**2. Structure-function of major ECM proteins of valvular tissue**

Proteins are the most abundant organic components of the human body. There are roughly 100,000 different kinds of proteins and they account for about 20% of the total body weight [2]. All proteins contain carbon, hydrogen, oxygen, and nitrogen and in some cases small quantities of sulfur. Proteins are classified according to the function they perform into structural, contractile and transport proteins, buffers, enzymes, antibodies and hormones. From these types, structural proteins such as collagen, elastin and keratin create a 3D framework for the body, providing strength, organization, and support for cells, tissues, and organs. Proteins consist of chains of amino acids, with each amino acid comprising a central carbon atom to which four groups are attached, including a hydrogen atom, an amino group (–NH2 ), a carboxylic acid group (–COOH) and a variable group, known as an R-group or side chain (**Figure 1a**). The amino and carboxylic acid groups are hydrophilic groups, and amino acids are relatively small, soluble molecules. Depending on the side chain the molecular structure changes drastically, giving each amino acid its individual chemical properties. The simplest R-group is hydrogen, which forms glycine (Gly). Within the physiological pH range, the carboxylic acid groups on many amino acids give up their hydrogen, going from –COOH to –COO− and getting negatively charged. Two amino acids can be linked by dehydration, which creates a covalent bond between the carboxylic acid group of one amino acid and the amino group of the other (**Figure 1b**). Such a bond is known as a peptide bond, and the molecule created dipeptide. The chain can be lengthened by the addition of more amino acids forming polypeptides. Polypeptides containing more than 100 amino acids are usually called proteins. Proteins contain negatively charged amino acids and, therefore, have a net negative charge [2, 3].

**Figure 1.** (a) Amino acid structure. (b) Peptide bond formation. (c) Schematic of the collagen triple helix (□ glycine; ♦ other amino acids). (d) Triple helices of procollagen I, II and III; Gal: galactose residue; Glu: glucose residue; redrawn

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from Nimni (1988) [5].

Proteins are very versatile and have a variety of different functional properties, which are determined not only by the R-groups on their constituent amino acids, but also by their shape. The primary determinant of shape is the sequence of amino acids (primary structure). The 20 major amino acids present in the human body, can be linked in a high number of combinations, creating proteins that vary in shape and function. Changing the identity of a single amino acid out of 10,000 or more in a protein may significantly alter its functional properties. Further levels of structural complexity include the secondary, tertiary, and quaternary structures. The secondary structure appears as parts of the polypeptide chain, which are bonded together by hydrogen bonding. This usually creates a simple spiral, known as α-helix, or less often a flat pleated sheet, or both. The tertiary structure results primarily from interactions between the polypeptide chain and the surrounding water molecules, and partly from interactions between the R-groups of amino acids in different parts of the molecule. Structural proteins contain several polypeptide chains; each one with its own secondary and tertiary structure. Interactions between these chains determine the quaternary structure of these proteins. The Structure-Function Relationship of Heart Valves in Health and Disease http://dx.doi.org/10.5772/intechopen.78280 3

as fibronectin and laminin, and glycosaminoglycans (GAGs) and proteoglycans. The ECM is involved in many normal and pathologic processes, playing an important role in tissue development and pathology, and mechanotransduction of the cells [1]. The ground substance is a hydrophilic gel that fills the spaces between the cells and the ECM. Dense connective tissues contain a small amount of ground substance and the cells are mostly fibroblasts. Loose connective tissues contain significantly more ground substance. Ground substance in normal connective tissues is clear and colorless and its composition varies with the tissue. It has a dense consistency similar to that of maple syrup, due to the presence of GAGs and proteoglycans.

2 Structural Insufficiency Anomalies in Cardiac Valves

The structure and hence the properties of a tissue are dependent on the chemical and physical nature of its constituents and their relative amounts. For example, nervous tissue consists almost entirely of cells, whereas bone is composed of collagen fibers and calcium phosphate minerals with minute quantities of cells and ground substance. Depending on the functional requirements on a particular tissue, the organization of its ECM macromolecules, cells and ground substance, as well as its resulting mechanical properties, vary. The simplest structure from the point of view of the collagen fibers consists of parallel fibers, as in tendons and ligaments. The 2D and 3D networks of the skin are more complex, whereas the structure of blood vessels and heart valves are the most complex ones. This chapter describes the basic biochemistry and function of the major structural proteins, collagen and elastic fibers, as well as the major GAGs and proteoglycans, present in the ECM of the heart valves, and illustrates their physiological and pathological significance. In addition it examines the structural basis, organization and structure-function relationship of valvular tissue, with particular focus on the aortic and mitral valves, by correlating its microscale histoarchitecture and biochemical constitution to its mesoscale biomechanics. Finally, the chapter examines the influence of pathological alterations, as a result of major valvular disease, on the histoarchitectural, constitutional and biomechanical characteristics of the valves.

**2. Structure-function of major ECM proteins of valvular tissue**

which four groups are attached, including a hydrogen atom, an amino group (–NH2

Proteins are the most abundant organic components of the human body. There are roughly 100,000 different kinds of proteins and they account for about 20% of the total body weight [2]. All proteins contain carbon, hydrogen, oxygen, and nitrogen and in some cases small quantities of sulfur. Proteins are classified according to the function they perform into structural, contractile and transport proteins, buffers, enzymes, antibodies and hormones. From these types, structural proteins such as collagen, elastin and keratin create a 3D framework for the body, providing strength, organization, and support for cells, tissues, and organs. Proteins consist of chains of amino acids, with each amino acid comprising a central carbon atom to

ylic acid group (–COOH) and a variable group, known as an R-group or side chain (**Figure 1a**). The amino and carboxylic acid groups are hydrophilic groups, and amino acids are relatively small, soluble molecules. Depending on the side chain the molecular structure changes drastically, giving each amino acid its individual chemical properties. The simplest R-group is hydrogen, which forms glycine (Gly). Within the physiological pH range, the carboxylic acid groups on many amino acids give up their hydrogen, going from –COOH to –COO−

getting negatively charged. Two amino acids can be linked by dehydration, which creates a covalent bond between the carboxylic acid group of one amino acid and the amino group

), a carbox-

and

**Figure 1.** (a) Amino acid structure. (b) Peptide bond formation. (c) Schematic of the collagen triple helix (□ glycine; ♦ other amino acids). (d) Triple helices of procollagen I, II and III; Gal: galactose residue; Glu: glucose residue; redrawn from Nimni (1988) [5].

of the other (**Figure 1b**). Such a bond is known as a peptide bond, and the molecule created dipeptide. The chain can be lengthened by the addition of more amino acids forming polypeptides. Polypeptides containing more than 100 amino acids are usually called proteins. Proteins contain negatively charged amino acids and, therefore, have a net negative charge [2, 3].

Proteins are very versatile and have a variety of different functional properties, which are determined not only by the R-groups on their constituent amino acids, but also by their shape. The primary determinant of shape is the sequence of amino acids (primary structure). The 20 major amino acids present in the human body, can be linked in a high number of combinations, creating proteins that vary in shape and function. Changing the identity of a single amino acid out of 10,000 or more in a protein may significantly alter its functional properties. Further levels of structural complexity include the secondary, tertiary, and quaternary structures. The secondary structure appears as parts of the polypeptide chain, which are bonded together by hydrogen bonding. This usually creates a simple spiral, known as α-helix, or less often a flat pleated sheet, or both. The tertiary structure results primarily from interactions between the polypeptide chain and the surrounding water molecules, and partly from interactions between the R-groups of amino acids in different parts of the molecule. Structural proteins contain several polypeptide chains; each one with its own secondary and tertiary structure. Interactions between these chains determine the quaternary structure of these proteins. The tertiary and quaternary structure of complex proteins depends not only on their amino acid sequence, but also on the local environmental characteristics, with small changes in the ionic composition, temperature, or pH affecting protein function [1–3].

trimers and align the three α-helix chains prior to the triple helix formation in the ER. In addition, specific Pro and Lys residues in the middle of the chains are hydroxylated by hydroxylases, and asparagine-linked oligosaccharides are added to the C-terminal propeptide, whereas galactose (Gal) or galactose-glucose (Gal-Glu) residues are attached to Hyl residues, in a process known as glycosylation [4]. Glycosylation is a common form of post-translational modification of proteins that occurs during processing in the ER and provides intrinsic stabilization of the protein structure and, thus, increases protein half-life and protects them against denaturation or proteolytic degradation [9–11]. The modifications of the α-helices in the ER facilitate their zipping from C- to N-terminus to form stable triple helices in the procollagen molecule. These modifications also allow the binding of the chaperone protein Hsp47 to the procollagen, which has been suggested to further stabilize the helices and/or prevent premature aggregation of the trimers [4]. The modification of the procollagen in the ER is crucial for the subsequent formation of mature collagen,

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**Table 1.** Amino acid residues per 1000 total residues in human collagen [6, 20] and elastin [7].

