Elastic Fibers and Biomechanics of the Aorta: Insights from Mouse Studies

Review

MATBIO-1552; No. of pages: 13; 4C:

Elastic fibers and biomechanics of the aorta: Insights from mouse studies

Hiromi Yanagisawa a and Jessica Wagenseil b a - Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba 305-8577, Japan b - Department of Mechanical Engineering and Materials Science, Washington University at St. Louis, St. Louis, MO, USA

Correspondence to Hiromi Yanagisawa and Jessica Wagenseil: [email protected], [email protected]. https://doi.org/10.1016/j.matbio.2019.03.001

Abstract

Elastic fibers are major components of the extracellular matrix (ECM) in the aorta and support a life-long cycling of stretch and recoil. Elastic fibers are formed from mid-gestation throughout early postnatal development and the synthesis is regulated at multiple steps, including coacervation, deposition, cross- linking, and assembly of insoluble elastin onto microfibril scaffolds. To date, more than 30 molecules have been shown to associate with elastic fibers and some of them play a critical role in the formation and maintenance of elastic fibers in vivo. Because the aorta is subjected to high pressure from the left ventricle, elasticity of the aorta provides the Windkessel effect and maintains stable blood flow to distal organs throughout the cardiac cycle. Disruption of elastic fibers due to congenital defects, inflammation, or aging dramatically reduces aortic elasticity and affects overall vessel mechanics. Another important component in the aorta is the vascular smooth muscle cells (SMCs). Elastic fibers and SMCs alternate to create a highly organized medial layer within the aortic wall. The physical connections between elastic fibers and SMCs form the elastin-contractile units and maintain cytoskeletal organization and proper responses of SMCs to mechanical strain. In this review, we revisit the components of elastic fibers and their roles in elastogenesis and how a loss of each component affects biomechanics of the aorta. Finally, we discuss the significance of elastin-contractile units in the maintenance of SMC function based on knowledge obtained from mouse models of human disease. © 2019 Elsevier B.V. All rights reserved.

Introduction lamella composed of elastic fibers alternate with vascular smooth muscle cells (SMCs) throughout Elastic fibers are required for expansion and recoil the medial region. Within each lamellar unit, elastic of the aortic wall and alteration in quantity or quality fibers and SMCs maintain close contacts through of elastic fibers exhibits profound effects on the connecting filaments that are formed by elastin integrity and biomechanics of the aorta. The aorta is extensions, thereby keeping SMCs and elastic the first segment of the arterial tree and receives lamella in a circumferential orientation [2]. These blood from the heart. In order to maintain a stable connections allow transmission of mechanical forces supply of blood to peripheral organs throughout the generated by the cyclic pumping of blood to the cardiac cycle, elasticity of the proximal ascending SMCs (outside-in signals), as well as transmission of aorta plays a critical role in storing a portion of blood tension generated by actomyosin within the SMCs during systole and sending blood out during diastole, to be delivered to the elastic lamella (inside-out exerting the Windkessel effect [1]. The aorta and signals), thereby forming “elastin-contractile units”. large elastic arteries are unique in that they contain Hence, elastic fibers serve as a mediator of dynamic well-defined elastic lamellar units, in which elastic mechanical signals in the blood vessel wall.

