**3. The molecular taxonomy: Genes, pathways, and proteins**

**Genetic syndromes provide important biologic insights.** Turner syndrome is associated with BAV and aorta abnormalities, and is the only monosomy compatible with life despite the fact that the vast majority of cases result in early spontaneous abortion. Turner syndrome can occur for a variety of reasons, including nondisjunction and mosaicism, and the exact genetic abnormality correlates with the severity of the malformations with 45,X more likely mosaicism less likely to have associated CVM. While there have been some studies examining possible maternal effects in nonsyndromic CVM [68], similar studies in Turner syndrome have not identified genomic imprinting in general or specifically with regard to BAV [69]. Interestingly, BAV morphology was RL in over 95% of cases, nearly uniform and significantly more disproportionate than the general ratio [70], suggesting a genotype-phenotype relationship of potential clinical significance. This is consistent with the observation that RL BAV is more commonly associated with aortic coarctation [5]. Little is known about long-term outcomes, e.g. the prevalence of CAVD requiring surgical aortic valve replacement or associated thoracic aneurysm that dissects, and there is not a mouse model to date that recapitulates the cardiac phenotype, but involvement of one of the sex chromosomes provides novel ways to explore specific genetic factors contributing to BAV.

may account for a very small proportion of cases of BAV and therefore may contribute to the pathogenesis underlying CAVD. As the focus has shifted from early to late (post endothelialmesenchymal transition) regulatory factors, the role of additional factors, such as Notch and Wnt have been studied in the context of ECM stratification in the mature cups [53,63,85]. The progression of CAVD includes activation of osteogenic gene regulatory pathways and calcification, generally localized to the fibrosa layer [25,86-88]. Atherosclerotic mechanisms have been implicated in valve calcification, and there are overlapping risk factors for CAVD and CAD as described above, suggesting endothelial injury and inflammation play a key role in disease progression [17,87,89]. However, it remains unclear if these are inciting causal factors or exacerbating factors. TGFB signaling dysregulation has been associated with CAVD and cardiovascular disease progression, especially as it pertains to fibrosis and inflammation [90-92]. During human aortic valve calcification, expression of several genes associated with osteogenesis, including *Runx2*, *osteocalcin*, *osteopontin*, *alkaline phophatase*, and *bone sialopro‐ tein,* is induced [93-97]. There is increasing evidence that CAVD recapitulates gene regulatory

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**The molecular basis of aberrant calcification is poorly understood.** While physiologic mineralization in the context of bone development and maintenance has been used success‐ fully as a paradigm to study aberrant calcification in CAVD [86,98], less is known about the genetic basis of disease phenotypes characterized by aberrant calcification. Vascular calcifica‐ tion and the calcification that can occur in advanced CAD has been studied extensively and forms some of the basis for the prevailing view that CAD and CAVD are related disease states. Using a rare genetic disease, alkaptonuria, Hannoush et al identified a metabolic link between vascular calcification and advanced CAVD in a cohort of nearly one hundred patients [99]. Importantly, CAVD in this population was present and advanced, often requiring surgery, independent of standard cardiac risk factors, suggesting a primary link in pathogenesis not related to common comorbidities. In vitro, studies have focused on vascular smooth muscle's role in calcification, especially in the context of clinical comorbidities of CAVD such as CAD and HTN, as well as the context of pathways regulating the associated inflammation and the renin-angiotensin system [92,100]. While vascular smooth muscle cells are not present in valve tissue, there are subsets of VICs that have smooth muscle cell-like properties [101,102] and the expression of smooth muscle actin is considered a marker of activated VICs, the cells impli‐

**Understanding valve tissue homeostasis or maintenance will require proteomics.** Focusing on valve injury or defects in valve homeostasis or maintenance requires increasing attention to processes downstream from the transcriptional regulation that dominates cardiac develop‐ ment paradigms of CAVD. Proteomics is one emerging field that provides a compelling strategy to address the challenges of dynamic post-translational biology in valve tissue and will undoubtedly have significant impact on our understanding of healthy valve maintenance and CAVD pathogenesis [104]. Proteomics involves a sophisticated technical approach that requires in vitro validation and substantial bioinformatics support. Angel et al. have demon‐ strated a number of seminal observations by defining the semilunar valve proteome in the adult mouse using MALDI mass spectrometry [105,106]. Specifically, this rigorous and

interactions characteristic of osteogenesis.

cated in CAVD progression [103].