#### **2.1. Collagen fibers**

Collagen is the major insoluble fibrous protein and the basic structural element for soft and hard tissues, supporting elements in the ECM. It is the main load-carrying element giving mechanical integrity and it is present in a variety of structural forms in different tissues. Collagen contains large domains of helical conformation, created by three α-helix polypeptide chains, each containing 1050 amino acids. The individual α-helix chains are left-handed helices with approximately three amino acid residues per turn. The chains are, in turn, coiled together to give a right-handed coiled triple helix (**Figure 1c**), which is the molecular basis of tropocollagen, the precursor of collagen. All collagens have been shown to contain three α-helix chains of similar structure, with each collagen type differentiating its individual properties mainly by incorporating segments that do not follow the triple helix conformation, and fold the collagen molecule into different kinds of three-dimensional structures [3]. The collagen molecule contains a high amount of three amino acids, including Gly, proline (Pro) and hydroxyproline (HYP), and since the latter is unique in collagen (elastin contains minute amount), the collagen content in a tissue can be easily determined by a HYP assay. The characteristic repeating amino acid sequence of the collagen molecule is Gly-X-Y, where X and Y can be any amino acid but are often proline and HYP and less often lysine (Lys) and hydroxylysine (Hyl) [4]. In the amino acid sequence of the collagen molecule every third residue is Gly (**Figure 1c**), whereas proline and HYP follow each other relatively frequently [3, 5, 6]. Hydrogen bonding between the peptide bond NH of Gly and the peptide carbonyl (C=O) group in an adjacent polypeptide (**Figure 1b**), holds the three α-helix peptide chains together in a three-stranded helix, which is formed due to the fixed angle of the C-N peptidyl-Pro or peptidyl-HYP bond [3]. In addition, the α-helixes are cross-linked via Lys, whereas collagen III also contains cysteine (Cys) that can be cross-linked within molecules through disulfide bonds [7]. The side chains of the amino acids of the collagen molecule are highly non-polar and, hence, hydrophobic, seeking the greatest number of contacts with the non-polar side chains of other amino acids. In cases that the hydrophobic contact is destroyed by a solution such as urea, ultrastructural changes are generated in the collagen fibers, such as shrinking [7, 8]. Collagen stability is also affected by the water content in its intra- and inter-chain structure. Specifically, when the water content is decreased the collagen structure gets destabilized, whereas lyophilized collagen also demonstrates decreased solubility [7, 8].

Depending on the source of tissues the collagen chains are different. A characteristic example is demonstrated in **Figure 1d**, which shows the triple helix conformation of three types of collagen, differing in α-helix composition and degree of glycosylation. The amino acid compositions of these chains are listed in **Table 1**. The collagen α-helix chains are synthesized by ribosomes attached to the rough endoplasmic reticulum (ER) as longer precursors called pro-α-helix chains. Subsequently, the pro-α-helix chains undergo a series of covalent modifications in the ER and fold into triple-helical procollagen molecules (**Figure 1d**) before they are released from the cells. Specifically, short non-triple-helical segments at either end (C– and N–terminus) of the pro-α-helix chains, called propeptides and containing Hyl, are covalently linked by disulfide bonds to form trimers and align the three α-helix chains prior to the triple helix formation in the ER. In addition, specific Pro and Lys residues in the middle of the chains are hydroxylated by hydroxylases, and asparagine-linked oligosaccharides are added to the C-terminal propeptide, whereas galactose (Gal) or galactose-glucose (Gal-Glu) residues are attached to Hyl residues, in a process known as glycosylation [4]. Glycosylation is a common form of post-translational modification of proteins that occurs during processing in the ER and provides intrinsic stabilization of the protein structure and, thus, increases protein half-life and protects them against denaturation or proteolytic degradation [9–11]. The modifications of the α-helices in the ER facilitate their zipping from C- to N-terminus to form stable triple helices in the procollagen molecule. These modifications also allow the binding of the chaperone protein Hsp47 to the procollagen, which has been suggested to further stabilize the helices and/or prevent premature aggregation of the trimers [4]. The modification of the procollagen in the ER is crucial for the subsequent formation of mature collagen,

tertiary and quaternary structure of complex proteins depends not only on their amino acid sequence, but also on the local environmental characteristics, with small changes in the ionic

Collagen is the major insoluble fibrous protein and the basic structural element for soft and hard tissues, supporting elements in the ECM. It is the main load-carrying element giving mechanical integrity and it is present in a variety of structural forms in different tissues. Collagen contains large domains of helical conformation, created by three α-helix polypeptide chains, each containing 1050 amino acids. The individual α-helix chains are left-handed helices with approximately three amino acid residues per turn. The chains are, in turn, coiled together to give a right-handed coiled triple helix (**Figure 1c**), which is the molecular basis of tropocollagen, the precursor of collagen. All collagens have been shown to contain three α-helix chains of similar structure, with each collagen type differentiating its individual properties mainly by incorporating segments that do not follow the triple helix conformation, and fold the collagen molecule into different kinds of three-dimensional structures [3]. The collagen molecule contains a high amount of three amino acids, including Gly, proline (Pro) and hydroxyproline (HYP), and since the latter is unique in collagen (elastin contains minute amount), the collagen content in a tissue can be easily determined by a HYP assay. The characteristic repeating amino acid sequence of the collagen molecule is Gly-X-Y, where X and Y can be any amino acid but are often proline and HYP and less often lysine (Lys) and hydroxylysine (Hyl) [4]. In the amino acid sequence of the collagen molecule every third residue is Gly (**Figure 1c**), whereas proline and HYP follow each other relatively frequently [3, 5, 6]. Hydrogen bonding between the peptide bond NH of Gly and the peptide carbonyl (C=O) group in an adjacent polypeptide (**Figure 1b**), holds the three α-helix peptide chains together in a three-stranded helix, which is formed due to the fixed angle of the C-N peptidyl-Pro or peptidyl-HYP bond [3]. In addition, the α-helixes are cross-linked via Lys, whereas collagen III also contains cysteine (Cys) that can be cross-linked within molecules through disulfide bonds [7]. The side chains of the amino acids of the collagen molecule are highly non-polar and, hence, hydrophobic, seeking the greatest number of contacts with the non-polar side chains of other amino acids. In cases that the hydrophobic contact is destroyed by a solution such as urea, ultrastructural changes are generated in the collagen fibers, such as shrinking [7, 8]. Collagen stability is also affected by the water content in its intra- and inter-chain structure. Specifically, when the water content is decreased the collagen structure gets destabilized, whereas lyophilized collagen also dem-

Depending on the source of tissues the collagen chains are different. A characteristic example is demonstrated in **Figure 1d**, which shows the triple helix conformation of three types of collagen, differing in α-helix composition and degree of glycosylation. The amino acid compositions of these chains are listed in **Table 1**. The collagen α-helix chains are synthesized by ribosomes attached to the rough endoplasmic reticulum (ER) as longer precursors called pro-α-helix chains. Subsequently, the pro-α-helix chains undergo a series of covalent modifications in the ER and fold into triple-helical procollagen molecules (**Figure 1d**) before they are released from the cells. Specifically, short non-triple-helical segments at either end (C– and N–terminus) of the pro-α-helix chains, called propeptides and containing Hyl, are covalently linked by disulfide bonds to form

composition, temperature, or pH affecting protein function [1–3].

**2.1. Collagen fibers**

4 Structural Insufficiency Anomalies in Cardiac Valves

onstrates decreased solubility [7, 8].


**Table 1.** Amino acid residues per 1000 total residues in human collagen [6, 20] and elastin [7].

whereas defects in this process have been reported to have serious consequences. For example, hydroxylation requires the facilitation of vitamin C, and cells deprived of it cannot hydroxylate the procollagen chains sufficiently to form stable triple helices. As a result, non-hydroxylated procollagen chains are degraded within the cell and no collagen fibers can be formed [12]. As a result, in tissues where the turnover (degradation and replacement) of the collagen occurs relatively rapidly, such as in blood vessels and heart valves that experience significant collagen fiber damage due to the repetitive deformation during normal function, the gradual loss of preexisting ECM collagen, which is not supplemented by newly-formed collagen, leads to tissues that are extremely fragile and prone to disintegration [13]. However, in other tissues, such as bone, the turnover of collagen is very low, with collagen molecules reportedly remaining intact for about 10 years [13]. Following the post-translational modification in the ER, the procollagen molecules are transported to the Golgi apparatus, where they are associated laterally to form small bundles, before they are secreted in the extracellular space (exocytosis). Following exocytosis, procollagen peptidases cleave the N-terminal and C-terminal propeptides, transforming the procollagen to tropocollagen molecules, approximately 300 nm long and 1.5 nm in diameter [14]. This allows the tropocollagen molecules to assemble into collagen fibrils by covalent cross-linking between two Lys or Hyl residues at the C-terminus of one tropocollagen molecule with two similar residues at the N-terminus of an adjacent molecule. These cross-links stabilize the packing of the tropocollagen molecules in a quarter-staggered array architecture (**Figure 2a**), generating strong fibrils with a diameter of 50–200 nm and several micrometers long, depending of the species and tissue, with a cross-striated appearance under transmission electron microscopy (**Figure 2b**) [3–6]. The striation has a period (D) of approximately 67 nm, with the lighter part of the striation representing a gap of approximately 0.6D between successive molecules (**Figure 2b**) [6, 15]. The covalent cross-linking between Lys and Hyl residues of adjacent collagen molecules confers significant strength to the collagen fibrils. This type of covalent bonding is only found in collagen and elastin, and its inhibition results in a dramatic reduction of the tensile strength of the fibrils, making tissues fragile and prone to tearing. The extent and type of cross-linking vary from tissue to tissue; in tissues where tensile strength is crucial, collagen is highly cross-linked [13]. Although the alignment of the tropocollagen molecules in the collagen fibril has been idealized as perfectly straight and parallel in **Figure 2a**, in reality they are bent in various degrees, depending on the attachment of water molecules, and have varying spacing between neighboring molecules [6]. Bundles of collagen fibrils form collagen fibers, with diameters ranging between 0.2 and 12 μm, and increasing with age, whereas their length depends on the tissue [16, 17]. Moreover, collagen fibers appear to be crimped (**Figure 2c**), and when the tissue is stretched the amplitude of the crimp decreases [6, 18, 19]. It has been suggested that the crimping of the collagen fibers is generated by the shrinking of the non-collagenous components of the ground substance, which causes collagen fiber buckling [19], whereas enzymatic digestion of the non-collagenous components of the ground substance alters the mechanical properties of the tissue [6].

