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

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. These microfibrils are predominantly fibrillin-1, but to a lesser extent Fibrillin-2. Microfibril associated glycoproteins (MAGPs), fibulins, and other proteins are also present in the micro‐ fibrillar sheath [38]. At the light microscope level, one can observe the fine elastic fibers by histological staining with Voerhoff's stain or related methods, but when tissue sections are viewed with transmission electron microscopy, there is a clear distinction between the elec‐ tron-dense elastin core and the microfibrillar sheath [39].

arterial wall, a process that involves the re-folding of the corrugations in of the fibrosa. This action restores the original shape and orientation of collagen quickly and consistently to pre‐ pare for the next cycle of valve closing. Although the elastic sheet in the ventricularis has fibers that are also oriented circumferentially as well as radially, elastin does not appear to play an important role in the mechanical behavior of the leaflet in the circumferential direc‐ tion. Valve leaflets exposed to cyclic circumferential stretch and cultured under flow for 48 hours maintained a constant concentration of elastic, suggesting that elastogenesis was not activated during the duration of stretch [45]. However, it is speculated that connections be‐ tween the elastic fiber and collagen networks facilitate the radial extensibility of the ventri‐ cularis layer and the overall leaflet [43]. There are also some elastic fibers in the fibrosa, which surround and connect the collagen fibers, thus preserving collagen crimp and the

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11

The elastic fiber structure in the spongiosa has been characterized much less than in the ven‐ tricularis, partly due to the difficulty in isolating its structure from the rest of the leaflet [30]. This elastic structure, however, has been observed during microdissection separating the leaflet [30,48], with scanning electron microscopy (SEM) [6,46], micro-computed tomogra‐ phy (micro-CT) [6], immunohistochemistry (IHC) [25], and autofluorescence imaging [49,50], which all have shown a fine elastic fiber network emanating from the ventricularis and connecting to the fibrosa. We have recently reported that the thickness of this elastic fi‐ ber network in the spongiosa is significantly thicker in the hinge and coaptation region than in the belly region of the aortic valve leaflet [8]. We also found two distinct patterns of spon‐ giosa elastic fibers within the leaflet: (i) a rectilinear pattern in the hinge and coaptation re‐ gion; and (ii) a radially oriented stripe pattern in the belly. Overall, it is believed that the elastic fibers in the spongiosa contribute to valve function in three ways. First, they connect the elastic fibers in the ventricularis to collagen in the fibrosa, which allows coupling of the mechanics of the two layers and matrix components, while using elastic recoil to exert pre‐ load on the fibrosa. Second, they distribute stress between collagen and elastic fibers, partic‐ ularly at low strains. Third, they passively allow relative movement and shear between the

Given the presence of a thick, rectilinearly-arranged structure of elastic fibers in the spongio‐ sa of the hinge and coaptation regions, it is speculated that this elastin structure plays a role in leaflet flexure [5,30]. Flexure of the leaflet towards the outflow direction compresses the fibrosa and applies tension to the ventricularis. Rather than undergoing compression, how‐ ever, the fibrosa may attempt to buckle separately from the leaflet, thereby exaggerating its corrugated configuration. The leaflet would subsequently bend at the troughs of this corru‐ gation, where the second moment of inertia would be locally reduced, albeit temporarily. Buckling would only occur with shearing between the fibrosa and ventricularis, which is al‐ lowed by both the compliant elastic fibers in the spongiosa connecting the two outer layers as well as by GAGs in the spongiosa lubricating the outer layer movement [5,30,51,52]. Re‐ coil from the elastic fibers in the spongiosa would then return the fibrosa to its original con‐ figuration so it could undergo the next cycle of loading [5,30]. At the hinge, where bending occurs in the opposite direction, it is speculated that the elastic fiber-rich ventricularis com‐

characteristic corrugated nature of the fibrosa [6,46,47].

outer layers [5,6,48].