The classic connective tissue disorders, Marfan and Ehlers-Danlos syndromes, caused by mutations in the FIBRILLIN-1 and COLLAGEN Type 3 genes respectively, are well-known to effect the aortic valve. While there is clearly reduced penetrance for BAV in these groups, there is a significantly increased incidence for BAV in both conditions of 10-30% [71,72]. Additional genetic syndromes that affect the connective tissue include Williams syndrome and osteogen‐ esis imperfecta, caused by mutations in the ELASTIN and COLLAGEN Type 1 genes respec‐ tively, which also have an increased incidence of valve malformation and disease [73,74]. In addition, there are a number of genetic syndromes that are associated with BAV, often in the context of complex CVM. These include aneuploidies such as deletion 4p, deletion 10p, deletion 11q (Jacobsen syndrome), trisomy 18 (Edwards syndrome), deletion 20p12 (Alagille syndrome), as well as other genetic syndromes, including Adams-Oliver syndrome and Kabuki syndrome [75,76]. Trisomy 18 is a particularly interesting entity that is associated with polyvalvular disease, an unusual type of valve disease that is characterized by malformation, including BAV, and dysplasia of the valves, a poorly understood process that does not have a clear association with CAVD but challenges the malformation-disease distinction [77]. In addition, BAV is often one of multiple CVMs in the same individual and the patterns of cooccurrence can inform cause [78]. Taken together, there is a multitude of ways that valve tissue can be affected, and a molecular understanding of these conditions will inform CAVD.

**Developmental signaling pathways identify basic regulatory factors in valvulogenesis.** From a cardiac development perspective, there are three transcription factors that are consid‐ ered the master regulators of basic heart development, NKX2.5, GATA4 and TBX5. Loss of function mutations in each of these genes has been associated with various forms of CVM [79-83]. While none of these genes has been associated with BAV, the Nkx2.5 mutant mouse is characterized by a variety of CVMs including BAV [84], suggesting like NOTCH1, NKX2.5 may account for a very small proportion of cases of BAV and therefore may contribute to the pathogenesis underlying CAVD. As the focus has shifted from early to late (post endothelialmesenchymal transition) regulatory factors, the role of additional factors, such as Notch and Wnt have been studied in the context of ECM stratification in the mature cups [53,63,85]. The progression of CAVD includes activation of osteogenic gene regulatory pathways and calcification, generally localized to the fibrosa layer [25,86-88]. Atherosclerotic mechanisms have been implicated in valve calcification, and there are overlapping risk factors for CAVD and CAD as described above, suggesting endothelial injury and inflammation play a key role in disease progression [17,87,89]. However, it remains unclear if these are inciting causal factors or exacerbating factors. TGFB signaling dysregulation has been associated with CAVD and cardiovascular disease progression, especially as it pertains to fibrosis and inflammation [90-92]. During human aortic valve calcification, expression of several genes associated with osteogenesis, including *Runx2*, *osteocalcin*, *osteopontin*, *alkaline phophatase*, and *bone sialopro‐ tein,* is induced [93-97]. There is increasing evidence that CAVD recapitulates gene regulatory interactions characteristic of osteogenesis.

**3. The molecular taxonomy: Genes, pathways, and proteins**

specific genetic factors contributing to BAV.