Collagens are differentiated in terms of their ability to form fibers and organize the fibers into networks. There are at least 20 types of collagen that have been identified and participate in the formation of the ECM of tissues [4]. Among these, types I, II, II, V and XI are fibrillar collagens, with types I, II, III comprising 80–90% of the total collagen in the body [3]. Collagen I is present in almost any tissue, but it predominantly present in bone, dermis, placental membranes, tendons, ligaments, blood vessels and heart valves. Collagen II is mainly located in hyaline cartilage and cartilage-like tissues and the vitreous body of the eye [20]. Similarly to collagen I, collagen III is also quite common in most tissues and represents a major constituent of blood vessels and heart valves, as well as other more distensible connective tissue. Collagen V has a similar distribution to collagen I, but it is a minor constituent in the tissues, whereas collagen XI is found mainly in cartilage and is distributed similarly to collagen II [21]. Collagen VI and IX represent a separate class of collagens that are associated to fibrillar collagens, linking them to each other or to other ECM components. The collagen VI tropocollagen comprises short triple-helical regions of approximately 60 nm long, separated by globular regions of approximately 40 nm long [3]. Collagen VI is common in placental villi [6] and has also been found in many other connective tissues, where it is bound to collagen I fibrils and has been suggested to enable the formation of thicker collagen I fibers [3]. Collagen IX is a proteoglycan in that one of its polypeptide subunits serves as the core protein for a chondroitin

showing the elastin in the elastic fiber network.

**Figure 2.** (a) Quarter-staggering of tropocollagen I molecules in collagen fibrils; redrawn from Lodish et al. (2003) [4]. (b) Transmission electron microscopy of the decellularised mitral valve leaflet showing the striations in the collagen I fibrils; adopted with permission from Granados et al. (2017) [68]. (c) Histological section of the anterior mitral valve leaflet stained with sirius red under polarized light, showing the crimping in the collagen I fibers; adopted with permission from Roberts et al. (2016) [18]. (d) Immunofluorescence staining for collagen IV produced by human endothelial cells seeded on culture plastic (yellow/orange: anti-collagen-IV; blue: Hoechst stain for nuclei); adopted with permission from Pflaum et al. (2017) [132]. (e) Two photon microscopy of the atrial surface of the native anterior mitral valve leaflet,

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whereas defects in this process have been reported to have serious consequences. For example, hydroxylation requires the facilitation of vitamin C, and cells deprived of it cannot hydroxylate the procollagen chains sufficiently to form stable triple helices. As a result, non-hydroxylated procollagen chains are degraded within the cell and no collagen fibers can be formed [12]. As a result, in tissues where the turnover (degradation and replacement) of the collagen occurs relatively rapidly, such as in blood vessels and heart valves that experience significant collagen fiber damage due to the repetitive deformation during normal function, the gradual loss of preexisting ECM collagen, which is not supplemented by newly-formed collagen, leads to tissues that are extremely fragile and prone to disintegration [13]. However, in other tissues, such as bone, the turnover of collagen is very low, with collagen molecules reportedly remaining intact for about 10 years [13]. Following the post-translational modification in the ER, the procollagen molecules are transported to the Golgi apparatus, where they are associated laterally to form small bundles, before they are secreted in the extracellular space (exocytosis). Following exocytosis, procollagen peptidases cleave the N-terminal and C-terminal propeptides, transforming the procollagen to tropocollagen molecules, approximately 300 nm long and 1.5 nm in diameter [14]. This allows the tropocollagen molecules to assemble into collagen fibrils by covalent cross-linking between two Lys or Hyl residues at the C-terminus of one tropocollagen molecule with two similar residues at the N-terminus of an adjacent molecule. These cross-links stabilize the packing of the tropocollagen molecules in a quarter-staggered array architecture (**Figure 2a**), generating strong fibrils with a diameter of 50–200 nm and several micrometers long, depending of the species and tissue, with a cross-striated appearance under transmission electron microscopy (**Figure 2b**) [3–6]. The striation has a period (D) of approximately 67 nm, with the lighter part of the striation representing a gap of approximately 0.6D between successive molecules (**Figure 2b**) [6, 15]. The covalent cross-linking between Lys and Hyl residues of adjacent collagen molecules confers significant strength to the collagen fibrils. This type of covalent bonding is only found in collagen and elastin, and its inhibition results in a dramatic reduction of the tensile strength of the fibrils, making tissues fragile and prone to tearing. The extent and type of cross-linking vary from tissue to tissue; in tissues where tensile strength is crucial, collagen is highly cross-linked [13]. Although the alignment of the tropocollagen molecules in the collagen fibril has been idealized as perfectly straight and parallel in **Figure 2a**, in reality they are bent in various degrees, depending on the attachment of water molecules, and have varying spacing between neighboring molecules [6]. Bundles of collagen fibrils form collagen fibers, with diameters ranging between 0.2 and 12 μm, and increasing with age, whereas their length depends on the tissue [16, 17]. Moreover, collagen fibers appear to be crimped (**Figure 2c**), and when the tissue is stretched the amplitude of the crimp decreases [6, 18, 19]. It has been suggested that the crimping of the collagen fibers is generated by the shrinking of the non-collagenous components of the ground substance, which causes collagen fiber buckling [19], whereas enzymatic digestion of the non-collagenous components of

6 Structural Insufficiency Anomalies in Cardiac Valves

the ground substance alters the mechanical properties of the tissue [6].

Collagens are differentiated in terms of their ability to form fibers and organize the fibers into networks. There are at least 20 types of collagen that have been identified and participate in the formation of the ECM of tissues [4]. Among these, types I, II, II, V and XI are fibrillar collagens, with types I, II, III comprising 80–90% of the total collagen in the body [3]. Collagen I is present in almost any tissue, but it predominantly present in bone, dermis, placental membranes, tendons, ligaments, blood vessels and heart valves. Collagen II is mainly located in

**Figure 2.** (a) Quarter-staggering of tropocollagen I molecules in collagen fibrils; redrawn from Lodish et al. (2003) [4]. (b) Transmission electron microscopy of the decellularised mitral valve leaflet showing the striations in the collagen I fibrils; adopted with permission from Granados et al. (2017) [68]. (c) Histological section of the anterior mitral valve leaflet stained with sirius red under polarized light, showing the crimping in the collagen I fibers; adopted with permission from Roberts et al. (2016) [18]. (d) Immunofluorescence staining for collagen IV produced by human endothelial cells seeded on culture plastic (yellow/orange: anti-collagen-IV; blue: Hoechst stain for nuclei); adopted with permission from Pflaum et al. (2017) [132]. (e) Two photon microscopy of the atrial surface of the native anterior mitral valve leaflet, showing the elastin in the elastic fiber network.

hyaline cartilage and cartilage-like tissues and the vitreous body of the eye [20]. Similarly to collagen I, collagen III is also quite common in most tissues and represents a major constituent of blood vessels and heart valves, as well as other more distensible connective tissue. Collagen V has a similar distribution to collagen I, but it is a minor constituent in the tissues, whereas collagen XI is found mainly in cartilage and is distributed similarly to collagen II [21]. Collagen VI and IX represent a separate class of collagens that are associated to fibrillar collagens, linking them to each other or to other ECM components. The collagen VI tropocollagen comprises short triple-helical regions of approximately 60 nm long, separated by globular regions of approximately 40 nm long [3]. Collagen VI is common in placental villi [6] and has also been found in many other connective tissues, where it is bound to collagen I fibrils and has been suggested to enable the formation of thicker collagen I fibers [3]. Collagen IX is a proteoglycan in that one of its polypeptide subunits serves as the core protein for a chondroitin sulfate side chain [6]. The collagen IX molecule comprises two long triple helices connected by a flexible link, on the α2(IX) chain of which the chondroitin sulfate glycosaminoglycan (GAG) chain is covalently linked to [3, 6]. Collagen IX does not assemble into fibrils, due to its interrupted triple-helical conformation. Nevertheless, it is bound along collagen II fibrils at regular intervals, binding them to the GAG- and proteoglycan-rich ECM, and also reportedly contributing to their assembly in collagen II fibers [22]. In addition to collagen IX, collagens XVIII and XV also function as core proteins in proteoglycans.