The molecular mechanism of elastic fiber formation tropoelastin occurs simultaneously with elastin depo- has begun to be elucidated through the identification sition onto microfibrils [15]. of molecules associated with elastic fibers and their Fibulin-4 (encoded by Efemp2) and fibulin-5 loss-of-function analyses in mice with distinct elastic (Fbln5) are secreted glycoproteins with a modular fiber defects [3–5]. Elastic fiber formation occurs via structure. They are expressed in the aorta and are multiple steps in a tightly regulated manner which essential for elastic fiber formation in vivo [3–5]. The actively takes place from mid-embryogenesis fibulin family is comprised of seven members and throughout early postnatal life [6,7]. Elastic fibers fibulin-4 through fibulin-7 belong to the short fibulins formed during this period last one's lifespan. Once (reviewed in [16,17]). Among short fibulins, fibulin-4 elastic fibers are degraded by aging or lost by injuries, and fibulin-5 are known as elastogenic fibulins. they do not regenerate spontaneously, thus Fibulin-5 was first reported as an elastin binding compromising the biomechanical properties of the protein and its deletion in vivo caused systemic aortic wall. Here, in attempt to provide knowledge on elastic fiber defects in mice [3,4]. Subsequently, it the detailed molecular mechanism of elastic fiber was shown that fibulin-5 potentiates coacervation of assembly and its role on vessel mechanics in vivo, we tropoelastin as well as limits the size of coacervated first review the components of elastic fibers and elastin [18,19]. Consistently, electron microscopy provide an updated view of elastic fiber formation, and analysis showed that Fbln5-null (Fbln5−/−)skin then we discuss the impact of the loss of major elastic contains large aggregates formed by insoluble fiber components on the biomechanics of the thoracic elastin [20]. Fibulin-4 shares high homology with aorta. Lastly, we discuss the importance of elastin- fibulin-5 and also binds tropoelastin in vitro [21]. contractile units and their association with aortic Deletion of fibulin-4 in mouse (Fbln4−/−) causes a diseases. more severe phenotype than the Fbln5−/− mouse, exhibiting aortic rupture, diaphragm herniation and perinatal lethality, which resembles Lox-null (Lox−/−) Molecular players in elastic fiber assembly pups [5,22–24]. Both fibulins have distinct roles in elastogenesis. Biochemical analyses showed that Elastic fibers are comprised of an elastin core and the N-terminal region of fibulin-4 binds a propeptide surrounding microfibril scaffold. Elastin is encoded of LOX and facilitates tropoelastin binding to LOX by a single ELN gene and is translated to a ~70 kDa [18], whereas fibulin-5 binds LOXL1 via the C- protein secreted as the monomeric form called terminal domain and facilitates tethering of LOXL1 tropoelastin from elastogenic cells in the skin, lung onto elastic fibers [25] and/or proteolytic activation of and vasculature [8]. Elastin contains alternating preproLOXL-1 to mature LOXL1 [20]. Taken together, hydrophobic and cross-linking domains that are fibulin-4 and fibulin-5 play a critical role in optimizing responsible for self-aggregation and formation of the conditions for tropoelastin cross-linking through cross-links, respectively [9]. Coacervation of tropoe- regulation of coacervation and interactions with LOX lastin, which induces self-aggregation and phase and LOXL1, respectively. separation, occurs spontaneously under physiologic Visible at the periphery of the elastin core are conditions and is important for alignment and cross- microfibrils that act as a pre-existing scaffold for linking of tropoelastin [10,11]. However, coacervation elastin polymer deposition as well as regulation of alone is not sufficient for polymerization of elastic growth factors and cytokines. Microfibrils are formed fibers, which requires the action of cross-linking by polymerization of large ECM proteins, mainly enzymes. Lysyl oxidase (LOX) and lysyl oxidase-like fibrillin-1 (Fbn1) and fibrillin-2 (Fbn2) [26–28]. Hep- 1 (LOXL1) belong to a multigene family consisting of aran sulfate and a fibronectin network are essential five members (LOX and LOXL1 to 4) and are copper- for formation of the microfibril scaffold [29–31]. and lysyl-tyrosyl-quinone (LTQ)-dependent secreted Fibronectin is also shown to be important for enzymes [12,13]. LOX and LOXL1 are the most microfibril homeostasis in early passage-human well-studied enzymes of the family that contain an N- skin fibroblasts [32]. A complete deletion of fibrillin- terminal propeptide, which is cleaved during proteo- 1 in mice causes neonatal lethality due to aortic lytic activation, and a conserved catalytic domain. aneurysm rupture and disruption of elastic fibers LOX and LOXL1 mediate cross-linking of elastin by [33]. Further deletion of Fbn2 in the Fbn1-null catalyzing the oxidative deamination of peptidyl lysine (Fbn1−/−) background in mice worsens the pheno- residues, subsequently forming the covalent elastin- type and induces embryonic lethality with poorly specific cross-links desmosine and isodesmosine. organized elastic fibers, indicating that microfibril LOX also catalyzes cross-linking of collagens. Recent scaffold formation is indeed a prerequisite for proper crystal structure analysis of human LOXL2 revealed deposition of polymerized elastin [33]. a conserved catalytic domain among the LOX family Fibrillins' role as a scaffold for elastic fiber assembly and showed generation of LTQ upon copper- is not only through homophilic interaction-based long mediated activation of the enzyme [14]. It has been fibril structures, but also by providing interactions with shown in vitro that LOX-mediated cross-linking of numerous binding proteins [34–36]. Recently, more