180 Calcific Aortic Valve Disease

**Genetic syndromes provide important biologic insights.** Turner syndrome is associated with BAV and aorta abnormalities, and is the only monosomy compatible with life despite the fact that the vast majority of cases result in early spontaneous abortion. Turner syndrome can occur for a variety of reasons, including nondisjunction and mosaicism, and the exact genetic abnormality correlates with the severity of the malformations with 45,X more likely mosaicism less likely to have associated CVM. While there have been some studies examining possible maternal effects in nonsyndromic CVM [68], similar studies in Turner syndrome have not identified genomic imprinting in general or specifically with regard to BAV [69]. Interestingly, BAV morphology was RL in over 95% of cases, nearly uniform and significantly more disproportionate than the general ratio [70], suggesting a genotype-phenotype relationship of potential clinical significance. This is consistent with the observation that RL BAV is more commonly associated with aortic coarctation [5]. Little is known about long-term outcomes, e.g. the prevalence of CAVD requiring surgical aortic valve replacement or associated thoracic aneurysm that dissects, and there is not a mouse model to date that recapitulates the cardiac phenotype, but involvement of one of the sex chromosomes provides novel ways to explore

The classic connective tissue disorders, Marfan and Ehlers-Danlos syndromes, caused by mutations in the FIBRILLIN-1 and COLLAGEN Type 3 genes respectively, are well-known to effect the aortic valve. While there is clearly reduced penetrance for BAV in these groups, there is a significantly increased incidence for BAV in both conditions of 10-30% [71,72]. Additional genetic syndromes that affect the connective tissue include Williams syndrome and osteogen‐ esis imperfecta, caused by mutations in the ELASTIN and COLLAGEN Type 1 genes respec‐ tively, which also have an increased incidence of valve malformation and disease [73,74]. In addition, there are a number of genetic syndromes that are associated with BAV, often in the context of complex CVM. These include aneuploidies such as deletion 4p, deletion 10p, deletion 11q (Jacobsen syndrome), trisomy 18 (Edwards syndrome), deletion 20p12 (Alagille syndrome), as well as other genetic syndromes, including Adams-Oliver syndrome and Kabuki syndrome [75,76]. Trisomy 18 is a particularly interesting entity that is associated with polyvalvular disease, an unusual type of valve disease that is characterized by malformation, including BAV, and dysplasia of the valves, a poorly understood process that does not have a clear association with CAVD but challenges the malformation-disease distinction [77]. In addition, BAV is often one of multiple CVMs in the same individual and the patterns of cooccurrence can inform cause [78]. Taken together, there is a multitude of ways that valve tissue can be affected, and a molecular understanding of these conditions will inform CAVD.

**Developmental signaling pathways identify basic regulatory factors in valvulogenesis.** From a cardiac development perspective, there are three transcription factors that are consid‐ ered the master regulators of basic heart development, NKX2.5, GATA4 and TBX5. Loss of function mutations in each of these genes has been associated with various forms of CVM [79-83]. While none of these genes has been associated with BAV, the Nkx2.5 mutant mouse is characterized by a variety of CVMs including BAV [84], suggesting like NOTCH1, NKX2.5 **The molecular basis of aberrant calcification is poorly understood.** While physiologic mineralization in the context of bone development and maintenance has been used success‐ fully as a paradigm to study aberrant calcification in CAVD [86,98], less is known about the genetic basis of disease phenotypes characterized by aberrant calcification. Vascular calcifica‐ tion and the calcification that can occur in advanced CAD has been studied extensively and forms some of the basis for the prevailing view that CAD and CAVD are related disease states. Using a rare genetic disease, alkaptonuria, Hannoush et al identified a metabolic link between vascular calcification and advanced CAVD in a cohort of nearly one hundred patients [99]. Importantly, CAVD in this population was present and advanced, often requiring surgery, independent of standard cardiac risk factors, suggesting a primary link in pathogenesis not related to common comorbidities. In vitro, studies have focused on vascular smooth muscle's role in calcification, especially in the context of clinical comorbidities of CAVD such as CAD and HTN, as well as the context of pathways regulating the associated inflammation and the renin-angiotensin system [92,100]. While vascular smooth muscle cells are not present in valve tissue, there are subsets of VICs that have smooth muscle cell-like properties [101,102] and the expression of smooth muscle actin is considered a marker of activated VICs, the cells impli‐ cated in CAVD progression [103].

