Notch Signaling in Congenital and Acquired Aortic Valve Disease

Calcific aortic valve disease represents the predominant pathology of tricuspid (trileaflet) and bicuspid aortic valves in developed countries (Ladich et al., 2011). Accounting for approxi‐ mately half of anatomically isolated aortic stenosis and 25 percent of patients with aortic regurgitation (Roberts, 1970), calcific bicuspid aortic valves requiring surgical intervention present at least two decades earlier than the tricuspid counterpart (Ward, 2000). Mechanisms important in cardiac and organ development — notably, the Notch pathway — have emerged as central players recapitulated and reused during the pathogenesis of calcific aortic valve disease, and support also a common etiology for bicuspid aortic valve and aortic valve calcification (Garg et al., 2005) (Table 1). Active engagement of inflammatory, remodeling, neovascularization and osteogenic (Aikawa et al., 2007a; Aikawa et al., 2007b; Miller et al., 2010; Rajamannan et al., 2003) pathways has conceptually replaced ‘degeneration’ in calcific aortic valve disease pathogenesis and progression (Dweck et al., 2012). Moreover, these pathways invoke similar mechanisms during cardiac morphogenesis. Dysregulated Notch activity has also been reported in vascular inflammation, macrophage activation (Fung et al., 2007), cardiometabolic disorder, and vascular and aortic valve calcification (Fukuda et al., 2012). Preclinical studies suggest that specific blockade of Notch ligand–receptor signaling potently suppresses vascular calcification and calcific aortic valve disease (Fukuda et al., 2012). In this chapter, we review the mechanisms of Notch signaling, aortic valve dysmor‐ phology pertinent to accelerated valve calcification, and discuss the pathways involving Notch that lead to aortic valve calcification and disease.


Introduction
Calcific aortic valve disease represents the predominant pathology of tricuspid (trileaflet) and bicuspid aortic valves in developed countries (Ladich et al., 2011). Accounting for approximately half of anatomically isolated aortic stenosis and 25 percent of patients with aortic regurgitation (Roberts, 1970), calcific bicuspid aortic valves requiring surgical intervention present at least two decades earlier than the tricuspid counterpart (Ward, 2000). Mechanisms important in cardiac and organ development -notably, the Notch pathway -have emerged as central players recapitulated and reused during the pathogenesis of calcific aortic valve disease, and support also a common etiology for bicuspid aortic valve and aortic valve calcification (Garg et al., 2005) (Table 1). Active engagement of inflammatory, remodeling, neovascularization and osteogenic (Aikawa et al., 2007a;Aikawa et al., 2007b;Miller et al., 2010;Rajamannan et al., 2003) pathways has conceptually replaced 'degeneration' in calcific aortic valve disease pathogenesis and progression (Dweck et al., 2012). Moreover, these pathways invoke similar mechanisms during cardiac morphogenesis. Dysregulated Notch activity has also been reported in vascular inflammation, macrophage activation (Fung et al., 2007), cardiometabolic disorder, and vascular and aortic valve calcification (Fukuda et al., 2012). Preclinical studies suggest that specific blockade of Notch ligand-receptor signaling potently suppresses vascular calcification and calcific aortic valve disease (Fukuda et al., 2012). In this chapter, we review the mechanisms of Notch signaling, aortic valve dysmorphology pertinent to accelerated valve calcification, and discuss the pathways involving Notch that lead to aortic valve calcification and disease.