tropoelastin fibulin-5 elastogenic cell fibulin-4 LOX LOXL1 LTBP-4L LTBP-4S EMILIN-1

microfibrils

fibronectin

Fig. 1. Proposed model of elastogenesis. Tropoelastin is secreted by elastogenic cells, binds to fibulins (predominantly fibulin-5), and undergoes self-aggregation (coacervation). Fibulin-5 binds to LOXL1 and fibulin-4 binds to LOX and the complexes are deposited onto microfibrils (green) with the aid of LTBP-4S and LTBP-4 L, respectively. Cross-linking and polymerization of elastic fibers proceed.

Please cite this article as: H. Yanagisawa and J. Wagenseil, Elastic fibers and biomechanics of the aorta: Insights from mouse studies, Matrix Biology, https://doi.org/10.1016/j.matbio.2019.03.001 4 Elastic fibers and biomechanics of the aorta only on material properties) between groups under amounts and predict reorganization of collagen fiber physiologic loading conditions. Structural stiffness is alignment and nonlinearity in Eln+/− aorta to maintain − − often determined from in vitro pressure-diameter tests the material stiffness near wild-type levels [56]. Eln / with the aorta held at the in vivo axial stretch ratio and mice that have been rescued by expression of human is the inverse of compliance or distensibility. We elastin through a bacterial artificial chromosome present pressure-diameter data for a range of mouse (hBACmNull) have about 30% of the normal elastin models and discuss the implications of elastic fiber levels and even more severe increases in aortic defects on structural stiffness under different loading structural stiffness and blood pressure than Eln+/− conditions. Calculation of material stiffness requires mice [57]. hBACmNull mice exhibit aortic narrowing information about the unloaded dimensions of the similar to humans with supravalvular aortic stenosis aortic wall, in addition to pressure-diameter data, and (SVAS) that is caused by elastin mutations leading to hence is not always reported. reduced levels of functional elastin [58]. Aortic Material stiffness depends on multiaxial deforma- narrowing in hBACmNull mice may be caused by tion of the aortic wall and should be calculated by deficient circumferential growth [59]. fitting a nonlinear strain energy function to biaxial mechanical testing data. However, an estimate of the Fibulin-5 circumferential material stiffness called the incremental elastic modulus can be obtained by determining the Fbln5−/− mice live a normal lifespan, but have linear slope of the stress-strain curve under physio- fragmented elastic fibers in elastin rich tissues [3,4]. logic loading conditions. The incremental elastic Fbln5−/− mice have a longer aorta [4] with increased modulus of the aorta for a range of animal species structural stiffness [3]. Biaxial mechanical testing from shark to toad to mouse to human is around confirms that Fbln5−/− aorta has increased structural 0.4 MPa and it has been suggested that this value stiffness, but similar material stiffness to wild-type represents a “universal elastic modulus” that provides aorta, as well as decreased stored strain energy and optimal mechanical function in a pulsatile cardiovas- increased energy dissipation [60]. The material − − cular