**Understanding valve tissue homeostasis or maintenance will require proteomics.** Focusing on valve injury or defects in valve homeostasis or maintenance requires increasing attention to processes downstream from the transcriptional regulation that dominates cardiac develop‐ ment paradigms of CAVD. Proteomics is one emerging field that provides a compelling strategy to address the challenges of dynamic post-translational biology in valve tissue and will undoubtedly have significant impact on our understanding of healthy valve maintenance and CAVD pathogenesis [104]. Proteomics involves a sophisticated technical approach that requires in vitro validation and substantial bioinformatics support. Angel et al. have demon‐ strated a number of seminal observations by defining the semilunar valve proteome in the adult mouse using MALDI mass spectrometry [105,106]. Specifically, this rigorous and unbiased approach has yielded the identification and characterization of global protein expression and protein-protein networking provides a specific cell-matrix definition of valve maintenance that can be used further to explore the impact of aging, physiologic hemodynamic stresses due to constant motion, and systemic pathologic insults on specific signaling and metabolic dynamics. Importantly, this study provides proof of concept in mouse that will allow the approach to leverage the power of targeted mutagenesis [107]. Despite the difficulty of obtaining healthy controls, early observations have been made in human valve disease specimens, that when compared with control tissue, demonstrate misexpression of critical matrix proteins, including specific lipoproteins, inflammatory proteins, and proteases [108]. One study has focused this approach on VICs exposed to pro-calcific stimuli and has shown that specific chaperone proteins alter transport and cytoskeletal organization, providing insight into both valve homeostasis and CAVD [109]. Taken together, proteomics promises to generate novel insight into disease progression as well as potentially develop a new clinical tool that uses novel global proteomic analyses in plasma as a noninvasive comprehensive biomarker panel.

and disease processes are shared [136]. Similar nodules are seen in the aortic valve annulus of the Adamts9 null mouse [137], confirming the importance of ECM remodeling enzymes. Elastolysis and associated elastic fiber fragments have been implicated as a trigger for myofibroblast mediated calcification [138,139]. Loss of balance between elastases and elastase inhibitors has been identified as one fundamental cause of elastolysis [140]. Interestingly, previous studies have shown that different elastic fiber fragments have different biologic functions, for example, some fragments induce calcification while others are chemo-attractants

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183

**The extracellular matrix is an interface between genetics and the environment.** The heart valves function essentially to maintain unobstructed unidirectional blood flow. Valve struc‐ ture-function relationships provide important insight in understanding mechanisms of valve homeostasis as well as developmental and disease processes. Valve ECM composition and biomechanics reflect underlying hemodynamics. There are three basic loading states that affect valve tissue during the cardiac cycle: flexure, shear and tension. Flexure occurs when the valve is actively opening or closing, shear occurs when blood is passing through the open valve, and tension occurs when the valve is closed [143]. Shear, compressive, and longitudinal stresses contribute to valve deformation, or displacement of the valve tissue during the constant motion of the cardiac cycle [144]. Valve tissue has exceptionally high strain because the tissue cycles to a completely unloaded state with each heart beat [145]. The heart beats more than 100,000 times per day handling approximately 5 liters of blood per minute. Over the average lifetime, there are greater than 3 billion heartbeats, or cardiac cycles. The long held appreciation of agerelated degeneration and latent valve disease may in fact represent subtle defects in valve

CAVD is characterized by VIC activation, which in turn results in increased ECM and increased remodeling enzyme gene expression [103,127,128], and hemodynamic factors may activate VICs and therefore contribute to pathology. VIC activation is apparent by induction of myofibroblast markers, such as vimentin, smooth muscle actin, and embryonic non-muscle myosin heavy chain [129]. Some VICs have been shown to be dynamic and play an active role in ECM maintenance, as well as potentially regeneration and repair, and these VICs are progenitor cells with smooth muscle like properties [101,102,103,123,146,147]. Recently, two studies have demonstrated the complex interaction between developmental programs that predispose tissue to disease and shear stresses that trigger inflammation [148,149], providing examples of how these factors when combined may cause AVD. Research efforts are beginning to reconcile developmental and biomechanical considerations in an effort to more closely examine CAVD in vivo. A better understanding of hemodynamic-induced cell-matrix

perturbations may inform the search for durable valve bioprostheses [150].

