**5. The middle layer, the spongiosa, is comprised mainly of glycosaminoglycans and proteoglycans**

Glycosaminoglycans and proteoglycans (GAGs and PGs, respectively) comprise a signifi‐ cant part of the aortic valve leaflets. PGs and GAGs are mainly found in the spongiosa layer of the valve, located between the ventricularis and fibrosa, where they play a vital role in maintaining normal valve function. Previous work has shown that GAGs and PGs serve to not only provide mechanical support to the tissue but also aid in the normal biological func‐ tions of the valve [54]. Therefore, it is crucial to fully understand the function of GAGs and PGs in both the normal and possible diseased states of tissues.

GAGs are composed of long and unbranched chains of repeating disaccharides, which con‐ sist of a hexosamine and either, depending on the GAG type, uronic acid or galactose. There exist the following families of GAGs with each group being defined by its composition: hya‐ luronan (HA), heparin, heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS) (Table 2) [55–58].

GAGs are primarily formed in the lumen of the Golgi apparatus. The formation process oc‐ curs, except in the case of HA, with glycosyltransferases alternatively adding a uronic acid or galactose with a hexosamine to a protein core. The attachment to the protein core varies based on the GAG type. Heparin, HS, CS, and DS are attached to a serine residue, connected to the protein core, via xylose. KS can attach to the protein core either by an asparagine resi‐ due at the N-terminus or linked to serine or threonine at the O-terminus. HA does not attach to a protein core. It is synthesized by the addition of sugars to the non-reducing termini of the forming polysaccharide by HA synthase, without a protein backbone. In all cases, modi‐ fications can be made to the resulting polysaccharides. Two noteworthy changes include sulfation of the chains and epimerization of the uronic acid. These changes do not occur, however, with HA. Sulfation and epimerization modifications can give a more distinct char‐ acteristic to the GAG chains. The epimerization of the uronic acid of CS leads to the produc‐ tion of DS. Epimerization also occurs on heparin and HS. Sulfation can occur in CS, DS, heparin, HS, and KS. N-sulfation takes place in heparin and HS; whereas, O-sulfation can take place in heparin, HS, CS, and DS. In addition to epimerization and sulfation, phosphor‐ ylation of the xylose linkage—occurring among CS, DS, heparin, and HS to their respective protein cores—can take place [54,58,59]. Through gel electrophoresis, it has been found that

2

HA comprises approximately half of the total GAG content in aortic valves [60]. It is impor‐ tant to note that all GAGs, with the exception of HA, exist *in vivo* as components of PGs. **9** 5 direction; valve direction. Valve

**8** 35 considered as dominating considered to dominate


**Table 2.** List of glycosaminoglycans and their composition [59] the same size, that would be great.

**Figure 2.** Proteoglycan structure

presses readily without buckling, most likely due to the tensile preload already exerted on the ventricularis, but that leaflet deflection may be limited by the stiff fibrosa, which would not allow the leaflet to bend [4,53]. Limited flexure at the hinge would allow the leaflet to absorb pressure from reverse blood flow in diastole, but prevents distention of the leaflet. Thus, our finding of a thicker spongiosa and elastic fiber structure in flexural regions pro‐ vides evidence of a significant role for elastin in flexure [8]. In addition, the thick network of elastic fibers that we have observed in the spongiosa of the coaptation region may play a

Glycosaminoglycans and proteoglycans (GAGs and PGs, respectively) comprise a signifi‐ cant part of the aortic valve leaflets. PGs and GAGs are mainly found in the spongiosa layer of the valve, located between the ventricularis and fibrosa, where they play a vital role in maintaining normal valve function. Previous work has shown that GAGs and PGs serve to not only provide mechanical support to the tissue but also aid in the normal biological func‐ tions of the valve [54]. Therefore, it is crucial to fully understand the function of GAGs and

GAGs are composed of long and unbranched chains of repeating disaccharides, which con‐ sist of a hexosamine and either, depending on the GAG type, uronic acid or galactose. There exist the following families of GAGs with each group being defined by its composition: hya‐ luronan (HA), heparin, heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate

GAGs are primarily formed in the lumen of the Golgi apparatus. The formation process oc‐ curs, except in the case of HA, with glycosyltransferases alternatively adding a uronic acid or galactose with a hexosamine to a protein core. The attachment to the protein core varies based on the GAG type. Heparin, HS, CS, and DS are attached to a serine residue, connected to the protein core, via xylose. KS can attach to the protein core either by an asparagine resi‐ due at the N-terminus or linked to serine or threonine at the O-terminus. HA does not attach to a protein core. It is synthesized by the addition of sugars to the non-reducing termini of the forming polysaccharide by HA synthase, without a protein backbone. In all cases, modi‐ fications can be made to the resulting polysaccharides. Two noteworthy changes include sulfation of the chains and epimerization of the uronic acid. These changes do not occur, however, with HA. Sulfation and epimerization modifications can give a more distinct char‐ acteristic to the GAG chains. The epimerization of the uronic acid of CS leads to the produc‐ tion of DS. Epimerization also occurs on heparin and HS. Sulfation can occur in CS, DS, heparin, HS, and KS. N-sulfation takes place in heparin and HS; whereas, O-sulfation can take place in heparin, HS, CS, and DS. In addition to epimerization and sulfation, phosphor‐ ylation of the xylose linkage—occurring among CS, DS, heparin, and HS to their respective protein cores—can take place [54,58,59]. Through gel electrophoresis, it has been found that

role in dampening vibrations that result from valve closing [5].

PGs in both the normal and possible diseased states of tissues.

**glycosaminoglycans and proteoglycans**

12 Calcific Aortic Valve Disease

(DS), and keratan sulfate (KS) (Table 2) [55–58].

**5. The middle layer, the spongiosa, is comprised mainly of**

**<sup>11</sup>**4 The protein core is translated to the lumen of the Golgi apparatus of the cells, where the GAGs are then added to the protein core [55]. During PG synthesis, a protein core moves from the endoplasmic reticulum of a cell to the Golgi apparatus, where GAGs are then added to the protein core [55]. **11** 7 hyalectins hyalectans **12** 1 hyalectin hyalectan **12** 3 hyalectin hyalectan PGs are formed when GAGs are added to a protein core through a covalent linkage (Figure 2). During PG synthesis, a protein core moves from the endoplasmic reticulum of a cell to the Golgi apparatus, where GAGs are then added to the protein core [55]. PGs can be found in in‐ tracellular organelles, on the cell surface, and in the extracellular matrix (ECM) [59]. PGs found in the ECM can be divided into three categories: PGs found within the basement mem‐ brane, hyalectans or PGs that interact with HA and lectins, and small leucine-rich PGs (SLRPs) or PGs that contain a leucine motif and have considerably low molecular weights. These PGs can be further classified by the type of protein backbone they contain, as well as the amount, type, and sulfation pattern of the GAGs that are attached to the backbone. More than thirty PGs have been characterized [61]. For example, well-characterized PGs that exist in cardiovas‐ cular tissue include decorin, biglycan, and versican. Decorin and biglycan have a core protein

**12** 17 guarantee the normal guarantee normal

**13** 26-27 alpha-V/beta-3 and alpha-V/beta-5 integrins α-V/β-3 and α-V/β-5 integrins

**13** 17 Fibronectin fibronectin

**15** 10 epidermitlysis epidermolysis

**15** 34 heparin heparan

**15** 39 from its five from five

**15** 41 enteokinase Enteokinase

**15** 14 and will maintain and can help maintain

**16** 9 immunoglobulin like immunoglobulin-like

size of 40 kDa and are a part of the SLRP family of PGs. They contain CS and DS GAG chains [61]. Versican is a large, chondroitin sulfate proteoglycan. It interacts with HA, and therefore is a hyalectan PG [62]. Other significant PGs in mammalian tissues include perlecan—a base‐ ment membrane protein that contains HS and CS, aggrecan—a hyalectan containing CS, and syndecans—a family of cell surface heparan sulfate proteoglycans containing HS and CS [61].

healthy tissues, the exact role of GAGs in diseased aortic valves needs further investigation,

Extracellular Matrix Organization, Structure, and Function

http://dx.doi.org/10.5772/52842

15

**6. Minor ECM components in heart valves also play significant roles in**

The extracellular matrix of heart valves contain a number of minor components that per‐ form a variety of functions. They are important in valve development, function, and pathol‐ ogy. The study and further characterization of these minor ECM components not only facilitates the development of targeted therapies but would also aid in the microenviron‐