system [48]. We highlight mouse models that are stiffness is maintained at similar values in Fbln5 / able to maintain the universal elastic modulus through and wild-type aorta throughout postnatal maturation remodeling of the aortic wall and those that cannot [61], suggesting a universal elastic modulus for the and are often associated with aneurysmal disease. aorta that is maintained even with genetic mutations Additional mechanical properties compared be- that alter the available amounts and structure of tween mouse models include residual strain (strain elastic fiber components in the wall [48]. Fbln5−/− remaining in the aortic wall in the unloaded state due aorta has decreased active contractility [62], which to growth and remodeling of the wall components), may be related to altered coupling between SMCs stored strain energy (the total energy stored due to and elastic fibers due to the absence of fibulin-5. multiaxial, physiologic deformation of the aortic wall and available to do work on the blood in vivo), and Fibulin-4 energy dissipation (energy loss due to viscous components in the aortic wall), which is the Fbln4−/− mice die at birth with severe vascular and − − difference between the stored strain energy with lung defects [5]. Newborn Fbln4 / mice have loading and returned strain energy with unloading. ascending aortic aneurysms and increased structural stiffness, but similar material stiffness to wild-type Elastin aorta [63] consistent with the hypothesized ability of the aorta to maintain material stiffness despite elastic Elastin null (Eln−/−) mice die at birth with obstructive fiber protein deficiencies. Similar to newborn Eln−/− aortic occlusion [49]. The ascending aorta from aorta, newborn Fbln4−/− aorta shows a large increase newborn Eln−/− mice has increased structural stiff- in energy dissipation with cyclic loading [51]demon- ness compared to Eln+/+ [50]. There is a large strating that correctly assembled elastic fibers, and increase in energy dissipation in Eln−/− aorta, which not just the elastin protein, are necessary for is consistent with the mechanical role of elastin in the reversible elasticity of the aorta. Mutations in fibulin- large arteries [51]. The aorta from elastin heterozy- 4 cause cutis laxa which is often accompanied by gous (Eln+/−) mice has increased structural stiffness aortic aneurysms. A mouse model with a fibulin-4 compared to Eln+/+ mice [52] and compensatory mutation (Fbln4E57K/E57K) identified in human patients remodeling of the axial and circumferential residual with cutis laxa develops ascending aortic aneurysms strains [53]. Eln+/− mice live a normal lifespan [54]. in approximately half of the mice homozygous for the During postnatal maturation, the increase in aortic mutation [64]. Fbln4E57K/E57K aorta has increased structural stiffness precedes and may cause hyper- structural stiffness compared to wild-type, even when tension in Eln+/− mice [55]. Results from constitutive an aneurysm is not present [65]. modeling of elastin and collagen contributions to aortic Mice with SMC specific loss of fibulin-4 (Fbln4SMKO) mechanics are consistent with reduced elastin were developed to further investigate the role of