**4. National Heart Lung and Blood Institute's research agenda for CAVD**

**New research agenda emphasizes genetics and development.** Recently, the National Heart Lung and Blood Institute Aortic Stenosis Working Group defined a comprehensive research

for endothelial cells [141,142].

tissue maintenance.

**Dysregulation of structural proteins and remodeling enzymes is a common pathway.** Normal valve function requires coordinated movement of complex structures. Gross and Kugel proposed nomenclature for valve tissue organization in 1931 that is now well established [110]. The mature valve structure is made up of highly organized ECM that is compartmen‐ talized into three layers, the fibrosa, spongiosa, and ventricularis [9,53,111]. The annulus, composed primarily of fibrous collagens, provides a buttress for dispersion of forces, and tethering of the cusp in a crown-shape for tissue stabilization [112,113]. Studies examining ECM in valve tissue have focused by convention on structural properties, specifically dura‐ bility (collagens) and flexibility (proteoglycans and elastic fibers). However, several studies have shown that ECM components reciprocally regulate growth factors and signaling pathways, in addition to causing architectural abnormalities, suggesting a primary rather than secondary role in pathogenesis [Reviewed in 53]. Studies in mouse models lacking ECM components critical for the mature aortic valve structure, including proteoglycans, collagens and elastic fiber components, demonstrate that the expression and organization of diverse ECM components are essential to the formation and structural integrity of the valves during development and after birth [114-123]. Further, mouse studies have shown that age and dietary manipulation can lead to ECM changes and CAVD [124-126].

During valve remodeling, the VICs regulate expression and organization of the valve ECM [127,128]. Additional ECM remodeling enzymes such as matrix metalloproteases (MMPs) and cathepsins also are expressed during valve maturation [128,129]. VICs from developing valves are highly synthetic, and extensive remodeling is required to achieve the mature organization [127,130]. In normal adult valves, the VICs are largely quiescent with little or no cell prolifer‐ ation and maintain baseline levels of ECM gene expression necessary for valve homeostasis [103]. ECM enzyme dysregulation is established in the valve disease literature [131-135]. The elastin insufficient mouse demonstrates cartilage-like nodules in the valve annulus reminis‐ cent of calcific nodules [119,136]. MMP misexpression malformation and more disease, suggesting malformation processes are due in part to remodeling defects and malformation and disease processes are shared [136]. Similar nodules are seen in the aortic valve annulus of the Adamts9 null mouse [137], confirming the importance of ECM remodeling enzymes. Elastolysis and associated elastic fiber fragments have been implicated as a trigger for myofibroblast mediated calcification [138,139]. Loss of balance between elastases and elastase inhibitors has been identified as one fundamental cause of elastolysis [140]. Interestingly, previous studies have shown that different elastic fiber fragments have different biologic functions, for example, some fragments induce calcification while others are chemo-attractants for endothelial cells [141,142].

unbiased approach has yielded the identification and characterization of global protein expression and protein-protein networking provides a specific cell-matrix definition of valve maintenance that can be used further to explore the impact of aging, physiologic hemodynamic stresses due to constant motion, and systemic pathologic insults on specific signaling and metabolic dynamics. Importantly, this study provides proof of concept in mouse that will allow the approach to leverage the power of targeted mutagenesis [107]. Despite the difficulty of obtaining healthy controls, early observations have been made in human valve disease specimens, that when compared with control tissue, demonstrate misexpression of critical matrix proteins, including specific lipoproteins, inflammatory proteins, and proteases [108]. One study has focused this approach on VICs exposed to pro-calcific stimuli and has shown that specific chaperone proteins alter transport and cytoskeletal organization, providing insight into both valve homeostasis and CAVD [109]. Taken together, proteomics promises to generate novel insight into disease progression as well as potentially develop a new clinical tool that uses novel global proteomic analyses in plasma as a noninvasive comprehensive

