**2. The aortic valve leaflets are layered structures**

Aortic valve leaflets consist of three main layers: the fibrosa, spongiosa, and ventricularis. Each layer has a distinct composition that aids in the normal mechanical and biochemical behavior of the valve. In diseased states, however, the composition of the layered structures can be altered compared to healthy tissues.

The fibrosa layer, close to the outflow surface, is mainly composed of collagen fibers with a small amount of elastic fibers, which are the major stress-bearing components and provide strength to maintain coaptation during diastole [2]. The circumferential alignment and ori‐ entation of collagen fibers contribute to the biological stress-strain relationship for aortic valve leaflets (Figure 1). This bilinear stress-strain curve represents the high extension with a low load and high elastic modulus with a high load applied [3]. Moreover, the particular ar‐ chitecture of collagen fibers contributes to the anisotropic mechanical behavior of the fibrosa layer. It has been found that the fibrosa is 4-6 times stiffer in the circumferential direction than in the radial direction [4].

The middle spongiosa layer of the leaflet predominantly consists of glycosaminoglycans and proteoglycans, particularly hyaluronan, which form a foam-like structure and bind a large amount of water. The spongiosa layer absorbs energy during compression, and facilitates the arrangement of collagen fibrils in the fibrosa and elastin in the ventricularis during the cardiac cycles [5].

and a growing body of research has demonstrated that the phenotype and function of cells, including valve cells, are influenced by the stiffness of the substrate to which they are ad‐ hered [1]. These two latter functions of the ECM are considered to be mechanobiological as opposed to merely biomechanical since they affect cell behavior. Fourth, ECM has binding sites for growth factors and other soluble molecules found in the extracellular space, and thus the ECM serves as a reservoir for numerous bioactive factors than can affect cell behav‐ ior if they are released (such as when the ECM is degraded) or if a cell migrates close to this

Overall, the heart valve field is beginning to appreciate that there are numerous interactions between the ECM, valve cells, and valve mechanics. Given the complicated relationships that are being demonstrated, it is not surprising that alterations to the normal arrangement or composition of ECM, which frequently occur in valve disease, significantly and detrimen‐ tally impact valve function in a rather vicious cycle. For this reason, there has been an in‐ creasing effort to characterize the ECM within normal heart valves not only to elucidate valve biomechanics and mechanobiology, but also to obtain a solid basis for comparison

This chapter will provide an overview of the ECM within heart valves, focusing on the aort‐ ic valve. After detailing the layered structure of the valve leaflets, each type of ECM compo‐ nent will be described and discussed in relation to its role in valve function and, in some

Aortic valve leaflets consist of three main layers: the fibrosa, spongiosa, and ventricularis. Each layer has a distinct composition that aids in the normal mechanical and biochemical behavior of the valve. In diseased states, however, the composition of the layered structures

The fibrosa layer, close to the outflow surface, is mainly composed of collagen fibers with a small amount of elastic fibers, which are the major stress-bearing components and provide strength to maintain coaptation during diastole [2]. The circumferential alignment and ori‐ entation of collagen fibers contribute to the biological stress-strain relationship for aortic valve leaflets (Figure 1). This bilinear stress-strain curve represents the high extension with a low load and high elastic modulus with a high load applied [3]. Moreover, the particular ar‐ chitecture of collagen fibers contributes to the anisotropic mechanical behavior of the fibrosa layer. It has been found that the fibrosa is 4-6 times stiffer in the circumferential direction

The middle spongiosa layer of the leaflet predominantly consists of glycosaminoglycans and proteoglycans, particularly hyaluronan, which form a foam-like structure and bind a large amount of water. The spongiosa layer absorbs energy during compression, and facilitates the arrangement of collagen fibrils in the fibrosa and elastin in the ventricularis during the

ECM reservoir.

4 Calcific Aortic Valve Disease

with diseased valves.

instances, valve dysfunction.

than in the radial direction [4].

cardiac cycles [5].