**2.2. Elastic fibers**

Elastin is an another structural insoluble fibrous protein, forming a large proportion of the ECM of arteries and veins, especially near the heart, and in other deformable tissues such as ligaments, heart valves, skin, lungs and areolar connective tissue. Elastin is the dominant ECM protein in arteries, contributing about 28–50% of the dry weight of the aorta [13, 23, 24], whereas in elastic ligaments and tendons the elastin content attributes 50 and 4% of the dry weight, respectively [23]. Elastin is the main component of the elastic fibers, which are major insoluble assemblies of the ECM that generate resilience into the tissues by providing a mechanism that permits tissues to deform under load and passively recoil to their original configuration after the load is released, preventing dynamic tissue creep [13, 24–26]. These properties are critical to the function of heart valves, which undergo repeated dynamic cycles of large extension and recoil during opening and closing. In addition to contributing to ECM resilience, the elastic fibers are also an important load-bearing structure, complementing the function of the collagen fibers at the sites where mechanical energy needs to be stored [25]. In particular, the elastic fibers dictate tissue mechanics at low strains before the stiffer collagen fibers are engaged at higher strains [24]. Elastic fibers are the most linearly elastic biosolids known, and are at least five times more extensible than rubber. They are generally twisted or straight with a diameter ranging between about 0.2 and 1.5 μm forming coarse networks (**Figure 2e**) [27]. In dense elastic tissues, such as the arteries, the elastic fibers fuse during development to form flattened co-centric sheets, or

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The elastic fibers are complex structures that comprise several components, with elastin and microfibrils constituting their two major ones [30]. Ultrastructurally, elastic fibers are composed of a homogeneous inner core of laterally packed, thin cross-linked elastin filaments that make up more than 90% of fibers, and an outer fibrillar mantle that consists of microfibrils and surrounds the elastin, accounting for 5–10% of the elastic fibers [23, 25, 26]. The amino acid compositions of these components are given in **Table 1** [7]. The microfibrils are organized into 8–16 nm beaded fibrils and are predominantly made of five distinct proteins, including two fibrillin glycoproteins (fibrillin-1 and fibrillin-2) and two microfibril-associated glycoproteins (MAGP-1 and MAGP-2) [13, 23, 31]. Additional components of the elastic fibers include lysyl oxidase, elastin-binding protein (EBP), proteoglycans, osteopontin, emilin, fibulin-1, and various microfibril-associated proteins [23]. Early development of the elastic fibers involves the assembly of fibrillin molecules in the extracellular space, in enfoldings of the cell surface, into head-to-tail microfibrillar arrays of approximately 160 nm in length that are cross-linked by transglutaminase to form mature beaded microfibrils. Transglutaminase forms γ-glutamyl-e-Lys isopeptide bonds within or between peptide chains [25, 29]. The mature microfibrils, which have a length of approximately 100 nm, form loosely-packed parallel bundles with an one-third-staggered architecture, that have been hypothesized to be stabilized by inter-microfibrillar crosslinking [25]. Non-stretched microfibrils have been reported to have a beaded periodicity of approximately 56 nm, whereas models have predicted up to 8 fibrillin molecules in the microfibril cross-section [25, 27, 31, 32]. Following the formation of the microfibril bundles, intracellularly-produced tropoelastin [33] is gradually deposited among the preformed template of fibrillin-rich microfibrils, until the elastic fibers reach full maturation [7, 23, 25, 26]. Some tissues, even at their mature state, contain bundles of microfibrils devoid of tropoelastin (oxytalan fibers), as well as sites where bundles

elastic laminae, with numerous fenestrations [17, 28, 29].

Collagen IV is a sheet-forming collagen, which is of particular importance for endothelialized and epithelialized tissues, since, together with laminin, is the primary component of all basal laminae, forming their basic two-dimensional fibrous network. Collagen IV, as well as most of the other ECM components that form the basal laminae, is synthesized by the cells that reside on it (**Figure 2d**) [4]. The collagen IV molecule is formed by a 400-nm-long triple helix with large globular domains at the C-terminus and smaller non-collagen ones at the N-terminus, whereas Gly-X-Y sequences of its α-helices are interrupted with segments that do not form a triple helix, and introduce flexible links and flexibility into the molecule [3, 4]. Following exocytosis into the extracellular space, adjacent collagen IV molecules assemble either into groups of 4 molecules by association of their globular N-terminus domains, yielding tetrameric units, or into pairs by association of their globular C-terminus domains, yielding dimeric units [4]. Subsequently, triple-helical regions from several tetrameric and dimeric units associate laterally, similarly to the case of fibril formation in fibrillar collagens, to form branching two-dimensional mesh-like fibrous networks to which other ECM components and adhesion receptors can bind (**Figure 2d**). In addition to the aforementioned types of collagen, other minor classes of collagens include anchoring collagens, such as collagen VII, which connects the basal lamina to the underlying connective tissue, transmembrane collagens, which function as cell adhesion receptors, and host defense collagens, which help the body to recognize and eliminate pathogens [4].

Collagen is encoded by approximately 30 genes, whereas its biosynthetic pathway involves a number of post-translational phases. Owning to this, collagen-related disease most commonly arise from genetic mutations affecting collagen encoding or post-translational modifications, whereas nutritional deficiencies might also affect the post-translational modifications, assembly, or secretion of collagen [1, 13]. Moreover, autoimmune conditions have also been reported to affect collagen fibers, whereas a number of different bacteria and viruses can degrade collagen or interfere with its production pathway [1]. Goodpasture's autoimmune disease causes self-attacking antibodies to bind to the α-helices of collagen IV, setting off an immune response that causes cellular damage [4]. The Ehlers-Danlos syndrome comprises at least 10 types of congenital disorders whose principal features include tissue hyper-extensibility and abnormal fragility. Type IV disorder is the most serious one, especially for the cardiovascular system, which affects collagen III and is associated with spontaneous rupture of arteries or the bowel [1, 13]. Type VI disorder, together with the Bruck syndrome and Menkes disease, are associated with lysyl hydroxylase deficiency, leading to defective cross-linking of collagen and elastin [1, 4, 23], whereas type VIIC disorder causes formation of abnormally thin and irregular collagen fibrils [1]. Moreover, the Alport syndrome is another genetic disorder that alters the C-terminal domain of the tropocollagen IV α-helices and affects the structure of collagen IV fibers, causing structural abnormalities in basal laminae [1, 4].

#### **2.2. Elastic fibers**

sulfate side chain [6]. The collagen IX molecule comprises two long triple helices connected by a flexible link, on the α2(IX) chain of which the chondroitin sulfate glycosaminoglycan (GAG) chain is covalently linked to [3, 6]. Collagen IX does not assemble into fibrils, due to its interrupted triple-helical conformation. Nevertheless, it is bound along collagen II fibrils at regular intervals, binding them to the GAG- and proteoglycan-rich ECM, and also reportedly contributing to their assembly in collagen II fibers [22]. In addition to collagen IX, collagens

Collagen IV is a sheet-forming collagen, which is of particular importance for endothelialized and epithelialized tissues, since, together with laminin, is the primary component of all basal laminae, forming their basic two-dimensional fibrous network. Collagen IV, as well as most of the other ECM components that form the basal laminae, is synthesized by the cells that reside on it (**Figure 2d**) [4]. The collagen IV molecule is formed by a 400-nm-long triple helix with large globular domains at the C-terminus and smaller non-collagen ones at the N-terminus, whereas Gly-X-Y sequences of its α-helices are interrupted with segments that do not form a triple helix, and introduce flexible links and flexibility into the molecule [3, 4]. Following exocytosis into the extracellular space, adjacent collagen IV molecules assemble either into groups of 4 molecules by association of their globular N-terminus domains, yielding tetrameric units, or into pairs by association of their globular C-terminus domains, yielding dimeric units [4]. Subsequently, triple-helical regions from several tetrameric and dimeric units associate laterally, similarly to the case of fibril formation in fibrillar collagens, to form branching two-dimensional mesh-like fibrous networks to which other ECM components and adhesion receptors can bind (**Figure 2d**). In addition to the aforementioned types of collagen, other minor classes of collagens include anchoring collagens, such as collagen VII, which connects the basal lamina to the underlying connective tissue, transmembrane collagens, which function as cell adhesion receptors, and host defense collagens, which help the body to recog-

Collagen is encoded by approximately 30 genes, whereas its biosynthetic pathway involves a number of post-translational phases. Owning to this, collagen-related disease most commonly arise from genetic mutations affecting collagen encoding or post-translational modifications, whereas nutritional deficiencies might also affect the post-translational modifications, assembly, or secretion of collagen [1, 13]. Moreover, autoimmune conditions have also been reported to affect collagen fibers, whereas a number of different bacteria and viruses can degrade collagen or interfere with its production pathway [1]. Goodpasture's autoimmune disease causes self-attacking antibodies to bind to the α-helices of collagen IV, setting off an immune response that causes cellular damage [4]. The Ehlers-Danlos syndrome comprises at least 10 types of congenital disorders whose principal features include tissue hyper-extensibility and abnormal fragility. Type IV disorder is the most serious one, especially for the cardiovascular system, which affects collagen III and is associated with spontaneous rupture of arteries or the bowel [1, 13]. Type VI disorder, together with the Bruck syndrome and Menkes disease, are associated with lysyl hydroxylase deficiency, leading to defective cross-linking of collagen and elastin [1, 4, 23], whereas type VIIC disorder causes formation of abnormally thin and irregular collagen fibrils [1]. Moreover, the Alport syndrome is another genetic disorder that alters the C-terminal domain of the tropocollagen IV α-helices and affects the structure of

collagen IV fibers, causing structural abnormalities in basal laminae [1, 4].