**Dysregulation of structural proteins and remodeling enzymes is a common pathway.** Normal valve function requires coordinated movement of complex structures. Gross and Kugel proposed nomenclature for valve tissue organization in 1931 that is now well established [110]. The mature valve structure is made up of highly organized ECM that is compartmen‐ talized into three layers, the fibrosa, spongiosa, and ventricularis [9,53,111]. The annulus, composed primarily of fibrous collagens, provides a buttress for dispersion of forces, and tethering of the cusp in a crown-shape for tissue stabilization [112,113]. Studies examining ECM in valve tissue have focused by convention on structural properties, specifically dura‐ bility (collagens) and flexibility (proteoglycans and elastic fibers). However, several studies have shown that ECM components reciprocally regulate growth factors and signaling pathways, in addition to causing architectural abnormalities, suggesting a primary rather than secondary role in pathogenesis [Reviewed in 53]. Studies in mouse models lacking ECM components critical for the mature aortic valve structure, including proteoglycans, collagens and elastic fiber components, demonstrate that the expression and organization of diverse ECM components are essential to the formation and structural integrity of the valves during development and after birth [114-123]. Further, mouse studies have shown that age and dietary

During valve remodeling, the VICs regulate expression and organization of the valve ECM [127,128]. Additional ECM remodeling enzymes such as matrix metalloproteases (MMPs) and cathepsins also are expressed during valve maturation [128,129]. VICs from developing valves are highly synthetic, and extensive remodeling is required to achieve the mature organization [127,130]. In normal adult valves, the VICs are largely quiescent with little or no cell prolifer‐ ation and maintain baseline levels of ECM gene expression necessary for valve homeostasis [103]. ECM enzyme dysregulation is established in the valve disease literature [131-135]. The elastin insufficient mouse demonstrates cartilage-like nodules in the valve annulus reminis‐ cent of calcific nodules [119,136]. MMP misexpression malformation and more disease, suggesting malformation processes are due in part to remodeling defects and malformation

manipulation can lead to ECM changes and CAVD [124-126].

biomarker panel.

182 Calcific Aortic Valve Disease

**The extracellular matrix is an interface between genetics and the environment.** The heart valves function essentially to maintain unobstructed unidirectional blood flow. Valve struc‐ ture-function relationships provide important insight in understanding mechanisms of valve homeostasis as well as developmental and disease processes. Valve ECM composition and biomechanics reflect underlying hemodynamics. There are three basic loading states that affect valve tissue during the cardiac cycle: flexure, shear and tension. Flexure occurs when the valve is actively opening or closing, shear occurs when blood is passing through the open valve, and tension occurs when the valve is closed [143]. Shear, compressive, and longitudinal stresses contribute to valve deformation, or displacement of the valve tissue during the constant motion of the cardiac cycle [144]. Valve tissue has exceptionally high strain because the tissue cycles to a completely unloaded state with each heart beat [145]. The heart beats more than 100,000 times per day handling approximately 5 liters of blood per minute. Over the average lifetime, there are greater than 3 billion heartbeats, or cardiac cycles. The long held appreciation of agerelated degeneration and latent valve disease may in fact represent subtle defects in valve tissue maintenance.

CAVD is characterized by VIC activation, which in turn results in increased ECM and increased remodeling enzyme gene expression [103,127,128], and hemodynamic factors may activate VICs and therefore contribute to pathology. VIC activation is apparent by induction of myofibroblast markers, such as vimentin, smooth muscle actin, and embryonic non-muscle myosin heavy chain [129]. Some VICs have been shown to be dynamic and play an active role in ECM maintenance, as well as potentially regeneration and repair, and these VICs are progenitor cells with smooth muscle like properties [101,102,103,123,146,147]. Recently, two studies have demonstrated the complex interaction between developmental programs that predispose tissue to disease and shear stresses that trigger inflammation [148,149], providing examples of how these factors when combined may cause AVD. Research efforts are beginning to reconcile developmental and biomechanical considerations in an effort to more closely examine CAVD in vivo. A better understanding of hemodynamic-induced cell-matrix perturbations may inform the search for durable valve bioprostheses [150].