Notch signaling
The human Notch receptor family comprises four members, Notch1 through Notch4, expressed as transmembrane molecules on the cell surface of neighboring cells that enable canonical signaling in a contact-dependent manner (Bray, 2006;Kopan and Ilagan, 2009). Canonical Notch signaling describes the 'classic' interaction between membrane-bound receptors and ligands expressed on the surface of neighboring (signaling and receiving) cells, whereas non-canonical signaling encompasses a diverse group of structurally unrelated ligands that contribute to the pleiotropic effect of Notch signaling (Kopan and Ilagan, 2009). In mammals, five members of the Delta-Serrate-LAG-2 (DSL) family have the capacity to activate or modify canonical Notch signaling -Delta-like 1 (Dll1), Dll3, Dll4, Jagged1, and Jagged2. Interaction between Notch receptor and ligand is tightly controlled, and the signaling outcome is determined by the receptor:ligand ratio (Artavanis-Tsakonas and Muskavitch, 2010; Gibert and Simpson, 2003;Heitzler and Simpson, 1991;Wilkinson et al., 1994) that critically determines asymmetry in cell fate and development of neighboring cells. This interaction between receptor and ligand can be modified posttranslationally through Notch glycosylation by lunatic, manic and radical glycosyltransferases (Bray, 2006). The receptor:ligand ratio is dependent on the differential expression of competing ligands on neighboring cells in trans, as opposed to cis interaction through which receptor and ligand expressed on the same cell can also modulate Notch signaling. The complexity of receptor-ligand interaction is further increased by the requirement of heterodimerization of the receptor (Kopan and Ilagan, 2009). Canonical interaction between Notch receptor and ligand leads to two sequential cleavage events at site 2 (S2) and S3. S2 is a 'permissive' extracellular juxtamembrane cleavage by a disintegrin and metalloprotease 17 (ADAM17, known also as tumor necrosis factor-α converting enzyme/TACE) and/or ADAM10 (Artavanis-Tsakonas and Muskavitch, 2010;

Notch dysfunction in aortic valve anomalies and other congenital heart diseases
Congenital aortic valve anomalies frequently associate with other abnormalities in neighboring structures, including the aortic root (e.g. dilatation, aneurysm), aorta (e.g. coarctation of aorta), ventricular outflow tract (e.g. septal defect, transposition of great vessels), and/or coronary arteries (e.g. coronary anomalies) (Perloff, 2003;Ward, 2000). The association of anomalies is due in part to the complexity and critical function of the endocardial cushion, and its formation during cardiac valve and septum development (Camenisch et al., 2010).
The tight regulation of Notch signaling during murine cardiac morphogenesis, particularly of the cardiac outflow tract and semilunar (aortic and pulmonary) valves, have been recently reviewed in detail by de la Pompa and Epstein (de la Pompa and Epstein, 2012). The evolutionarily conserved nature of Notch across mammalian species is generally recognized to be applicable to human. The highly coordinated action of Notch in progenitor cell proliferation and differentiation is instrumental during development. Earliest signs of cardiac morphogenesis occur with formation of the cardiac crescent by midline fusion of first and second heart fields that feature expression of Notch1, Dll4 and Jagged1 in the primitive endocardium  The complexity of gene-phenotype effects in human is highlighted by variable penetrance of JAG1 mutation (e.g. G274D missense mutation) and phenotypic expression, as demonstrated by differences in the degree of glycosylation, protein trafficking and cell-surface protein expression given the same mutation (Lu et al., 2003). This heterogeneity is reminiscent of the variable effects of NOTCH1 in the pathogenesis of bicuspid aortic valve and other cardiovascular anomalies (Garg et al., 2005), and epigenetic factors such as intracardiac fluid forces may be important contributors that couple with transcription factors to affect cardiogenesis and valve development (Hove et al., 2003;Lee et al., 2006;Vermot et al., 2009).