XVIII and XV also function as core proteins in proteoglycans.

8 Structural Insufficiency Anomalies in Cardiac Valves

nize and eliminate pathogens [4].

Elastin is an another structural insoluble fibrous protein, forming a large proportion of the ECM of arteries and veins, especially near the heart, and in other deformable tissues such as ligaments, heart valves, skin, lungs and areolar connective tissue. Elastin is the dominant ECM protein in arteries, contributing about 28–50% of the dry weight of the aorta [13, 23, 24], whereas in elastic ligaments and tendons the elastin content attributes 50 and 4% of the dry weight, respectively [23]. Elastin is the main component of the elastic fibers, which are major insoluble assemblies of the ECM that generate resilience into the tissues by providing a mechanism that permits tissues to deform under load and passively recoil to their original configuration after the load is released, preventing dynamic tissue creep [13, 24–26]. These properties are critical to the function of heart valves, which undergo repeated dynamic cycles of large extension and recoil during opening and closing. In addition to contributing to ECM resilience, the elastic fibers are also an important load-bearing structure, complementing the function of the collagen fibers at the sites where mechanical energy needs to be stored [25]. In particular, the elastic fibers dictate tissue mechanics at low strains before the stiffer collagen fibers are engaged at higher strains [24]. Elastic fibers are the most linearly elastic biosolids known, and are at least five times more extensible than rubber. They are generally twisted or straight with a diameter ranging between about 0.2 and 1.5 μm forming coarse networks (**Figure 2e**) [27]. In dense elastic tissues, such as the arteries, the elastic fibers fuse during development to form flattened co-centric sheets, or elastic laminae, with numerous fenestrations [17, 28, 29].

The elastic fibers are complex structures that comprise several components, with elastin and microfibrils constituting their two major ones [30]. Ultrastructurally, elastic fibers are composed of a homogeneous inner core of laterally packed, thin cross-linked elastin filaments that make up more than 90% of fibers, and an outer fibrillar mantle that consists of microfibrils and surrounds the elastin, accounting for 5–10% of the elastic fibers [23, 25, 26]. The amino acid compositions of these components are given in **Table 1** [7]. The microfibrils are organized into 8–16 nm beaded fibrils and are predominantly made of five distinct proteins, including two fibrillin glycoproteins (fibrillin-1 and fibrillin-2) and two microfibril-associated glycoproteins (MAGP-1 and MAGP-2) [13, 23, 31]. Additional components of the elastic fibers include lysyl oxidase, elastin-binding protein (EBP), proteoglycans, osteopontin, emilin, fibulin-1, and various microfibril-associated proteins [23]. Early development of the elastic fibers involves the assembly of fibrillin molecules in the extracellular space, in enfoldings of the cell surface, into head-to-tail microfibrillar arrays of approximately 160 nm in length that are cross-linked by transglutaminase to form mature beaded microfibrils. Transglutaminase forms γ-glutamyl-e-Lys isopeptide bonds within or between peptide chains [25, 29]. The mature microfibrils, which have a length of approximately 100 nm, form loosely-packed parallel bundles with an one-third-staggered architecture, that have been hypothesized to be stabilized by inter-microfibrillar crosslinking [25]. Non-stretched microfibrils have been reported to have a beaded periodicity of approximately 56 nm, whereas models have predicted up to 8 fibrillin molecules in the microfibril cross-section [25, 27, 31, 32].

Following the formation of the microfibril bundles, intracellularly-produced tropoelastin [33] is gradually deposited among the preformed template of fibrillin-rich microfibrils, until the elastic fibers reach full maturation [7, 23, 25, 26]. Some tissues, even at their mature state, contain bundles of microfibrils devoid of tropoelastin (oxytalan fibers), as well as sites where bundles of microfibrils are only partially intermixed with tropoelastin (elaunin fibers), and never developed into fully mature elastic fibers [26]. These microfibril states represent interruptions in successive phases of elastic fiber development, indicating that microfibrils can be both progenitors of fully mature elastic fibers in fetal tissues, and independent connective tissue components (oxytalan fibers) [26, 34]. Tropoelastin is the soluble precursor of elastin, which is synthesized by fibroblasts and smooth muscle cells (SMCs) and secreted as a soluble and highly hydrophobic monomer of about 750 amino acids long [13, 24, 35]. Similarly to tropocollagen, tropoelastin contains an abundance of Pro and Gly, but is not glycosylated and contains minute amounts of HYP and no Hyl [13]. Human tropoelastin contains more than 30% Gly, whereas about 75% of its amino acid sequence is made up of just four hydrophobic amino acids, including Gly, valine (Val), alanine (Ala) and Pro [23]. Structurally, tropoelastin comprises two types of alternating segments, including hydrophobic segments and Ala-/Lys-rich α-helical segments, which form cross-links between adjacent molecules [13]. During tropoelastin deposition among the microfibrils, the latter get displaced to the periphery of the growing fiber, allowing the formation of the elastin core by the accumulation of tropoelastin molecules [13, 31]. The accumulated tropoelastin molecules couple covalently to each other by lysyl oxidase-derived cross-links, to form the polymeric elastin, stabilizing the elastin core of the elastic fibers [25, 31]. Lysyl oxidase catalyzes the selective oxidative deamination of Lys residues in tropoelastin, leading to the formation of bi-functional (dehydrolysinonorleucine and allysine aldol), tri-functional (dehydromerodesmosine) and tetra-functional (desmosine and isodesmosine) crosslinks [25]. The resulting cross-linked mature elastin is a very stable and persistent structure that is extremely hydrophobic and insoluble due to the extensive cross-linking at Lys residues [23]. The high stability of elastin has been attributed to the low content of polar amino acid residues, including the anionic Lys, histidine (His) and arginine (Arg), as well as the cationic aspartic acid (Asp) and glutamic acid (Glu) [7].

the chains adopt a relatively aligned conformation, limiting their conformational freedom and, thus, decreasing their entropy. Under relaxation, the chains resume their random conformation, increasing the number of different conformational states and, thus, their entropy. This change in the entropy of the cross-linked chains has been suggested to provide the free energy for the elastic recoil [6, 23, 32]. Along these lines, it has been suggested that intramolecular folding of fibrillin molecules at their termini and Pro-rich regions, and at flexible sites between 8-Cys motifs, provides the extension/recoil mechanism of microfibrils [25, 27]. Similarly, cross-linked mature elastin has been suggested to derive its high elasticity from the

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cross-linking of Lys residues into desmosine, isodesmosine, and lysinonorleucine [7].

with calcium and lipid accumulation in the elastic fibers [23, 46].

perinatally from congestive heart failure and valvular deficiency [38].

ies, and SVAS [23, 25], due to the excessive proliferation of SMCs [13, 24].

In contrast to collagens that can be encoded by large gene families, there is only one gene (ELN) that is responsible for synthesizing tropoelastin [40, 41]. Tropoelastin is mainly synthesized *in utero* and early childhood, whereas by middle-age only a small amount of this molecule is produced [23, 42]. Owing to this, elastin is the longest lasting protein in the body, with a half-life of approximately 74 years [23, 43]. However, in the event of elastin damage due to acquired disease or aging, the low levels of tropoelastin produced mean that elastic fibers cannot be repaired sufficiently. Along these lines, loss of elasticity, due to degradative changes in elastic fibers, is a major contributing factor in connective tissue aging, and aortic aneurysm development [23, 25, 44, 45]. Elastic fiber degradation has also been reported to occur in atherosclerosis, with atherosclerotic vessels presenting increased stiffness, together

In the case of genetic disorders, mutations in the genes of the components of the elastic fibers have been associated with congenital disorders in elastic fiber-rich connective tissues. Specifically, Marfan syndrome and other overlapping disorders, such as MASS (mitral valve prolapse, aortic dilation, and skin/skeletal manifestations) syndrome, and autosomal TAAD (thoracic aortic aneurysms and dissections) [47], termed fibrillinopathies [48], have been associated with fibrillin-1 mutations, leading to increased fragmentation of the elastic fibers that, in turn, lead to life-threatening cardiovascular disease and severe skeletal and ocular defects [13, 25, 38]. Progressive aortic valve root dilation, aortic dissection and rupture, and aortic or mitral valve regurgitation are the most serious conditions associated with Marfan syndrome, with a 90% mortality rate in these patients. Neonatal patients with Marfan syndrome die