**2. The aortic valve leaflets are layered structures**

can be altered compared to healthy tissues.

**Figure 1.** Schematic drawing of stress-strain relationships for collagen and elastin fibers during valve motion, repro‐ duced with permission [3]

The ventricularis layer, close to the inflow surface, is rich in elastin with a moderate amount of collagen, which extends in diastole and recoils during systole [6]. The recoil of elastin re‐ stores the crimp of collagen fibrils and decreases the surface area of the stretched tissue from the closing phase [5]. The thickness of the three layers varies from the base to the free edge of the cusp [7].

It is worth noting that elastic fibers were found to span the whole leaflet, and connect or an‐ chor three discrete layers together [6,8]. In addition, elastin provides intrafibrillar connec‐ tions between collagen bundles in the fibrosa layer, whereas it forms a three-dimensional interconnected network in the spongiosa layer [8]. During unloading, the intrafiber elastin, which has high extensibility, helps the collagen fibers return to their wavy and crimped state [6]. These interconnected structures of elastic fibers anchor the discrete layers together, and prevent delamination, which therefore improves the continuity of material behavior of the whole leaflet. Table 1 summarizes the key ECM components in the layers and their ma‐ jor functions.


**Table 1.** The key ECM components in each layer of the leaflet and their major functions

The structures of the leaflets described above provide the following critical functions [6,9– 11]: 1) anisotropic mechanical behavior withstanding circumferential stress and extending radially; 2) bilinear biological stress-strain behavior allowing the leaflet to extend before bearing load in the closed phase; 3) elastic recoil to fully open the valve and restore the layer structures for the next cycle. The particular shape of the leaflets and their unique macro- and micro-structures cause the anisotropic mechanical behavior along the circumferential and radial directions of the leaflets [9–11].

**3. Collagen comprises a significant portion of the aortic valve leaflet**

mechanical properties that support aortic valve function.

allows for the formation of a triple helix structure [21].

fibrils ensures the stability of the complex [21].

mechanical properties suited for maintaining unidirectional blood flow.

Collagen is an essential component of the aortic valve's layered structure and is vital for maintaining the tissue's mechanical integrity. Mainly responsible for tensile strength, colla‐ gen is a strong load-bearing protein created and regulated by VICs. Although present throughout the entire valve, collagen is largely located in the fibrosa where it reduces high tensile stresses. In addition to its central role in valve mechanics, collagen acts as a regulator of VIC phenotype and calcification. Insight into the structure of collagen reveals its unique

Extracellular Matrix Organization, Structure, and Function

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

7

Fibrillar collagens are high strength fibers that comprise nearly all of the valve's collagen content. Fibrillar collagens are groups of 3 coiled polypeptide chains that assemble together in tightly packed parallel arrangements. These coils are approximately 300 nm long and join together in a staggered banding pattern with a periodicity of 67 nm [20]. The aortic valve is mainly composed of fibrillar collagen types I, III, and V. Each of these collagens is construct‐ ed from different types of alpha chains that govern the overall function of the collagen mole‐ cule. Together, these three collagen types work to provide the aortic valve with unique

Synthesis of fibrillar collagen is an essential mechanism for maintaining the valve's mechan‐ ical integrity. This complex process originates within VICs and is completed in the valve ECM. Production of collagen begins with the intracellular creation of polypeptide alpha chains. There exist ten distinct polypeptide chains that consist of approximately 300 consec‐ utive Gly-X-Y amino acid sequences flanked by small terminal domains. The secondary structure of collagen is created by folding alpha chains into a right-handed alpha helix with the peptide bonds localized at the backbone of the helix and the amino acid side chains fac‐ ing outward. With slightly less than three residues per turn and a pitch of approximately 8.6 nm, glycine residues are positioned in such a way that the side chains of these residues al‐ low for the formation of the helix. The single hydrogen side chains of these glycine residues

