**6. Minor ECM components in heart valves also play significant roles in normal valve function and in pathological states**

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

Vitronectin is a glycoprotein that is approximately 75 kDa in size and is present in both se‐ rum and the ECM as an adhesive substrate [68]. It is involved in the inhibition of the com‐ plement system [68] and is associated with the regulation of hemostasis [69]. Vitronectin also promotes cellular attachment to ECM and is involved in cellular migration [68]. This glycoprotein, along with fibronectin, is found in moderate amounts in aortic, pulmonary, and mitral valves, localizing around valve endothelial cells (VEC) on the inflow layer [25]. In addition, both fibronectin and vitronectin have been shown to associate with collagen fibers in chordae tendinae [70].

Fibronectin is a dimer glycoprotein which consists of two ~250 kDa subunits and is a com‐ ponent of the extracellular matrix [71]. There are many various isoforms of fibronectin, which is the result of alternative mRNA splicing [71]. In addition to being an insoluble ECM component secreted primarily by fibroblasts, soluble fibronectin is also found in the plasma [71]. Fibronectin acts by binding to integrins, collagens, fibrin, and heparin sulfate proteo‐ glycans [71], which allows it to participate in wound healing [72,73] and act as a critical player in embryogenesis [74]. Although not a major ECM component in heart valves, valve interstitial cells (VIC) secrete fibronectin in response to valve damage, providing a means for cell migration [75].

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

Osteopontin is a phosphoprotein, meaning that it contains chemically bound phosphoric acid. Originally found in bone, it also contains the arginine-glycine-aspartate (RGD) motif more commonly attributed to fibronectin and is also a constituent of ECM in other tissues [82]. It is secreted by various tissues such as fibroblasts [82] and immune cells, including dendritic cells, macrophages, and neutrophils [83]. Osteopontin is known to interact with various surface receptors that make it a crucial player in bone remodeling [84], wound heal‐ ing, inflammation, and immune responses [83]. It is also known to be involved in vascular remodeling during endothelial injury [82]. Osteopontin is present in valves calcified as a re‐ sult of disease as well as in calcified bioprosthetic heart valves [85]. The calcification process of aortic valves closely resembles osteoblast differentiation in regards to expression of genes characteristic of bone formation, such as osteopontin and osteocalcin [86].

tion of the basement membrane. In addition, MMPs contribute to the biological activity that occurs within the basement membrane. Understanding basement membrane composition and behavior, during both healthy and diseased states of the aortic valve, may lead to a bet‐

Extracellular Matrix Organization, Structure, and Function

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

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Laminins belong to a family of heterotrimeric glycoproteins composed of combinations of α, β, and γ chains that form a cross-like structure averaging between 400-900 kDa in size [94,95]. Laminins play an integral role in the formation of the supportive ECM network. The unique cross-like shape allows laminin molecules to bind with neighboring laminins and ECM via the three short chains, and use the long alpha chain as a cell anchoring site [96]. In addition to their structural contributions to the basement membrane, laminins are essential for proper biological activity. These glycoproteins have been shown to promote cell adhe‐ sion, migration, differentiation, and maintenance of cellular phenotype [94,97,98]. Dysfunc‐ tion in laminin expression has been linked to diseases with improper tissue formation such as muscular dystrophy, epidermolysis bullosa, and various nephritic syndromes [94,99].

Although laminin is not as ubiquitous as collagen, this basement membrane component has been highly investigated as an ECM substrate for *in vitro* cultures. However, this glycopro‐ tein may influence valve cell types differently. *In vivo* and *in vitro* studies have shown that laminin interacts with endothelial and epithelial cells, and can help maintain physiological functionality of the cells [97,100,101]. However, VICs cultured on laminin have been found to support high quantities of calcific nodule formation in the presence of TGF-β, when com‐ pared to subendothelial ECM components collagen type I and fibronectin [19,102]. The vari‐ ous regions of laminin protein have been reported to mediate specific cell responses. The Gdomains of laminin α chains are associated with heparin binding and cell adhesion, whereas regions along the laminin β chains promote cell differentiation [98,100,101]. The peptide se‐ quence YIGSR from the laminin β-1 chain has been shown to promote endothelial cell adhe‐ sion and proliferation, however, it also influences other cell types including smooth muscle and tumor cells [98,101]. VICs cultured on YIGSR were also shown to promote calcific nod‐ ule formation, although less than those seeded on fibronectin derived RGDS peptides. How‐ ever, when the 67-kDa laminin cell receptor was blocked, the YIGSR seeded VIC cultures significantly increased in nodule formation and gene expression for various myogenic and osteogenic markers, suggesting that disruption in laminin binding may be linked to valve calcification [103]. IKVAV, another peptide sequence derived from the laminin α1-chain, has been linked to promoting angiogenesis, cell migration and spreading [97,98,104]. Though most work with this peptide has been done with endothelial and tumor cells, its ability to promote angiogenesis may also be a future area of interest in studying how angiogenesis mediates valve tissue calcification. Furthermore, laminin influence on cell activity varies be‐ tween cell types, and may promote VIC activation and tissue calcification in diseased states.

Perlecan (Pln) is one of the more abundant heparan sulfate proteoglycans and is found in several tissues including in the endochondral barrier in bones [105]; however, it is primarily localized in vascular basement membranes. It has a major role in regulating the develop‐ ment of blood vessels, the heart, cartilage, and the nervous system. Physiologically, perlecan plays a prominent role in regulating cellular proliferation, differentiation, organization, and

ter understanding of calcific aortic valve disease.

Osteocalcin is a small, non-collagenous protein that is considered a late-stage marker for bone formation and is one of a small group of proteins that are osteoblast-specific [87,88]. It is present in general circulation [87] and its capacity for binding hydroxyapatite and calcium suggests that it is largely involved in mineral deposition [88], but it also has recently been shown to act in a hormone-like manner by enhancing insulin secretion [87]. Its traditional role as a product of bone indicates that valve calcification may actually be a result of active bone formation in the valve tissue [86]. This bone formation may be the result of VEGF se‐ cretion by endothelial cells during neoangiogenesis occurring in response to inflammation, as seen in rheumatic valve calcification [89]. Additionally, increased serum levels of osteo‐ calcin were shown to be indicative of aortic valve disease in patients [90].

In addition to the matrix proteins, matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are also found in heart valves. They assist in tissue development and remodeling and can be used as indicators of disease. It is also believed that the ECM degradation result‐ ing from MMP activity serves to release growth factors bound to ECM components and thus alter the microenvironment chemically, as well as structurally [91]. Calcified leaflets from stenotic valves have been shown to express levels of MMP-2 that are similar to those of nor‐ mal valves but express higher levels of MMP-3, MMP-9, and TIMP-1 [14]. MMP-1, produced by activated myofibroblasts and macrophages, is also prevalent in calcific aortic valve steno‐ sis and may be related to high TNF-α levels resulting from inflammation [92].