The unique mechanical behavior of the elastic fiber is conferred primarily by the mechan‐ ical function of elastin and fibrillin. Crosslinked elastin is remarkable for its ability to un‐ dergo high amounts of deformation when subjected to small amounts of load, as well as to recoil back to its original dimensions, when the load is removed, with very little loss of energy. Fibrillin-1, the most widely studied of the microfibrillar components, is also high‐ ly extensible. Fibrillin and the other microfibrillar components also coordinate, in a com‐ plicated manner still under investigation [38], to aid in the cross-linking of tropoelastin and assemble the final elastic fiber. Interestingly, fibrillin is not always associated with elastic fibers. Fibrillin can often be found by itself, in which it may independently func‐ tion as a mechanical, load-bearing but highly extensible scaffold [40]. Numerous domains in fibrillin exist for binding integrins, heparan sulafate proteoglycans, and growth factors, which point to substantial roles for alone fibrillin and mature elastic fibers in mediating cell signaling and adhesion [41].

In semilunar heart valves, elastin is found primarily within the ventricularis layer on the in‐ flow side of the leaflet, but is also abundant in the middle spongiosa layer. A thin, frequent‐ ly imperceptible layer of elastic fibers, the aterialis, is found atop the collagen-rich fibrosa layer. These elastic fibers merge with the intima of the adjacent arterial well, but the overall function of the arterialis has not been well characterized [42].

In the ventricularis, elastic fibers are present in dense and continuous sheets across the whole of the leaflet. These fiber sheets are the most significant contributor to the mechanical properties of the ventricularis [6,30], which can be demonstrated when all ECM components but elastin are removed when using NaOH digestion. After this treatment, the digested ven‐ tricularis matches the mechanical behavior of the undigested ventricularis radially, indicat‐ ing a strong presence of elastin in the radial direction [30]. The elastic fibers within the ventricularis undergo considerable, continual stretch from the initial stage of closure, when blood flow vortices are starting to push the leaflets towards the valve orifice, to the final coapted position of the leaflets. The extension of these elastic fibers accommodates the un‐ folding of the fibrosa layer, which is normally corrugated in the unloaded position. During this unfolding process, the elastic fibers are bearing the loading of the entire leaflet [43]. Even at high strains, when the collagen in the fibrosa is considered to dominate mechanical properties, the elastin in the ventricularis still plays a significant role. This effect was shown when separated ventricularis was preloaded to mimic its intact configuration; the separated ventricularis was shown to bear load before the separated fibrosa [44]. It has been speculat‐ ed that this response acts as a safety mechanism to prevent radial overextension of the aortic valve leaflet. Then, when the pressure across the valve is reduced, the elastic fiber sheet in the ventricularis recoils and retracts the leaflets back toward the annular attachment to the arterial wall, a process that involves the re-folding of the corrugations in of the fibrosa. This action restores the original shape and orientation of collagen quickly and consistently to pre‐ pare for the next cycle of valve closing. Although the elastic sheet in the ventricularis has fibers that are also oriented circumferentially as well as radially, elastin does not appear to play an important role in the mechanical behavior of the leaflet in the circumferential direc‐ tion. Valve leaflets exposed to cyclic circumferential stretch and cultured under flow for 48 hours maintained a constant concentration of elastic, suggesting that elastogenesis was not activated during the duration of stretch [45]. However, it is speculated that connections be‐ tween the elastic fiber and collagen networks facilitate the radial extensibility of the ventri‐ cularis layer and the overall leaflet [43]. There are also some elastic fibers in the fibrosa, which surround and connect the collagen fibers, thus preserving collagen crimp and the characteristic corrugated nature of the fibrosa [6,46,47].

