**4. Acquired aortic valve disease**

#### **4.1. 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 aris‐ ing from active inflammatory and oxidative processes (Kaden et al., 2004; New and Aika‐ wa, 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 biomi‐ neralization, leading to formation of calcium nodules that distort valve geometry and function, culminating in outflow-limiting aortic stenosis with or without regurgitation (Dweck et al., 2012; Rajamannan et al., 2011). Conditions that promote systemic inflamma‐ tion, such as atherosclerosis, dyslipidemia and diabetes mellitus, have been shown to ex‐ acerbate the development of calcific aortic valve disease (Rajamannan et al., 2011). While statins may stabilize atheromatous plaques, reduce vascular calcification and clinical ad‐ verse outcomes, they have unfortunately not been shown to benefit calcific aortic valve disease in disease progression or patient outcomes (Chan et al., 2010; Cowell et al., 2005; Rossebo et al., 2008).

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

Notch Signaling in Congenital and Acquired Aortic Valve Disease

http://dx.doi.org/10.5772/56533

113

**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 hypercho‐ lesterolemic diet, and independently treated with IgG isotype control or anti-Dll4 monoclonal antibody (Dll4 Ab). 750 nm 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 an‐ tibody treated specimen is visualized using alkaline phosphatase (ALP) staining (middle row). Adapted from Fukuda

and colleagues (Fukuda et al., 2012).

exert inhibitory effects on inflammation and calcification.

Studies exploring Notch signaling beyond congenital disorders and developmental biology identified Dll4 in macrophage-mediated inflammation (Fung et al., 2007). Recently, Fukuda and colleagues demonstrated that blockade of Dll4-Notch signaling using anti-Dll4 monoclo‐ nal antibody decreased BMP2, a central regulator of osteogenesis and bone mineralization (Fukuda et al., 2012), in line with other studies showing reduced aortic valve calcification with BMP2 knockdown by siRNA (Nigam and Srivastava, 2009), and the proinflammatory cytokine, TNF-α, accelerated BMP2-mediated calcification of human aortic valve interstitial cells from patients with calcific aortic valve stenosis (Yu et al., 2011). BMP2 mediates aortic valve calcification via Runx2 (Osf2/Cbfa1), a transcriptional activator of osteoblast development or gene expression (Ducy et al., 1997; Kaden et al., 2004; Mohler et al., 2001), and is suppressible by activation of Notch1 via Hey (HRT) (Acharya et al., 2011; Nigam and Srivastava, 2009). Moreover, the marked attenuation of aortic valvular calcification and stenosis through the blockade of angiogenesis-promoting Dll4 in a mouse model of hypercholesterolemia (Figure 2) also supports the current theory that angioneogenesis is a crucial stage in the natural history of calcific aortic valve disease (Dweck et al., 2012), recapitulating cardiogenesis and valve development (de la Pompa and Epstein, 2012; van den Akker et al., 2012). Thus, Dll4 critically bridges inflammation and angioneogenesis to osteogenesis in calcific aortic valve disease (Fukuda et al., 2012). These effects are probably independent of Notch 1 (Nus et al., 2011), since activation of the receptor presumably leads to inhibition of valve calcification (Acharya et al., 2011), whereas evidence on the benefits of Dll4 blockade (i.e. interruption of Dll4–Notch signaling) suggests that a Notch receptor other than Notch1, when activated, potentiates the development and progression of valve calcification. A shift in the Notch receptor:ligand ratio 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.

**4. Acquired aortic valve disease**

112 Calcific Aortic Valve Disease

Rossebo et al., 2008).

**4.1. 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 aris‐ ing from active inflammatory and oxidative processes (Kaden et al., 2004; New and Aika‐ wa, 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 biomi‐ neralization, leading to formation of calcium nodules that distort valve geometry and function, culminating in outflow-limiting aortic stenosis with or without regurgitation (Dweck et al., 2012; Rajamannan et al., 2011). Conditions that promote systemic inflamma‐ tion, such as atherosclerosis, dyslipidemia and diabetes mellitus, have been shown to ex‐ acerbate the development of calcific aortic valve disease (Rajamannan et al., 2011). While statins may stabilize atheromatous plaques, reduce vascular calcification and clinical ad‐ verse outcomes, they have unfortunately not been shown to benefit calcific aortic valve disease in disease progression or patient outcomes (Chan et al., 2010; Cowell et al., 2005;

Studies exploring Notch signaling beyond congenital disorders and developmental biology identified Dll4 in macrophage-mediated inflammation (Fung et al., 2007). Recently, Fukuda and colleagues demonstrated that blockade of Dll4-Notch signaling using anti-Dll4 monoclo‐ nal antibody decreased BMP2, a central regulator of osteogenesis and bone mineralization (Fukuda et al., 2012), in line with other studies showing reduced aortic valve calcification with BMP2 knockdown by siRNA (Nigam and Srivastava, 2009), and the proinflammatory cytokine, TNF-α, accelerated BMP2-mediated calcification of human aortic valve interstitial cells from patients with calcific aortic valve stenosis (Yu et al., 2011). BMP2 mediates aortic valve calcification via Runx2 (Osf2/Cbfa1), a transcriptional activator of osteoblast development or gene expression (Ducy et al., 1997; Kaden et al., 2004; Mohler et al., 2001), and is suppressible by activation of Notch1 via Hey (HRT) (Acharya et al., 2011; Nigam and Srivastava, 2009). Moreover, the marked attenuation of aortic valvular calcification and stenosis through the blockade of angiogenesis-promoting Dll4 in a mouse model of hypercholesterolemia (Figure 2) also supports the current theory that angioneogenesis is a crucial stage in the natural history of calcific aortic valve disease (Dweck et al., 2012), recapitulating cardiogenesis and valve development (de la Pompa and Epstein, 2012; van den Akker et al., 2012). Thus, Dll4 critically bridges inflammation and angioneogenesis to osteogenesis in calcific aortic valve disease (Fukuda et al., 2012). These effects are probably independent of Notch 1 (Nus et al., 2011), since activation of the receptor presumably leads to inhibition of valve calcification (Acharya et al., 2011), whereas evidence on the benefits of Dll4 blockade (i.e. interruption of Dll4–Notch signaling) suggests that a Notch receptor other than Notch1, when activated, potentiates the development and progression of valve calcification. A shift in the Notch receptor:ligand ratio

**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 hypercho‐ lesterolemic diet, and independently treated with IgG isotype control or anti-Dll4 monoclonal antibody (Dll4 Ab). 750 nm 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 an‐ tibody treated specimen is visualized using alkaline phosphatase (ALP) staining (middle row). Adapted from Fukuda and colleagues (Fukuda et al., 2012).
