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

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

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‐

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‐

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

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‐

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

tropic, i.e., the strains differ in the radial and circumferential directions [11].

suggests the osteoblastic differentiation of valve interstitial cells (VICs).

tion *in vitro* have been shown to be dependent on ECM composition [19].

these structures may lead to calcific heart valve disease.

radial directions of the leaflets [9–11].

6 Calcific Aortic Valve Disease

lamination between layers [6,8].

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 mechanical properties that support aortic valve function.

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 mechanical properties suited for maintaining unidirectional blood flow.

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

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 fibrils ensures the stability of the complex [21].

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 containing variably aligned collagen. Ranging from 10 to 50 μm in size, these membranes are believed to be much more extensible than the collagen fiber bundles they connect. These multilayer structures can easily slide past one another during valve movement, providing the combination of flexibility and tensile strength necessary for the required mechanics dur‐ ing valve opening and closing [22].

lagen molecule directly corresponds to the elastic modulus. However, this relationship does not apply to the radial direction, possibly due to the presence of elastin [31]. When there is no mechanical stress on the leaflet, the fibrosa exists as a number of folds in the radial direc‐ tion known as corrugations. Large extensibility is achieved through these collagen corruga‐ tions in combination with collagen crimp. When stress is applied to the leaflet, initial extension is accomplished by straightening of the collagen crimp. Further stress causes the corrugations to unfold in the radial direction [30]. Together, collagen crimp and corruga‐ tions allow the fibrosa to extend further in the radial direction when compared to the cir‐

Extracellular Matrix Organization, Structure, and Function

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

9

Throughout the lifetime of the aortic valve, collagen synthesis and degradation are responsi‐ ble for maintaining adequate valve strength and extensibility. Constant turnover of collagen allows the valve to adapt to regional changes in tensile strength. *In vitro* studies show that VICs respond to cyclic mechanical loading as a way to balance collagen synthesis and degra‐ dation. Cyclic stretch of valve leaflets stimulates VIC collagen type III production. In partic‐ ular, the amount and duration of the stretching can have an effect on the amount of collagen produced [27]. Additionally, VICs in culture express collagen type I and collagen type III mRNA for new matrix synthesis [33]. New collagen production is localized to specific re‐ gions of the valve depending on the collagen type that is produced. Collagen type I synthe‐ sis occurs in the fibrosa around, but not within, areas of mature collagen. Collagen type III synthesis, however, mainly occurs outside of the fibrosa [34]. Collagen degradation is also an important function of VICs and acts as an essential control to collagen production. Stud‐ ies have shown that VICs seeded into collagen scaffolds express MMPs that degrade the scaffold in a heterogeneous manner [33]. Thus, VICs continuously regulate the mechanical

properties of the surrounding ECM through collagen synthesis and degradation.

Aside from its mechanical functions, collagen has been shown to regulate VIC phenotype and calcification potential. *In vitro* studies were unable to induce calcification in VICs cul‐ tured on collagen proteins in standard media. It is believed that collagen actively inhibits VIC calcification [19]. Other studies have shown that scaffold collagen content also affects VIC proliferation. Specifically, one study reported that VICs adhered and spread on colla‐ gen surfaces but were not able to proliferate [35]. Another study showed that VIC prolifera‐ tion decreased on scaffolds containing higher collagen content [36]. An *in vitro* study indicated that matrix stiffness regulates VIC differentiation to myofibrogenic or osteogenic

**4. Elastic fibers comprise a significant portion of the ventricularis layer of**

Elastic fibers are macromolecular assemblies of several different molecules. The majority of the elastic fiber consists of elastin, an insoluble protein generated by lysyl oxidase crosslink‐ ing of soluble tropoelastin monomers (approximately 70 kDa). The elastin tends to be locat‐ ed in the inner core of the elastic fiber and is surrounded by a fine mesh of microfibrils.

cumferential direction.

phenotypes in calcific conditions [37].