The tertiary structure of collagen involves the formation of a left-handed triple helix con‐ structed in the C to N direction. These triple helices exist as both homotrimers and hetero‐ trimers of alpha chains. Collagen type III is a homotrimer of α1(III) while collagen type I is a heterotrimer of α1(I) and α2(I). Additionally, collagen type V is a heterotrimer of α1(V) and α2(V). Known as procollagen, the tertiary structure molecule is approximately 1.5 nm wide and longer than 300 nm. For creation of the final supramolecular structure, the procollagen molecule is transported into the ECM for crosslinking and fibril formation. After modifica‐ tion in the extracellular space, procollagen is converted into tropocollagen, which undergoes fibrillogenesis where the triple helices are packed together into bundles. Crosslinking of the

The arrangement of collagen fiber bundles is crucial to the proper functioning of the aortic valve. Collagen fibers are organized into multilayer structures linked by thin membranes

**fibrosa**

During the closed phase (diastole), the leaflets experience the maximum load. Collagen bun‐ dles in the fibrosa layer are the major stress-bearing component withstanding approximately 80 mm Hg pressure while the valve is closed and bulging back towards the ventricle [3]. Collagen fibrils are assembled into parallel collagen fiber bundles oriented along the circum‐ ferential direction in the leaflet, which are able to withstand such high tensile forces. How‐ ever, collagen fibers cannot be compressed, making the alignment of collagen (waviness and crimping) important for decreasing the area of the stretched fibrosa layer. Although the col‐ lagen fibrils have limited extensibility (approximately 1-2% yield strain), the waviness and crimping allows the fibrosa to withstand roughly 40% strain under loading. Straightening of wavy fibers provides approximately 17% strain, whereas the crimping allows additional ap‐ proximately 23% strain [6]. In addition, the strains of the cusps in the closed phase are aniso‐ tropic, i.e., the strains differ in the radial and circumferential directions [11].

During valve opening, cusps become relaxed through recoil of the elongated, taut elastin. This restores the wavy and crimped state of collagen fibers while decreasing the surface area of the cusps. The GAG-rich spongiosa layer facilitates the rearrangements of the collagen and elastic fibers during the cardiac cycle, dampens vibration from closing, and resists de‐ lamination between layers [6,8].

It is evident that normal aortic valve function is maintained, in part, by not only the compo‐ sition but also the arrangement and orientation of ECM components, particularly collagen, elastin, and GAGs, in the leaflets. Furthermore, it is important to note that alteration of the composition [12] and mechanics [13] of ECM in the aortic valve leaflets was found in dis‐ eased conditions. In calcific aortic valve disease (CAVD), collagen bundles and elastin fibers in the fibrosa layer were disrupted and disorganized [14]; meanwhile, there was increased proteoglycan deposition [12]. Matrix metalloproteinases (MMPs) [14,15] and the potent elas‐ tase cathepsin S [16], which are produced by macrophages, contribute to this ECM remodel‐ ing. Moreover, ECM proteins related to bone, i.e., osteocalcin and osteonectin, were present in the calcified fibrosa layer [17]. These proteins promote mineralization, and their presence suggests the osteoblastic differentiation of valve interstitial cells (VICs).

In addition, excessive myofibroblast differentiation from VICs, leading to ECM accumula‐ tion and fibrosis, was influenced by remodeling of ECM in the fibrosa and facilitated by elastin degradation [18]. Furthermore, myofibroblast differentiation from VICs and calcifica‐ tion *in vitro* have been shown to be dependent on ECM composition [19].

Taken together, the macroscopic layered structure and the microscopic structure in each lay‐ er of the leaflets impart pronounced anisotropic mechanical behavior that allows the valve to open and close during a great number of cardiac cycles throughout life. These structures are tailored to fulfill the normal functions and maintain the homeostasis of the leaflets in a healthy condition. However, abnormal alteration of composition and mechanics of ECM in these structures may lead to calcific heart valve disease.