Increased fragmentation of the elastic fibers has also been reported in cutis laxa and Menkes disease, the latter of which is associated with tortuous blood vessels and abnormalities in other tissues [23]. Moreover, the pseudoxanthoma elasticum and Buschke-Ollendorff syndromes have been associated with fragmentation, clumping and calcification of elastic fibers, leading to cardiovascular defects [23, 25]. Elastin mutations that cause elastin deficiency have been reported to cause reduced elastin content and disruption in the architecture of aorta and heart valves in Williams syndrome and supravalvular stenosis (SVAS) [23, 25, 29]. Elastin has been reported to induce actin stress fiber organization and inhibit SMC proliferation [24], indicating that apart from its structural role, elastin has a role in arterial morphogenesis [23]. Owing to this, reduction in the elastin content causes narrowing of the aorta and other arter-

The elastic recoil generated by the elastic fibers is a critical attribute for tissues that are required to undergo repetitive stretch/relaxation cycles. Tropoelastin is among the most elastic proteins, with a capacity to stretch eight times its resting molecular length (≈20 nm) and recoil without damage, while showing no hysteresis after repeated stretch and relaxation cycles, demonstrating a near perfect spring behavior with minimal energy loss [36, 37]. Atomic force microscopy studies have estimated the Young's modulus of tropoelastin to approximately 3 kPa [37], which is two order of magnitudes smaller (more flexible) than mature cross-linked elastin, estimated between 0.6 and 1.1 MPa [6, 35], four orders of magnitude smaller than microfibrils, estimated between 78 and 96 MPa [38], and six orders of magnitude smaller than mature cross-linked collagen I, estimated between 1 and 1.2 GPa [6, 35]. Given the fact that elastin and elastic fibers are robust structures that effectively last for the lifetime of the organism, studies have suggested that the elastic recoil generated in the elastic fibers cannot be a result of the stressing of the chemical bonds, since this would lead to the fiber deterioration [32]. Several studies have proposed that elastic recoil could be potentially attributed to the change in the number of conformation states of the cross-linked polypeptide chains during stretching and relaxation [6, 7, 13, 39]. Elaborating, under relaxed conditions the cross-linked polypeptide chains adopt a loose random coil conformation, which results in an increased number of different intramolecular conformational states in the chains. Under stretching, the chains adopt a relatively aligned conformation, limiting their conformational freedom and, thus, decreasing their entropy. Under relaxation, the chains resume their random conformation, increasing the number of different conformational states and, thus, their entropy. This change in the entropy of the cross-linked chains has been suggested to provide the free energy for the elastic recoil [6, 23, 32]. Along these lines, it has been suggested that intramolecular folding of fibrillin molecules at their termini and Pro-rich regions, and at flexible sites between 8-Cys motifs, provides the extension/recoil mechanism of microfibrils [25, 27]. Similarly, cross-linked mature elastin has been suggested to derive its high elasticity from the cross-linking of Lys residues into desmosine, isodesmosine, and lysinonorleucine [7].

of microfibrils are only partially intermixed with tropoelastin (elaunin fibers), and never developed into fully mature elastic fibers [26]. These microfibril states represent interruptions in successive phases of elastic fiber development, indicating that microfibrils can be both progenitors of fully mature elastic fibers in fetal tissues, and independent connective tissue components (oxytalan fibers) [26, 34]. Tropoelastin is the soluble precursor of elastin, which is synthesized by fibroblasts and smooth muscle cells (SMCs) and secreted as a soluble and highly hydrophobic monomer of about 750 amino acids long [13, 24, 35]. Similarly to tropocollagen, tropoelastin contains an abundance of Pro and Gly, but is not glycosylated and contains minute amounts of HYP and no Hyl [13]. Human tropoelastin contains more than 30% Gly, whereas about 75% of its amino acid sequence is made up of just four hydrophobic amino acids, including Gly, valine (Val), alanine (Ala) and Pro [23]. Structurally, tropoelastin comprises two types of alternating segments, including hydrophobic segments and Ala-/Lys-rich α-helical segments, which form cross-links between adjacent molecules [13]. During tropoelastin deposition among the microfibrils, the latter get displaced to the periphery of the growing fiber, allowing the formation of the elastin core by the accumulation of tropoelastin molecules [13, 31]. The accumulated tropoelastin molecules couple covalently to each other by lysyl oxidase-derived cross-links, to form the polymeric elastin, stabilizing the elastin core of the elastic fibers [25, 31]. Lysyl oxidase catalyzes the selective oxidative deamination of Lys residues in tropoelastin, leading to the formation of bi-functional (dehydrolysinonorleucine and allysine aldol), tri-functional (dehydromerodesmosine) and tetra-functional (desmosine and isodesmosine) crosslinks [25]. The resulting cross-linked mature elastin is a very stable and persistent structure that is extremely hydrophobic and insoluble due to the extensive cross-linking at Lys residues [23]. The high stability of elastin has been attributed to the low content of polar amino acid residues, including the anionic Lys, histidine (His) and arginine (Arg), as well as the cationic aspartic acid (Asp)

The elastic recoil generated by the elastic fibers is a critical attribute for tissues that are required to undergo repetitive stretch/relaxation cycles. Tropoelastin is among the most elastic proteins, with a capacity to stretch eight times its resting molecular length (≈20 nm) and recoil without damage, while showing no hysteresis after repeated stretch and relaxation cycles, demonstrating a near perfect spring behavior with minimal energy loss [36, 37]. Atomic force microscopy studies have estimated the Young's modulus of tropoelastin to approximately 3 kPa [37], which is two order of magnitudes smaller (more flexible) than mature cross-linked elastin, estimated between 0.6 and 1.1 MPa [6, 35], four orders of magnitude smaller than microfibrils, estimated between 78 and 96 MPa [38], and six orders of magnitude smaller than mature cross-linked collagen I, estimated between 1 and 1.2 GPa [6, 35]. Given the fact that elastin and elastic fibers are robust structures that effectively last for the lifetime of the organism, studies have suggested that the elastic recoil generated in the elastic fibers cannot be a result of the stressing of the chemical bonds, since this would lead to the fiber deterioration [32]. Several studies have proposed that elastic recoil could be potentially attributed to the change in the number of conformation states of the cross-linked polypeptide chains during stretching and relaxation [6, 7, 13, 39]. Elaborating, under relaxed conditions the cross-linked polypeptide chains adopt a loose random coil conformation, which results in an increased number of different intramolecular conformational states in the chains. Under stretching,

and glutamic acid (Glu) [7].

10 Structural Insufficiency Anomalies in Cardiac Valves

In contrast to collagens that can be encoded by large gene families, there is only one gene (ELN) that is responsible for synthesizing tropoelastin [40, 41]. Tropoelastin is mainly synthesized *in utero* and early childhood, whereas by middle-age only a small amount of this molecule is produced [23, 42]. Owing to this, elastin is the longest lasting protein in the body, with a half-life of approximately 74 years [23, 43]. However, in the event of elastin damage due to acquired disease or aging, the low levels of tropoelastin produced mean that elastic fibers cannot be repaired sufficiently. Along these lines, loss of elasticity, due to degradative changes in elastic fibers, is a major contributing factor in connective tissue aging, and aortic aneurysm development [23, 25, 44, 45]. Elastic fiber degradation has also been reported to occur in atherosclerosis, with atherosclerotic vessels presenting increased stiffness, together with calcium and lipid accumulation in the elastic fibers [23, 46].

In the case of genetic disorders, mutations in the genes of the components of the elastic fibers have been associated with congenital disorders in elastic fiber-rich connective tissues. Specifically, Marfan syndrome and other overlapping disorders, such as MASS (mitral valve prolapse, aortic dilation, and skin/skeletal manifestations) syndrome, and autosomal TAAD (thoracic aortic aneurysms and dissections) [47], termed fibrillinopathies [48], have been associated with fibrillin-1 mutations, leading to increased fragmentation of the elastic fibers that, in turn, lead to life-threatening cardiovascular disease and severe skeletal and ocular defects [13, 25, 38]. Progressive aortic valve root dilation, aortic dissection and rupture, and aortic or mitral valve regurgitation are the most serious conditions associated with Marfan syndrome, with a 90% mortality rate in these patients. Neonatal patients with Marfan syndrome die perinatally from congestive heart failure and valvular deficiency [38].

Increased fragmentation of the elastic fibers has also been reported in cutis laxa and Menkes disease, the latter of which is associated with tortuous blood vessels and abnormalities in other tissues [23]. Moreover, the pseudoxanthoma elasticum and Buschke-Ollendorff syndromes have been associated with fragmentation, clumping and calcification of elastic fibers, leading to cardiovascular defects [23, 25]. Elastin mutations that cause elastin deficiency have been reported to cause reduced elastin content and disruption in the architecture of aorta and heart valves in Williams syndrome and supravalvular stenosis (SVAS) [23, 25, 29]. Elastin has been reported to induce actin stress fiber organization and inhibit SMC proliferation [24], indicating that apart from its structural role, elastin has a role in arterial morphogenesis [23]. Owing to this, reduction in the elastin content causes narrowing of the aorta and other arteries, and SVAS [23, 25], due to the excessive proliferation of SMCs [13, 24].