**the aortic valve leaflets**

Collagen constitutes approximately 90% of the protein content of the valve insoluble matrix [23]. The vast majority of the valve's content is composed of collagens type I, III, and V. To‐ gether, these fibrillar collagens account for 60% of the valve's dry weight [24]. There is ap‐ proximately 74% collagen type I, 24% collagen type III, and 2% collagen type V distributed throughout the valve [25–27]. Whereas collagen type I mainly exists in the fibrosa, collagen type III is expressed ubiquitously throughout all three layers [25].

Collagen fibers mainly function to reduce stress on the leaflets during systole and diastole. While elastin controls initial valve opening and closing, collagen fibers reduce peak stresses in the leaflet matrix by an estimated 60%. These fibers have an important role in stabilizing leaflet motion [28]. Throughout leaflet movement, collagen fibers adjust position to resist tensile forces. As transvalvular pressure increases, the ventricularis expands in the circum‐ ferential direction, causing collagen fibers to become highly aligned. This is believed to in‐ crease the cuspal stiffness of the valve during diastole and prevent overextension of the valve [29].

The heterogeneous distribution of collagen throughout the aortic valve provides high strength in areas of greater stress while also allowing the valve to achieve a large degree of flexibility. Within the fibrosa, the primary tensile load-bearing layer, collagen fibers are highly aligned in the circumferential direction, resulting in tissue anisotropy. The arrange‐ ment of these fibers corresponds to the direction of highest tensile stress. In contrast, the ventricularis endures smaller tensile forces involved with initial opening and closing of the valve [30]. In addition to circumferentially oriented collagen, the largest and strongest colla‐ gen fiber bundles are localized in the areas of greatest tensile stress along the lower part of the commissure and coapting regions [22]. This unique arrangement and positioning of col‐ lagen reduces high tensile loads on the valve while allowing flexibility to open and close.

Comparisons between the fibrosa and ventricularis indicate that the fibrosa has a greater elastic modulus in the circumferential direction but a similar elastic modulus in the radial direction. These mechanical differences are largely the result of the number of aligned colla‐ gen fibers in each direction. With fewer collagen fibers, the ventricularis is approximately half as stiff as the fibrosa in the circumferential direction. In the radial direction, however, each layer contains approximately the same amount of collagen fibers and has similar elastic moduli [31]. Taken together, the multilayer valve structure causes aortic valves to be less stiff and more extensible radially than circumferentially [32].

Collagen achieves high strength and extensibility with the aid of additional mechanisms that contribute to the valve's mechanical properties. These include collagen cross-links, col‐ lagen crimp, and layer corrugations. Collagen cross-links function to increase the strength of aligned collagen. In the circumferential direction, the number of collagen cross-links per col‐ lagen molecule directly corresponds to the elastic modulus. However, this relationship does not apply to the radial direction, possibly due to the presence of elastin [31]. When there is no mechanical stress on the leaflet, the fibrosa exists as a number of folds in the radial direc‐ tion known as corrugations. Large extensibility is achieved through these collagen corruga‐ tions in combination with collagen crimp. When stress is applied to the leaflet, initial extension is accomplished by straightening of the collagen crimp. Further stress causes the corrugations to unfold in the radial direction [30]. Together, collagen crimp and corruga‐ tions allow the fibrosa to extend further in the radial direction when compared to the cir‐ cumferential direction.

containing variably aligned collagen. Ranging from 10 to 50 μm in size, these membranes are believed to be much more extensible than the collagen fiber bundles they connect. These multilayer structures can easily slide past one another during valve movement, providing the combination of flexibility and tensile strength necessary for the required mechanics dur‐

Collagen constitutes approximately 90% of the protein content of the valve insoluble matrix [23]. The vast majority of the valve's content is composed of collagens type I, III, and V. To‐ gether, these fibrillar collagens account for 60% of the valve's dry weight [24]. There is ap‐ proximately 74% collagen type I, 24% collagen type III, and 2% collagen type V distributed throughout the valve [25–27]. Whereas collagen type I mainly exists in the fibrosa, collagen