Please cite this article as: H. Yanagisawa and J. Wagenseil, Elastic fibers and biomechanics of the aorta: Insights from mouse studies, Matrix Biology, https://doi.org/10.1016/j.matbio.2019.03.001 Elastic fibers and biomechanics of the aorta 5 fibulin-4 in vascular biology. Fbln4SMKO mice develop behavior of the aorta due to reduced elastic fiber ascending aortic aneurysms [24]. Aneurysms are crosslinking may alter SMC mechanobiology and detectable by 2 weeks of age in Fbln4SMKO mice, but contribute to aneurysm formation and dissection [75]. increases in structural stiffness are detectable by 1 week of age [66], indicating that increases in Fibrillin-1 structural stiffness precede and may play a causal role in aneurysm formation. Biaxial mechanical testing Homozygous mice with deletion of a large portion showed an increase in aortic material stiffness of of the fibrillin-1 gene and insertion of a neomycin Fbln4SMKO mice and other mouse models of aneu- resistance cassette (Fbn1mgΔ/mgΔ) die before three rysmal disease [67] suggesting that aneurysms may weeks of age from cardiovascular complications [76]. manifest in adult animals when the aorta is not able to Mice that underexpress fibrillin-1 due to insertion of a maintain the universal elastic modulus. In support of neomycin resistance cassette, without deletion of any this hypothesis, crossing Fbln4SMKO mice with fibrillin-1 gene segments (Fbn1mgR/mgR) die at about thrombospondin-null (Thbs1−/−) mice rescues the 4 months of age with large aortic aneurysms [77]. aneurysm phenotype along with the material stiffness Fbn1mgR/mgR ascending aorta has increased structural [68]. A growth and remodeling model of aortic and material stiffnesses, even when an aneurysm is development was used to understand how changes not present [78], consistent with the hypothesis that in elastin, collagen, and SMC amounts and behavior aneurysms may manifest when the adult mouse aorta could lead to the resulting mechanical behavior of is not able to maintain the universal elastic modulus. Fbln4SMKO aortas. Reductions in elastin stress Similar to Fbln5−/− aorta, Fbn1mgR/mgR aorta has contribution, increases in collagen stress contribution, reduced stored strain energy and increased energy and altered SMC response to developmental changes dissipation [78]. in hemodynamic forces were required for the model to reproduce Fbln4SMKO aorta mechanical behavior [69]. Comparison of aortic biomechanics and elastin/ The changes in SMC response are consistent with collagen contributions in different mouse models changes in SMC phenotype observed in Fbln4SMKO mice [24,66]. Pressure-diameter tests at the in vivo axial stretch were performed in many of the mouse models Lysyl oxidase discussed above and are summarized in Fig. 2. The structural stiffness (or local slope of the curve) is Lox−/− mice die at birth with ruptured arterial often affected by elastic fiber defects, but is highly aneurysms and diaphragm [22,70]. Newborn Lox−/− dependent on the applied pressure. Due to the mice have ascending aortic aneurysms, increased pressure dependence, an aorta from a hypertensive structural stiffness, and increased material stiffness mouse (such as Eln+/−) will have increased structural compared to wild-type aortas [71]. The increased stiffness in vivo, even with no changes in the material stiffness in Lox−/− aortas contrasts with the geometry and material properties of the aortic wall normal material stiffness in Fbln4−/− aortas [63]. [79]. The applied pressure at which the curve Since lysyl oxidase crosslinks both elastin and transitions between low and high structural stiffness collagen, it is possible that the compensatory is also affected by elastic fiber defects. The structural changes in collagen arrangement and nonlinearity stiffness at low pressure is associated with elastin hypothesized to maintain the universal elastic resistance to deformation, while the structural modulus in other mouse models of elastic fiber stiffness at high pressure is associated with collagen defects (i.e. Eln+/− [56] and Fbln5−/− [61]) cannot resistance to deformation [80]. For many of the occur in Lox−/− aortas. Like newborn Eln−/− and mouse models, there are not large differences in the Fbln4−/− aortas, newborn Lox−/− aortas show a large structural stiffness at low pressure where elastin increase in energy dissipation confirming that dominates, implying that many genetic modifications effective elastic fiber crosslinking is necessary for (including Eln+/− and Fbln5−/−) have little effect on reversible elasticity in the aorta [51]. Mutations in the low-pressure mechanical behavior attributed to lysyl oxidase have been identified in human patients elastin. The more severe mouse models that show with thoracic aortic aneurysms [72]. A mouse model highly fragmented elastic fibers and develop aortic with a mutation identified in humans (LOX p.M298R) aneurysms (including Fbln4SMKO and Fbn1mgR/mgR) shows perinatal lethality and large aneurysms in mice have decreased stiffness at low pressure, consistent homozygous for the mutation. Mice heterozygous for with reduced mechanical contributions of elastin. the mutation live a normal lifespan, but have increased Interestingly, all of the mouse models with elastic structural stiffness in the aorta [73]. Chemical inhibition fiber defects have increased stiffness in the high- of LOX with BAPN induces thoracic aortic aneurysm pressure region attributed to collagen. They also and dissection in mice [74]. SMCs in BAPN treated transition to the stiffer region at lower pressures than aorta are characterized by mechanical stress induced wild-type aorta. Some models, such as Fbln4SMKO apoptosis, indicating that changes in mechanical [81], have collagen defects in addition to elastin