#### **3. Glycosaminoglycans and proteoglycans**

GAGs (also termed mucopolysaccharides) are the most abundant unbranched heteropolysaccharides in the body, composed of repeating disaccharide units [1]. Disaccharides are formed by two simple sugars (monosaccharides; basic units of carbohydrates), which are joined by dehydration synthesis. Polysaccharide chains are large, stiff and complex molecules that exist in tissues as highly viscous materials that interact with proteins and readily bind both water and cations. The GAGs derive their name from the fact that one of the two sugars in their repeating disaccharide unit is always an amino sugar, either N-acetylglucosamine (N-acetyl-D-glucosamine; GlcNAc) or N-acetylgalactosamine (N-acetyl-D-galactosamine; GalNAc) [1, 7, 13]. The other of the two sugars is usually either an uronic acid (glucuronic acid or iduronic acid) or D-galactose [1, 4, 13], whereas one or both of the sugars contain at least one anionic group (carboxyl or sulfate) [4], rendering GAGs highly negatively charged [13]. Owing to the relatively high stiffness and hydrophilicity of the polysaccharide chains, GAGs form extended conformations with a large volume to mass ratio, forming hydrated gels even at very low concentrations, and imparting high viscosity and, thus, low compressibility, to the ECM. The gel-forming character of the GAGs is due to their high negative charge that attracts cations, especially Na<sup>+</sup> , which causes large osmotic inflow of water into the ECM, creating a swelling pressure that enables the ECM to withstand compressive loads [1, 13]. The GAG gels can form with varying pore size and charge density, and it has been suggested that they act as selective sieves to regulate molecule and cell traffic according to their size and charge [13]. Although GAGs constitute less than 10% of the weight of the fibrous proteins in the ECM of connective tissues, their high volume to weight ratio and gel-forming ability enables them to fill most of the extracellular space, providing mechanical support to the tissue [7, 13], as well as lubrication between tissue (joints) and elastic and collagen fibers [2, 6].

With the exception of hyaluronic acid, all GAGs are found covalently linked to a polypeptide chain, or core protein, forming proteoglycans that are synthesized by most cell types [4]. Proteoglycans is a diverse group of highly glycosylated glycoproteins, which are distinguishable from other glycoproteins by the nature, quantity, and arrangement of their sugar side chains [13]. Proteoglycans feature at least one GAG side chain, whereas their carbohydrate content can reach up to 95% of their weight [1, 13]. On the other hand, the carbohydrate content of glycoproteins is between 1 and 60% of their weight, an is present in the form of numerous relatively short, branched oligosaccharide chains [2, 13]. Proteoglycans have the potential of almost limitless heterogeneity [13], differentiating in the type of core protein, number and types of GAG chains, and number of disaccharide modifications by sulfate groups [1, 4, 13]. Some of the most common proteoglycans that have been characterized are listed in **Table 2**. Similarly to other proteins, the core proteins of proteoglycans are synthesized by ribosomes attached to the ER, and translocated into the lumen of the ER. Subsequently, the core protein is transported to the Golgi apparatus where it is glycosylated [1, 2]. During glycosylation, the GAGs are linked to their core proteins in one of three different modes. The first two modes involve the formation of O-glycosidic bonds between the hydroxyl side chains of serine (Ser) residues in the core protein and a xylose (Xyl) residue, which is unique to proteoglycans, or between Ser residues in the core protein and GalNAc residues in the GAG chain (keratan sulfate II). In the first case, two Gal residues are subsequently added to the Xyl residue sequentially, forming a link trisaccharide (-Gal-Gal-Xyl-) that serves as a polysaccharide growth primer, followed by further linear growth of the GAG on the Gal terminal of the link trisaccharide (**Figure 3**). The third mode involves the formation of N-glycosylamine bonds, as found in N-linked glycoproteins, between the amide nitrogen of asparagine (Asn) residues in the core protein and the GlcNAc residues of the GAG chains [4, 13]. Subsequently, the polysaccharide chains are often modified in the Golgi apparatus by the covalent linkage of

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**Table 2.** Prevalent localization of different GAG types and their associated proteoglycans.

Depending on the type of the repeating disaccharide unit, type of linkage between the sugars, and number and location of sulfate groups, GAGs are classified into four groups, including (i) hyaluronic acid, (ii) heparan sulfate and heparin, (iii) keratan sulfates (I and II), and (iv) chondroitin sulfates (chondroitin 4-sulfate and chondroitin 6-sulfate) and dermatan sulfate (**Table 2**) [1, 4, 13]. Hyaluronic acid is the simplest GAG and the only one that it does not contain any sulfate or attach covalently to proteins as a proteoglycan. However, it does form complexes with proteoglycans in the ECM, non-covalently [7]. All other GAGs contain sulfate groups, either as O-esters or as N-sulfate (in heparin and heparan sulfate) [1]. Heparin is a hyper-sulfated form of heparan sulfate that produced by mast cells and is an important anticoagulant that can activate the antithrombin III clotting inhibitor in plasma [1, 4]. It can also bind to the lipoprotein lipase, which is a membrane-associated enzyme present in capillary walls that hydrolyzes triglycerides to fatty acids, causing its release into the circulation [1, 4]. Hyaluronic acid is abundant in embryonic tissues and has been suggested to play an important role in facilitating cell migration during morphogenesis and wound repair, whereas high concentrations of hyaluronic acid, as well as chondroitin sulfates, in tissues contribute to their compressibility. Keratan sulfates and dermatan sulfate lie between collagen fibrils and play a critical role in their alignment. Heparan sulfate is associated with cell membranes and has been suggested to act as a receptor mediating in cell growth and cell-cell and cell-ECM communication [1]. In adult tissues, GAGs have a slow turnover, with half-lives between days and weeks [1].

With the exception of hyaluronic acid, all GAGs are found covalently linked to a polypeptide chain, or core protein, forming proteoglycans that are synthesized by most cell types [4]. Proteoglycans is a diverse group of highly glycosylated glycoproteins, which are distinguishable from other glycoproteins by the nature, quantity, and arrangement of their sugar side chains [13]. Proteoglycans feature at least one GAG side chain, whereas their carbohydrate content can reach up to 95% of their weight [1, 13]. On the other hand, the carbohydrate content of glycoproteins is between 1 and 60% of their weight, an is present in the form of numerous relatively short, branched oligosaccharide chains [2, 13]. Proteoglycans have the potential of almost limitless heterogeneity [13], differentiating in the type of core protein, number and types of GAG chains, and number of disaccharide modifications by sulfate groups [1, 4, 13]. Some of the most common proteoglycans that have been characterized are listed in **Table 2**. Similarly to other proteins, the core proteins of proteoglycans are synthesized by ribosomes attached to the ER, and translocated into the lumen of the ER. Subsequently, the core protein is transported to the Golgi apparatus where it is glycosylated [1, 2]. During glycosylation, the GAGs are linked to their core proteins in one of three different modes. The first two modes involve the formation of O-glycosidic bonds between the hydroxyl side chains of serine (Ser) residues in the core protein and a xylose (Xyl) residue, which is unique to proteoglycans, or between Ser residues in the core protein and GalNAc residues in the GAG chain (keratan sulfate II). In the first case, two Gal residues are subsequently added to the Xyl residue sequentially, forming a link trisaccharide (-Gal-Gal-Xyl-) that serves as a polysaccharide growth primer, followed by further linear growth of the GAG on the Gal terminal of the link trisaccharide (**Figure 3**). The third mode involves the formation of N-glycosylamine bonds, as found in N-linked glycoproteins, between the amide nitrogen of asparagine (Asn) residues in the core protein and the GlcNAc residues of the GAG chains [4, 13]. Subsequently, the polysaccharide chains are often modified in the Golgi apparatus by the covalent linkage of

**3. Glycosaminoglycans and proteoglycans**

12 Structural Insufficiency Anomalies in Cardiac Valves

attracts cations, especially Na<sup>+</sup>

GAGs (also termed mucopolysaccharides) are the most abundant unbranched heteropolysaccharides in the body, composed of repeating disaccharide units [1]. Disaccharides are formed by two simple sugars (monosaccharides; basic units of carbohydrates), which are joined by dehydration synthesis. Polysaccharide chains are large, stiff and complex molecules that exist in tissues as highly viscous materials that interact with proteins and readily bind both water and cations. The GAGs derive their name from the fact that one of the two sugars in their repeating disaccharide unit is always an amino sugar, either N-acetylglucosamine (N-acetyl-D-glucosamine; GlcNAc) or N-acetylgalactosamine (N-acetyl-D-galactosamine; GalNAc) [1, 7, 13]. The other of the two sugars is usually either an uronic acid (glucuronic acid or iduronic acid) or D-galactose [1, 4, 13], whereas one or both of the sugars contain at least one anionic group (carboxyl or sulfate) [4], rendering GAGs highly negatively charged [13]. Owing to the relatively high stiffness and hydrophilicity of the polysaccharide chains, GAGs form extended conformations with a large volume to mass ratio, forming hydrated gels even at very low concentrations, and imparting high viscosity and, thus, low compressibility, to the ECM. The gel-forming character of the GAGs is due to their high negative charge that

creating a swelling pressure that enables the ECM to withstand compressive loads [1, 13]. The GAG gels can form with varying pore size and charge density, and it has been suggested that they act as selective sieves to regulate molecule and cell traffic according to their size and charge [13]. Although GAGs constitute less than 10% of the weight of the fibrous proteins in the ECM of connective tissues, their high volume to weight ratio and gel-forming ability enables them to fill most of the extracellular space, providing mechanical support to the tissue [7, 13], as well as lubrication between tissue (joints) and elastic and collagen fibers [2, 6].