Vitronectin is a glycoprotein that is approximately 75 kDa in size and is present in both se‐ rum and the ECM as an adhesive substrate [68]. It is involved in the inhibition of the com‐ plement system [68] and is associated with the regulation of hemostasis [69]. Vitronectin also promotes cellular attachment to ECM and is involved in cellular migration [68]. This glycoprotein, along with fibronectin, is found in moderate amounts in aortic, pulmonary, and mitral valves, localizing around valve endothelial cells (VEC) on the inflow layer [25]. In addition, both fibronectin and vitronectin have been shown to associate with collagen fibers

Fibronectin is a dimer glycoprotein which consists of two ~250 kDa subunits and is a com‐ ponent of the extracellular matrix [71]. There are many various isoforms of fibronectin, which is the result of alternative mRNA splicing [71]. In addition to being an insoluble ECM component secreted primarily by fibroblasts, soluble fibronectin is also found in the plasma [71]. Fibronectin acts by binding to integrins, collagens, fibrin, and heparin sulfate proteo‐ glycans [71], which allows it to participate in wound healing [72,73] and act as a critical player in embryogenesis [74]. Although not a major ECM component in heart valves, valve interstitial cells (VIC) secrete fibronectin in response to valve damage, providing a means for

Additionally, fibronectin, along with osteonectin and periostin, confers stiffness to the fibro‐ sa layer [76]. Periostin is a component of the ECM that acts as a ligand for α-V/β-3 and α-V/β-5 integrins and is known to support adhesion and epithelial migration [77]. It is present in the extracellular matrix of several types of tissues and is upregulated in several types of cancers [78]. Recombinant periostin has been shown to promote cardiomyocyte proliferation and angiogenesis after a myocardial infarction [79]. It has been shown previously that peri‐ ostin plays a role in murine embryonic valve development and remains present in the valves throughout the lifespan even when there is no pathological calcification [80]. A recent study involving chick cardiac development suggests that the presence of periostin in the de‐ veloping heart may provide a means of organizing other ECM molecules in order to facili‐ tate early epithelial-mesenchymal transition (EMT) [81]. However, the overexpression of

periostin and osteopontin can lead to valve calcification.

**normal valve function and in pathological states**

mental mimicry needed for potential tissue engineering applications.

as well.

in chordae tendinae [70].

cell migration [75].

GAGs, and in turn PGs, have a significant role in aortic valve tissue behavior. GAGs have been shown to enhance the viscoelastic properties of the valve leaflets through binding of water molecules [63]. The sulfation and carboxylation on the GAGs make them highly nega‐ tively-charged polysaccharides. This negative charge draws in water molecules. Once the tissue becomes hydrated, it acts like a sponge for the valve leaflets. As noted previously, GAGs and PGs are highly abundant in the middle layer of the aortic valve leaflet. One of the main functions of this cushioned layer, the spongiosa, is to provide a barrier between two other layers, the ventricularis and fibrosa, of the valve. This barrier allows for proper shear‐ ing between the layers as well as compressibility of the leaflet without compromising the leaflet's overall structural or biological integrity when mechanical stimuli are applied to aortic valve leaflets [63,64]. The mechanical competency that GAGs provide is crucial to the aortic valve leaflets. The aortic valve leaflets serve to ensure unidirectional blood flow from the left ventricle to the aorta. In order to guarantee normal blood flow, the leaflets must open and close properly. Therefore, the flexibility that GAGs provide to the leaflet is crucial to the normal valve's function. In addition, the space that GAGs occupy and form in the ma‐ trix serve to organize other molecules within the structure. The structure and hydration that GAGs provide also allow for biological cues to occur within the valve. Moreover, GAGs are known to aid in cell migration, proliferation, act as receptors for signaling molecules, bind growth factors, and serve in the recruitment of various cell types [54].