Aortic valve dysmorphology, bicuspid aortic valve and calcification
Anomalies of the aortic valve can be classified based on size, shape, the number of valve leaflets, cuspal inequality, nature of commissures (e.g. unicomissural, acquired fusion), and location of a false raphé if present (Perloff, 2003;Ward, 2000). Unicuspid, quadricuspid and six-cuspid aortic valves occur rarely (Perloff, 2003), and associated mutations have not been reported, unlike bicuspid aortic valves resulting from impaired Notch1 signaling (Garg et al., 2005). Unicuspid and bicuspid aortic valves often prematurely develop valve calcification at least two decades earlier than their normal trileaflet counterpart (Pachulski and Chan, 1993). Although maldistribution of mechanical stress contributes to the fibrocalcific process, additional factors apart from biomechanical forces including inflammatory and profibrotic processes direct the differentiation of valve fibroblasts into myofibroblasts and osteoblasts that promote osteogenesis (Dweck et al., 2012;Rajamannan et al., 2003).
Maldistribution of shear stress on valve cusps is thought to promote calcification of the aortic valve seen in unicuspid, bicuspid, and tricuspid aortic valve with cuspal inequality (Perloff, 2003). Bicuspid aortic valve is found in 1-2% of the general population in the United States, with a slight male predominance reported in some studies (Roberts et al., 2012;Ward, 2000). Maldistribution of diastolic force among valve cusps and sinus attachment is thought also to promote ascending aortic dilatation or aneurysm (Burks et al., 1998;Perloff, 2003;Roberts, 1970). However, it remains unclear whether these aortic manifestations are genetically determined or represent a byproduct of mechanical stress, given that aortic dilatation is indistinct among regurgitant, stenotic and functionally normal bicuspid aortic valves (Hahn et al., 1992). Emerging evidence supports increased proteolytic activity in the aortic valve and adjacent areas including the aorta that may enhance the remodeling processes (Aikawa et al., 2007b).
Valvular calcification in the early stages causes aortic sclerosis, which predicts increased risks for cardiovascular morbidity and mortality (Otto et al., 1999). As the process progresses, the aortic valve orifice narrows while the valve anatomy and function become gradually distorted to produce valvular aortic stenosis with or without regurgitation, myocardial hypertrophic response, myocardial fibrosis, heart failure, and hemodynamic instability (Dweck et al., 2012). In recent years, the concept of degeneration in the pathogenesis of calcific aortic valve disease has been superseded by that of phenotypic modulation recapitulating embryonic development, angiogenesis, acquired and innate immune activation, wound healing and bone formation (Hakuno et al., 2009).

Aortic valve calcification and systemic inflammation
Aortic valve sclerosis has been estimated to affect at least 20% of adults over 65 years of age in the general population (Lindroos et al., 1993;Stewart et al., 1997). Calcific aortic valve disease represents a continuum of maladapted calcification in the aortic valve arising from active inflammatory and oxidative processes (Kaden et al., 2004;New and Aikawa, 2011;Towler, 2008), as well as a shift in the valve interstitial phenotype from chondrogenic to osteogenic. Early calcification of the aortic valve leads to increased valve leaflet thickness and stiffness in a condition termed aortic valve sclerosis (Otto et al., 1999). Continuation of the inflammatory process propagates angioneogenesis and biomineralization, leading to formation of calcium nodules that distort valve geometry and function, culminating in outflow-limiting aortic stenosis with or without regurgitation and/or the DLL:Jagged (Notch ligands) ratio may plausibly alter the cell-to-cell signalling strength and modality in cis and/or in trans, thus, modifying the final functional outcome. Much work remains to be done to fully delineate the mechanisms through which anti-Dll4 antibody exert inhibitory effects on inflammation and calcification. Figure 2. Ex vivo mapping using fluorescence reflectance imaging to grossly visualize the biomineralization of the hearts and vessels of atherosclerosis-prone (low-density lipoprotein receptor-deficient, Ldlr -/-) animals fed a hypercholesterolemic diet, and independently treated with IgG isotype control or anti-Dll4 monoclonal antibody (Dll4 Ab). 750nm CLIO750 nanoparticles were used to image macrophages, and 680-nm VisEn OsteoSense680 was used for the detection of osteogenic activity (top and bottom rows). Decreased osteogenic activity in the anti-Dll4 monoclonal antibody treated specimen is visualized using alkaline phosphatase (ALP) staining (middle row). Adapted from Fukuda and colleagues (Fukuda et al., 2012).

Clinical implications
Calcific aortic valve disease in individuals with severe aortic stenosis can progress quickly after presentation with symptoms, usually portending limited short-term survival (Turina et al., 1987). Clinical trials on medical therapy including statins have found little benefit and utility in forestalling disease progression, with no demonstrated impact on survival. Since the evidence suggests that inflammatory cells, particularly macrophages, play a crucial role in calcification, anti-inflammatory therapies may prevent development of arterial and valvular calcification. We and others have demonstrated that lipid lowering reduces inflammation . This may suggest that anti-inflammatory therapies need to be initiated early (Aikawa and Otto, 2012), and thus clinical trials involving patients who had been diagnosed with aortic stenosis due to advanced calcification did not show substantial benefits of lipid lowering therapy. To establish more effective therapies, it is crucial to better understand the complex mechanisms for aortic valve calcification. To identify individuals with subclinical aortic valve calcification and those with high probability or propensity of developing severe aortic valvular stenosis, methods for early detection of calcific changes (e.g., molecular imaging, biomarkers) need to be developed.