These microfibrils are predominantly fibrillin-1, but to a lesser extent Fibrillin-2. Microfibril associated glycoproteins (MAGPs), fibulins, and other proteins are also present in the micro‐ fibrillar sheath [38]. At the light microscope level, one can observe the fine elastic fibers by histological staining with Voerhoff's stain or related methods, but when tissue sections are viewed with transmission electron microscopy, there is a clear distinction between the elec‐

The unique mechanical behavior of the elastic fiber is conferred primarily by the mechan‐ ical function of elastin and fibrillin. Crosslinked elastin is remarkable for its ability to un‐ dergo high amounts of deformation when subjected to small amounts of load, as well as to recoil back to its original dimensions, when the load is removed, with very little loss of energy. Fibrillin-1, the most widely studied of the microfibrillar components, is also high‐ ly extensible. Fibrillin and the other microfibrillar components also coordinate, in a com‐ plicated manner still under investigation [38], to aid in the cross-linking of tropoelastin and assemble the final elastic fiber. Interestingly, fibrillin is not always associated with elastic fibers. Fibrillin can often be found by itself, in which it may independently func‐ tion as a mechanical, load-bearing but highly extensible scaffold [40]. Numerous domains in fibrillin exist for binding integrins, heparan sulafate proteoglycans, and growth factors, which point to substantial roles for alone fibrillin and mature elastic fibers in mediating

In semilunar heart valves, elastin is found primarily within the ventricularis layer on the in‐ flow side of the leaflet, but is also abundant in the middle spongiosa layer. A thin, frequent‐ ly imperceptible layer of elastic fibers, the aterialis, is found atop the collagen-rich fibrosa layer. These elastic fibers merge with the intima of the adjacent arterial well, but the overall

In the ventricularis, elastic fibers are present in dense and continuous sheets across the whole of the leaflet. These fiber sheets are the most significant contributor to the mechanical properties of the ventricularis [6,30], which can be demonstrated when all ECM components but elastin are removed when using NaOH digestion. After this treatment, the digested ven‐ tricularis matches the mechanical behavior of the undigested ventricularis radially, indicat‐ ing a strong presence of elastin in the radial direction [30]. The elastic fibers within the ventricularis undergo considerable, continual stretch from the initial stage of closure, when blood flow vortices are starting to push the leaflets towards the valve orifice, to the final coapted position of the leaflets. The extension of these elastic fibers accommodates the un‐ folding of the fibrosa layer, which is normally corrugated in the unloaded position. During this unfolding process, the elastic fibers are bearing the loading of the entire leaflet [43]. Even at high strains, when the collagen in the fibrosa is considered to dominate mechanical properties, the elastin in the ventricularis still plays a significant role. This effect was shown when separated ventricularis was preloaded to mimic its intact configuration; the separated ventricularis was shown to bear load before the separated fibrosa [44]. It has been speculat‐ ed that this response acts as a safety mechanism to prevent radial overextension of the aortic valve leaflet. Then, when the pressure across the valve is reduced, the elastic fiber sheet in the ventricularis recoils and retracts the leaflets back toward the annular attachment to the

tron-dense elastin core and the microfibrillar sheath [39].

function of the arterialis has not been well characterized [42].

cell signaling and adhesion [41].

10 Calcific Aortic Valve Disease

The elastic fiber structure in the spongiosa has been characterized much less than in the ven‐ tricularis, partly due to the difficulty in isolating its structure from the rest of the leaflet [30]. This elastic structure, however, has been observed during microdissection separating the leaflet [30,48], with scanning electron microscopy (SEM) [6,46], micro-computed tomogra‐ phy (micro-CT) [6], immunohistochemistry (IHC) [25], and autofluorescence imaging [49,50], which all have shown a fine elastic fiber network emanating from the ventricularis and connecting to the fibrosa. We have recently reported that the thickness of this elastic fi‐ ber network in the spongiosa is significantly thicker in the hinge and coaptation region than in the belly region of the aortic valve leaflet [8]. We also found two distinct patterns of spon‐ giosa elastic fibers within the leaflet: (i) a rectilinear pattern in the hinge and coaptation re‐ gion; and (ii) a radially oriented stripe pattern in the belly. Overall, it is believed that the elastic fibers in the spongiosa contribute to valve function in three ways. First, they connect the elastic fibers in the ventricularis to collagen in the fibrosa, which allows coupling of the mechanics of the two layers and matrix components, while using elastic recoil to exert pre‐ load on the fibrosa. Second, they distribute stress between collagen and elastic fibers, partic‐ ularly at low strains. Third, they passively allow relative movement and shear between the outer layers [5,6,48].