175 175 WT 150 150 Eln+/- o o )

125 g 125 Fbln5-/- o o 100 o o 100 o o Fbln4E57K/E57K no aneurysm

75 o re (mmH 75 o E57K/E57K o su o Fbln4 aneurysm o o o o es pressure (mmHg) 50 50 o pr o Fbln4SMKO aneurysm 25 25 LoxM298R/+ 0 0 mgR/mgR 700 1100 1500 1900 2300 2700 1 1.3 1.6 1.9 Fbn1 aneurysm

mgR/mgR ABouter diameter (µm) normalized outer diameter (µm/µm) Fbn1 no aneurysm

Fig. 2. Pressure-diameter curves for ascending aorta from adult mice with elastic fiber defects. Absolute values of the outer diameter at each pressure (A) and normalized values with respect to the starting outer diameter at 0 mm Hg (B) are shown. The local slope of the curve represents the structural stiffness. The black circles on each curve mark the approximate transition from low stiffness (elastin dominated) to high stiffness (collagen dominated) behavior. Aortas from mice with elastic fiber defects transition to high stiffness at lower pressures than wild-type, with aneurysmal models having the lowest transition pressure. Data was approximated from [55] for Eln+/−,[61] for Fbln5−/−,[65] for Fbln4E57K/E57K,[66] for Fbln4SMKO,[73] for Lox+/M298R, and [78] for Fbn1mgR/mgR. The wild-type (WT) curve was estimated from the average behavior of three different studies [55,61,66]. defects that may explain the changes in mechanical regulated mainly by a ligand-induced, receptor- − behavior at high pressure. Others, such as Eln+/ mediated signaling pathway involving the mobiliza- [52,56], do not have measurable changes in tion of intracellular calcium and contractile filaments collagen amounts or organization. Experiments (reviewed in [84]). SMCs organize contractile fila- with elastase degradation and results from constitu- ments comprised of smooth muscle (SM) actin and tive modeling of aortic wall components suggest that SM myosin in a diagonal direction and they connect elastic fibers hold collagen fibers in compression, to the dense plaque (DP), where elastic fibers attach resulting in wavy collagen fibers, and allowing them to integrins from outside of the cell and form focal to deform significantly before contributing to me- adhesions (FAs) underneath the cell membrane [85]. chanical resistance [82,83]. The loss of functional Actin isoforms that form contractile filaments in large elastic fibers may decrease collagen fiber waviness, arteries are mainly α SM actin [86], whereas non- so the collagen fibers are straighter and contribute to muscle γ actin forms cytoskeleton near the cortex of the mechanical resistance at a lower point in the SMCs [87]. Actin molecules in non-contractile pressure-diameter curves. The loss of functional filaments are more dynamic compared to contractile elastic fibers may also reduce the variation in filaments, but both contractile filaments and the collagen fiber waviness so that most collagen fibers cortically-distributed actin cytoskeleton generate are recruited at the same deformation, leading to a tension in SMCs [88]. sharp rather than gradual increase in structural Recently, it has been shown that genetic muta- stiffness. These observations imply that changes to tions in the components of elastin-contractile units elastin and collagen (and SMCs) must be consid- are responsible for thoracic aortic aneurysms (TAAs) ered together to fully understand the mechanical (reviewed in [89]). The mutations are largely divided implications of elastic fiber defects. into two groups; the first group includes genes encoding for extracellular proteins that constitute or are associated with microfibrils such as FBN1 [90], Biological significance of elastin LOX [72,73], EFEMP2 [91], microfibril associated contractile units protein 5 (MFAP5)[92], elastin microfibril interface 1 (EMILIN1)[93] and LTBP3 [94]. The bundles of SMCs possess an intrinsic contractile machinery microfibrils are initially linked to SMCs, then elastin to respond to mechanical loads. The contractile progressively infiltrates the microfibrils to form machinery is indirectly connected to elastic fibers to elastin extensions [2]. Fbn1 interacts with integrin form an elastin-contractile unit, which is the physical α5β1andαvβ3 via its RGD domain [95]and and functional unit from elastic fibers to actin downregulation of Fbn1 in mouse (Fbn1mgR/mgR) contractile filaments that allows the coordinated markedly reduces phosphorylation of focal adhesion response of aortic wall components to mechanical kinase (FAK) in cardiac myocytes and leads to stress [2](Fig. 3). SMCs do not form sarcomeres like dilated cardiomyopathy [96]. In addition, compound cardiac or skeletal muscle cells and contraction is heterozygous mice for Fbn1 and β1integrin(Fbn1+/−;

elastin extension mechanical force EELL

dense plaque contractile filaments

TTalinalin

ILK PPxx cell membrane FAK ACTA2

dense plaque MYH11 Regulators: PKG-1 contractile filaments MLCK

Fig. 3. Schematic presentation of the elastin-contractile unit in SMCs. Elastic fibers bind to α- and β-heteromeric integrins through elastin extensions and form dense plaques (orange), where focal adhesion proteins such as Talin, paxillin (Px), focal adhesion kinase (FAK) and integrin linked kinase (ILK) bind and regulate contractile filaments (red, major isoform is ACTA2) as well as activate various downstream signaling. Cellular tension is generated by the contraction of actin and myosin (pink, major isoform is MYH11). The regulators of muscle contraction such as PKG-1 and MLCK also play a crucial role in response to mechanical stimuli. The defects in elastin-contractile units result in the formation of thoracic aortic aneurysms. EL: elastic lamina.