Depending on the type of the repeating disaccharide unit, type of linkage between the sugars, and number and location of sulfate groups, GAGs are classified into four groups, including (i) hyaluronic acid, (ii) heparan sulfate and heparin, (iii) keratan sulfates (I and II), and (iv) chondroitin sulfates (chondroitin 4-sulfate and chondroitin 6-sulfate) and dermatan sulfate (**Table 2**) [1, 4, 13]. Hyaluronic acid is the simplest GAG and the only one that it does not contain any sulfate or attach covalently to proteins as a proteoglycan. However, it does form complexes with proteoglycans in the ECM, non-covalently [7]. All other GAGs contain sulfate groups, either as O-esters or as N-sulfate (in heparin and heparan sulfate) [1]. Heparin is a hyper-sulfated form of heparan sulfate that produced by mast cells and is an important anticoagulant that can activate the antithrombin III clotting inhibitor in plasma [1, 4]. It can also bind to the lipoprotein lipase, which is a membrane-associated enzyme present in capillary walls that hydrolyzes triglycerides to fatty acids, causing its release into the circulation [1, 4]. Hyaluronic acid is abundant in embryonic tissues and has been suggested to play an important role in facilitating cell migration during morphogenesis and wound repair, whereas high concentrations of hyaluronic acid, as well as chondroitin sulfates, in tissues contribute to their compressibility. Keratan sulfates and dermatan sulfate lie between collagen fibrils and play a critical role in their alignment. Heparan sulfate is associated with cell membranes and has been suggested to act as a receptor mediating in cell growth and cell-cell and cell-ECM communication [1]. In adult tissues, GAGs

have a slow turnover, with half-lives between days and weeks [1].

, which causes large osmotic inflow of water into the ECM,


**Table 2.** Prevalent localization of different GAG types and their associated proteoglycans.

which are enzymes involved in GAG degradation [56, 57, 62]. Absence or malfunctioning of lysosomal hydrolases lead to GAG accumulation in various tissues, including liver, spleen, bone, skin, and central nervous system [1, 60, 61]. Moreover, severe congenital deficiency in dermatan sulfate synthesis leads to a short stature, prematurely aged appearance, and generalized defects in the skin, joints, muscles, and bones of individuals [13]. GAGs and proteoglycans have also been associated with major diseases, such as cancer and atherosclerosis, and aging. The intima of arterial walls contains, among others, dermatan sulfate proteoglycans, which are synthesized by arterial SMCs and bind plasma low-density lipoproteins that are involved in atherosclerotic plaque development. Since atherosclerotic lesions feature increased proliferation of SMCs, the dermatan sulfate content is increased in these sites, suggesting a potential role of this GAG in atherosclerotic plaque development [63]. Moreover, the amount of chondroitin sulfate diminishes with age, whereas the amounts of keratan sulfate and hyaluronic acid increase. These changes may contribute to conditions, such as osteoarthritis, and other degenerative diseases, as well as the characteristic changes in aged skin in the elderly [1, 2].

Structure-Function Relationship of Heart Valves in Health and Disease

http://dx.doi.org/10.5772/intechopen.78280

15

Depending on the functional requirements for a particular tissue, the organization of fibers, cells and other ECM macromolecules in the tissue and, thus, the mechanical properties of the tissue vary. The simplest structure from the point of view of the collagen fibers consists of parallel fibers as in tendons and ligaments. The 2D and 3D networks of the skin are more complex, whereas the structures of aortic, pulmonary, mitral and tricuspid valves in the heart are the most complex ones. The structure of the heart valves is adapted to allow unidirectional and unobstructed passage of blood without regurgitation, trauma to blood elements, thromboembolism, and excessive stress concentrations in the valve leaflets and supporting tissue. The cellular and extracellular elements of normal valves accommodate repetitive changes in shape and dimension throughout the cardiac cycle. They provide effective stress transfer to the annulus and adjacent tissue, and mediate functional remodeling and repair of injury caused by the large repetitive deformations. The aortic and pulmonary valves comprise three similar-size leaflets, and are located between the left ventricle and aorta, and the right ventricle and pulmonary artery, respectively. Their leaflets resemble half-moons and, thus, they are referred to as semilunar (SL) valves. The mitral valve (MV) is located between the left ventricle and left atrium, and comprises two noticeably different leaflets that resemble a bishop's miter when they are closed. The tricuspid valve is located between the right ventricle and right atrium, and comprises three leaflets. Owing to their location between the atria and the ventricles, the mitral and tricuspid valves are referred to as the atrioventricular (AV) valves. In addition to their leaflets, the AV valves also comprise fan-shaped tendinous chord (chordae tendinae) that link the AV valve leaflets to protrusions on the ventricular wall, the papillary muscles, and act similarly to the parachute chords, preventing

**4. Structure and constituents of normal heart valves**

the AV valve leaflets to prolapse into the atria when they are fully closed.

The aortic and pulmonary valve leaflets open against the aorta and pulmonary artery, respectively, during ventricular systole, whereas the mitral and tricuspid valve leaflets open against the myocardial wall of the right and left ventricle, respectively, during ventricular diastole. The semilunar valves close during ventricular diastole and the atrioventricular valves

**Figure 3.** Biosynthesis of proteoglycans, showing the case of the linking of chondroitin sulfate chains to their core protein. Ser: serine residue; Gal: galactose residue; GlcUA: glucuronic acid residue; Xyl: xylose residue. Modified from Alberts et al. (2014) [13] and Lodish et al. (2003) [4].

small molecules, such as sulfate groups onto GalNAc and other moieties and the epimerization of GlcUA to IdUA residues. Following glycosylation, the completed proteoglycan is then exported in secretory vesicles to the ECM [1, 4, 13].

In the extracellular space, proteoglycans bind various secreted signal molecules, such as growth factors, enhancing or inhibiting their signaling activity [13]. They also bind to other types of secreted proteins, including proteases and protease inhibitors, regulating their activities [1, 4]. Moreover, GAGs and proteoglycans can associate to each other to form big aggregates. Such a case is the aggrecan proteoglycans, which comprise chondroitin sulfate and keratan sulfate, and assemble with hyaluronic acid. GAGs and proteoglycans also associate with fibrous proteins, such as collagen and basal laminae, creating extremely complex structures. Such a proteoglycan is decorin, which comprises chondroitin sulfate and dermatan sulfate, and binds to collagen fibrils, aiding in collagen fiber formation. Decorin deficiency has been reported to lead to reduced tensile strength in tissues [13, 49–52]. Another proteoglycan of this type is perlecan, which is major proteoglycan of basal laminae, consisting of heparan sulfate and chondroitin sulfate chains linked to a large multi-domain core protein. Perlecan is incorporated in the basal laminae by binding to both laminin and collagen IV, connecting the two networks, and defining the structure and function of basal laminae. Owing to its multi-domain components, perlecan can also crosslink cell surface molecules to ECM components [4]. In addition to proteoglycans that fully reside in the extracellular space, some proteoglycans form integral components of the cell membranes by having their core protein spanning or attached to the lipid bilayer of the cell membrane. Such proteoglycans are the syndecans, which comprise three chondroitin sulfate and heparan sulfate chains and are expressed by many cell types, including epithelial cells and fibroblasts [13, 53–55]. Syndecans bind to ECM collagens and other ECM proteins such as the fibronectins, anchoring cells to the ECM, while interacting with the intracellular actin cytoskeleton [4].

The importance of GAGs and proteoglycans is emphasized by the severe developmental defects that can occur when specific proteoglycans are inactivated by mutation. A number of congenital enzyme deficiencies have been linked to GAG metabolic disorders (mucopolysaccharidoses) [13]. Among mucopolysaccharidoses, the Hurler [56–59] and Hunter [60–62] syndromes are the most widely studied. Mucopolysaccharidoses are associated with mutations in the gene encoding lysosomal hydrolases, including endoglycosidases and exoglycosidases, which are enzymes involved in GAG degradation [56, 57, 62]. Absence or malfunctioning of lysosomal hydrolases lead to GAG accumulation in various tissues, including liver, spleen, bone, skin, and central nervous system [1, 60, 61]. Moreover, severe congenital deficiency in dermatan sulfate synthesis leads to a short stature, prematurely aged appearance, and generalized defects in the skin, joints, muscles, and bones of individuals [13]. GAGs and proteoglycans have also been associated with major diseases, such as cancer and atherosclerosis, and aging. The intima of arterial walls contains, among others, dermatan sulfate proteoglycans, which are synthesized by arterial SMCs and bind plasma low-density lipoproteins that are involved in atherosclerotic plaque development. Since atherosclerotic lesions feature increased proliferation of SMCs, the dermatan sulfate content is increased in these sites, suggesting a potential role of this GAG in atherosclerotic plaque development [63]. Moreover, the amount of chondroitin sulfate diminishes with age, whereas the amounts of keratan sulfate and hyaluronic acid increase. These changes may contribute to conditions, such as osteoarthritis, and other degenerative diseases, as well as the characteristic changes in aged skin in the elderly [1, 2].