Given the presence of a thick, rectilinearly-arranged structure of elastic fibers in the spongio‐ sa of the hinge and coaptation regions, it is speculated that this elastin structure plays a role in leaflet flexure [5,30]. Flexure of the leaflet towards the outflow direction compresses the fibrosa and applies tension to the ventricularis. Rather than undergoing compression, how‐ ever, the fibrosa may attempt to buckle separately from the leaflet, thereby exaggerating its corrugated configuration. The leaflet would subsequently bend at the troughs of this corru‐ gation, where the second moment of inertia would be locally reduced, albeit temporarily. Buckling would only occur with shearing between the fibrosa and ventricularis, which is al‐ lowed by both the compliant elastic fibers in the spongiosa connecting the two outer layers as well as by GAGs in the spongiosa lubricating the outer layer movement [5,30,51,52]. Re‐ coil from the elastic fibers in the spongiosa would then return the fibrosa to its original con‐ figuration so it could undergo the next cycle of loading [5,30]. At the hinge, where bending occurs in the opposite direction, it is speculated that the elastic fiber-rich ventricularis com‐ presses readily without buckling, most likely due to the tensile preload already exerted on the ventricularis, but that leaflet deflection may be limited by the stiff fibrosa, which would not allow the leaflet to bend [4,53]. Limited flexure at the hinge would allow the leaflet to absorb pressure from reverse blood flow in diastole, but prevents distention of the leaflet. Thus, our finding of a thicker spongiosa and elastic fiber structure in flexural regions pro‐ vides evidence of a significant role for elastin in flexure [8]. In addition, the thick network of elastic fibers that we have observed in the spongiosa of the coaptation region may play a role in dampening vibrations that result from valve closing [5].

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

**11** Table 2 center (only) the column with the heading Galactose, all of the other columns remain with left


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During PG synthesis, a protein core moves from the endoplasmic reticulum of a cell to the Golgi apparatus, where GAGs are then added to the

protein core [55].

2

**Glycosaminoglycan Uronic acid Galactose Hexosamine**

**11** 2 exist in vivo as PGs. exist in vivo as components of PGs.

Iduronic

**9** 5 direction; valve direction. Valve

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

Iduronic

include the word "core" on "protein core"

Iduronic

**Table 2.** List of glycosaminoglycans and their composition [59]

the same size, that would be great.

also, please use the revised figure

Heparin Glucuronic

alignment

Heparan sulfate Glucuronic

Dermatan sulfate Glucuronic

**<sup>11</sup>**4 The protein core is translated to the lumen of

**Figure 2.** Proteoglycan structure

the Golgi apparatus of the cells, where the GAGs are then added to the protein core [55].

**11** 7 hyalectins hyalectans

**12** 1 hyalectin hyalectan

**12** 3 hyalectin hyalectan

**13** 17 Fibronectin fibronectin

**15** 10 epidermitlysis epidermolysis

**15** 34 heparin heparan

**15** 39 from its five from five

**15** 41 enteokinase Enteokinase

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

**16** 9 immunoglobulin like immunoglobulin-like

**12** 17 guarantee the normal guarantee normal

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

Hyaluronan Glucuronic - N-acetylglucosamine

Chondroitin sulfate Glucuronic - N-acetylgalactosamine

**11** Figure 2 move the word "Glycosaminoglycan" down so it does not overlap with the lower arrow

Keratan sulfate - + N-acetylglucosamine

The sizing on the previous figure (Figure 2 in the proof) was very good; if you could make this

Protein core

Glycosaminoglycan

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