Collagen fibers mainly function to reduce stress on the leaflets during systole and diastole. While elastin controls initial valve opening and closing, collagen fibers reduce peak stresses in the leaflet matrix by an estimated 60%. These fibers have an important role in stabilizing leaflet motion [28]. Throughout leaflet movement, collagen fibers adjust position to resist tensile forces. As transvalvular pressure increases, the ventricularis expands in the circum‐ ferential direction, causing collagen fibers to become highly aligned. This is believed to in‐ crease the cuspal stiffness of the valve during diastole and prevent overextension of the

The heterogeneous distribution of collagen throughout the aortic valve provides high strength in areas of greater stress while also allowing the valve to achieve a large degree of flexibility. Within the fibrosa, the primary tensile load-bearing layer, collagen fibers are highly aligned in the circumferential direction, resulting in tissue anisotropy. The arrange‐ ment of these fibers corresponds to the direction of highest tensile stress. In contrast, the ventricularis endures smaller tensile forces involved with initial opening and closing of the valve [30]. In addition to circumferentially oriented collagen, the largest and strongest colla‐ gen fiber bundles are localized in the areas of greatest tensile stress along the lower part of the commissure and coapting regions [22]. This unique arrangement and positioning of col‐ lagen reduces high tensile loads on the valve while allowing flexibility to open and close.

Comparisons between the fibrosa and ventricularis indicate that the fibrosa has a greater elastic modulus in the circumferential direction but a similar elastic modulus in the radial direction. These mechanical differences are largely the result of the number of aligned colla‐ gen fibers in each direction. With fewer collagen fibers, the ventricularis is approximately half as stiff as the fibrosa in the circumferential direction. In the radial direction, however, each layer contains approximately the same amount of collagen fibers and has similar elastic moduli [31]. Taken together, the multilayer valve structure causes aortic valves to be less

Collagen achieves high strength and extensibility with the aid of additional mechanisms that contribute to the valve's mechanical properties. These include collagen cross-links, col‐ lagen crimp, and layer corrugations. Collagen cross-links function to increase the strength of aligned collagen. In the circumferential direction, the number of collagen cross-links per col‐

type III is expressed ubiquitously throughout all three layers [25].

stiff and more extensible radially than circumferentially [32].

ing valve opening and closing [22].

8 Calcific Aortic Valve Disease

valve [29].

Throughout the lifetime of the aortic valve, collagen synthesis and degradation are responsi‐ ble for maintaining adequate valve strength and extensibility. Constant turnover of collagen allows the valve to adapt to regional changes in tensile strength. *In vitro* studies show that VICs respond to cyclic mechanical loading as a way to balance collagen synthesis and degra‐ dation. Cyclic stretch of valve leaflets stimulates VIC collagen type III production. In partic‐ ular, the amount and duration of the stretching can have an effect on the amount of collagen produced [27]. Additionally, VICs in culture express collagen type I and collagen type III mRNA for new matrix synthesis [33]. New collagen production is localized to specific re‐ gions of the valve depending on the collagen type that is produced. Collagen type I synthe‐ sis occurs in the fibrosa around, but not within, areas of mature collagen. Collagen type III synthesis, however, mainly occurs outside of the fibrosa [34]. Collagen degradation is also an important function of VICs and acts as an essential control to collagen production. Stud‐ ies have shown that VICs seeded into collagen scaffolds express MMPs that degrade the scaffold in a heterogeneous manner [33]. Thus, VICs continuously regulate the mechanical properties of the surrounding ECM through collagen synthesis and degradation.

Aside from its mechanical functions, collagen has been shown to regulate VIC phenotype and calcification potential. *In vitro* studies were unable to induce calcification in VICs cul‐ tured on collagen proteins in standard media. It is believed that collagen actively inhibits VIC calcification [19]. Other studies have shown that scaffold collagen content also affects VIC proliferation. Specifically, one study reported that VICs adhered and spread on colla‐ gen surfaces but were not able to proliferate [35]. Another study showed that VIC prolifera‐ tion decreased on scaffolds containing higher collagen content [36]. An *in vitro* study indicated that matrix stiffness regulates VIC differentiation to myofibrogenic or osteogenic phenotypes in calcific conditions [37].
