibition/NSAID use could reduce CAVD progression. How‐ ever, one caveat is that COX2 inhibitor therapy can be associated with some rare but signifi‐ cant adverse cardiovascular events such as myocardial infarction and stroke [252, 253]. Perhaps therapeutics designed toward downstream targets of PG signaling, such as p38 MAPK, could improve outcomes of CAVD patients without the cardiovascular side effects of selective coxibs [254].

#### **5.7. Development of new therapeutic approaches based on valvulogenic and osteogenic molecular mechanisms.**

As reviewed above, Notch, Wnt, and BMP signaling have been implicated in the progres‐ sion of CAVD. Pharmacotherapies designed to act as Wnt and BMP inhibitors, or Notch ag‐ onists, could be a potential avenue for new therapeutics to treat the progression of CAVD. BMP signaling is thought to be a specific indicator of aortic valve calcification as active BMP signaling is observed in adult diseased valves with prominent calcification and is not found in pediatric diseased valves void of calcification [13]. Furthermore, BMP2 signaling stimu‐ lates VIC calcific nodule formation and induces osteogenic gene expression [138, 171]. It is possible that therapies designed to inhibit BMP signaling will block osteogenic-like calcifica‐ tion in diseased aortic valves. Likewise, inhibition of the Wnt/β-catenin signaling pathway may also serve to reduce aortic valve calcification during disease, which is supported by evi‐ dence from animal studies in ApoE knockout mice. When fed an atherogenic diet, ApoE knockout mice reportedly develop aortic valve calcification, however, when the Wnt co-re‐ ceptor Lrp5 is genetically deleted in these mice, the amount of aortic valve calcification is significantly reduced [180]. Therefore, Wnt inhibition may be another potential therapeutic approach for treating CAVD. Lastly, strategies to maintain Notch signaling in the valves may be another potential way to inhibit calcification in CAVD. Notch inhibition of calcifica‐ tion and osteogenic gene expression has been demonstrated in aortic VICs in culture and re‐ duced Notch signaling in vivo leads to CAVD in mice [65, 153, 175]. Furthermore, Notch1 haploinsufficiency in humans is associated with CAVD, indicating that maintaining Notch signaling is important for valve homeostasis [64]. Thus, therapeutic strategies designed to affect one or more of these pathways may serve to prevent valve calcification in CAVD. A potential limitation of this approach is that BMP, Wnt, and Notch signaling pathways are involved in many homeostatic and disease processes. For example, Wnt signaling is in‐ creased in many types of cancer, and all three pathways are involved in bone homeostasis. Therefore the development of therapeutics based on these molecular mechanisms must take into account potential effects on multiple organ systems. Nevertheless, targeted approaches based on these pathways could represent a new therapeutic avenue in the development of pharmacologic based approaches to CAVD.