It is believed that GAGs/PGs likely play an active role in aortic valve tissue disease. Re‐ search has shown regional variation of decorin, biglycan, versican and HA in, near, and dis‐ tal to regions of calcification in diseased aortic valves, suggesting the occurrence of remodeling in the tissue during an unhealthy state [65]. In addition, although the exact cau‐ sation of calcific aortic valve disease is unknown, it is speculated that it may be due, at least in part, to an inflammatory process [17]. Interestingly, GAGs are thought to play an active role, quite often in the case of cellular injury, in many inflammatory processes for a variety of cell types and have shown to alter in structure and localization in these processes [66]. In addition, some researchers believe that lipid binding due to the unique structure of GAGs may be critical to the accumulation of lipids in calcified aortic valves, a characteristic that is hypothesized to aid in valvular calcification [67]. Although the specific mechanisms under‐ lying calcific aortic valve disease are not quite understood, the complex nature and distin‐ guishable differences of GAGs in both healthy and diseased tissue give rise to the possibility of GAGs being a key factor in valve calcification.

GAGs are very complex disaccharides that highly dictate the behavior of PGs. These poly‐ saccharides are vital in maintaining mechanical, structural, and biological integrity of the aortic valve. Although there is growing interest in further elucidating the role of GAGs in healthy tissues, the exact role of GAGs in diseased aortic valves needs further investigation, as well.

size of 40 kDa and are a part of the SLRP family of PGs. They contain CS and DS GAG chains [61]. Versican is a large, chondroitin sulfate proteoglycan. It interacts with HA, and therefore is a hyalectan PG [62]. Other significant PGs in mammalian tissues include perlecan—a base‐ ment membrane protein that contains HS and CS, aggrecan—a hyalectan containing CS, and syndecans—a family of cell surface heparan sulfate proteoglycans containing HS and CS [61].

14 Calcific Aortic Valve Disease

GAGs, and in turn PGs, have a significant role in aortic valve tissue behavior. GAGs have been shown to enhance the viscoelastic properties of the valve leaflets through binding of water molecules [63]. The sulfation and carboxylation on the GAGs make them highly nega‐ tively-charged polysaccharides. This negative charge draws in water molecules. Once the tissue becomes hydrated, it acts like a sponge for the valve leaflets. As noted previously, GAGs and PGs are highly abundant in the middle layer of the aortic valve leaflet. One of the main functions of this cushioned layer, the spongiosa, is to provide a barrier between two other layers, the ventricularis and fibrosa, of the valve. This barrier allows for proper shear‐ ing between the layers as well as compressibility of the leaflet without compromising the leaflet's overall structural or biological integrity when mechanical stimuli are applied to aortic valve leaflets [63,64]. The mechanical competency that GAGs provide is crucial to the aortic valve leaflets. The aortic valve leaflets serve to ensure unidirectional blood flow from the left ventricle to the aorta. In order to guarantee normal blood flow, the leaflets must open and close properly. Therefore, the flexibility that GAGs provide to the leaflet is crucial to the normal valve's function. In addition, the space that GAGs occupy and form in the ma‐ trix serve to organize other molecules within the structure. The structure and hydration that GAGs provide also allow for biological cues to occur within the valve. Moreover, GAGs are known to aid in cell migration, proliferation, act as receptors for signaling molecules, bind

It is believed that GAGs/PGs likely play an active role in aortic valve tissue disease. Re‐ search has shown regional variation of decorin, biglycan, versican and HA in, near, and dis‐ tal to regions of calcification in diseased aortic valves, suggesting the occurrence of remodeling in the tissue during an unhealthy state [65]. In addition, although the exact cau‐ sation of calcific aortic valve disease is unknown, it is speculated that it may be due, at least in part, to an inflammatory process [17]. Interestingly, GAGs are thought to play an active role, quite often in the case of cellular injury, in many inflammatory processes for a variety of cell types and have shown to alter in structure and localization in these processes [66]. In addition, some researchers believe that lipid binding due to the unique structure of GAGs may be critical to the accumulation of lipids in calcified aortic valves, a characteristic that is hypothesized to aid in valvular calcification [67]. Although the specific mechanisms under‐ lying calcific aortic valve disease are not quite understood, the complex nature and distin‐ guishable differences of GAGs in both healthy and diseased tissue give rise to the possibility

GAGs are very complex disaccharides that highly dictate the behavior of PGs. These poly‐ saccharides are vital in maintaining mechanical, structural, and biological integrity of the aortic valve. Although there is growing interest in further elucidating the role of GAGs in

growth factors, and serve in the recruitment of various cell types [54].

of GAGs being a key factor in valve calcification.