− Itgb1+/ ) recapitulate the phenotype of Fbn1mgR/mgR [99,100]). Integrin linked kinase (ILK) is a scaffold mice, suggesting that Fbn1 may be crucial to signal protein that associates with the cytoplasmic tail of β activation in SMCs mediated by elastin extensions integrin and forms the IPP complex with cysteine- and integrins. Interestingly, mutations in Eln [58]or rich protein (PINCH) and parvin and links to actin Fbln5 [97] that form the elastin core do not cause filaments [101]. Deletion of Ilk in SMCs results in aortic aneurysms; rather, they develop supravalvular aneurysmal dilatation of the aorta [102] and deletion aortic stenosis (SVAS) and tortuous elongated aorta, in neural crest cells shows reduced phosphorylation respectively, exhibiting morphogenetic defects due to of pSmad3 and downregulation of SMC markers impaired elastic fibers in the aorta. with aneurysm development [103]. Talin serves as Focal adhesions were described as electron- a mechanosensitive molecular hub transducing dense regions in SMCs where elastin extensions mechanical force to actin filaments and converting attach to the outside of the cell and actin filaments it into biochemical signals (reviewed in [104]). anchor obliquely at the cytoplasmic side underneath Activation of talin, which binds the β integrin tail, the cell membrane [98]. Although mutations in the leads to conformational change of the integrin into components of focal adhesions have not been the active form, and tension generated by actin reported in aortic aneurysms, studies in mice have filaments can stabilize integrins together with talin in implicated their role in aortic homeostasis. Mechan- its extended open form [105]. Recently, downregu- ical force in the aorta is transmitted to focal lation of talin has been reported in SMCs harvested adhesions via heterodimeric α and β integrins and from patients with aortic dissection, along with regulates actin filaments. Several key molecules increased proliferation and migration [106]. have been identified within focal adhesions that are The second group for which elastin-contractile unit involved in mechanotransduction (reviewed by mutations are reported in TAAs includes genes

Please cite this article as: H. Yanagisawa and J. Wagenseil, Elastic fibers and biomechanics of the aorta: Insights from mouse studies, Matrix Biology, https://doi.org/10.1016/j.matbio.2019.03.001 8 Elastic fibers and biomechanics of the aorta encoding for the components of actomyosin contrac- that inside out and outside in mechanical signaling tile filaments and their regulators in SMCs (reviewed in likely contribute to the regulation of elastic fiber [107]). Mutations in SM-myosin heavy chain (encoded synthesis in development and dysfunction with by MYH11), which causes deletion in the C-terminal disease. coiled-coil domain, is predicted to exert a dominant The mechanical data on different mouse models of negative effect on myosin motor activity [108]. In elastic fiber defects show that increased aortic addition, 3 missense variants (L1264P, R1275L, structural stiffness is a common consequence of R712Q) and 1 rare variant (R247) in MYH11 have reduced elastin levels or misassembled elastic been identified and suggested to damage coiled-coil fibers, but that increased aortic material stiffness domain (L1264P), affect the ATPase region (R712Q) generally manifests in extreme cases that are and disrupt the myosin motor activity (R247C) [109]. associated with aneurysmal disease. The results More than 40 mutations in ACTA2 have been suggest that failure of the aortic wall to maintain the identified in familial TAA and dissection [110]and universal elastic modulus has downstream effects R179H and R258C mutant actin have been shown to on SMC phenotype that may be caused by the cause polymerization defects and slower movement microenvironment within the stiffened ECM. Future compared with wild-type actin filaments in in vitro work to quantify the material behavior of the aorta motility assays [111,112]. Mutations in regulators of under physiologic loading conditions and the resulting SMC contraction have also been reported, including SMC phenotype in different mouse models with the MLCK gene encoding for myosin light chain kinase elastic fiber and other ECM defects is needed to that induces muscle contraction [113] and gain-of- further investigate this hypothesis and understand the function mutation of PKG1 gene encoding for type I limits and consequences of maintaining the universal cyclic guanosine monophosphate (cGMP)-dependent elastic modulus. When reported, stored strain energy protein kinase that controls muscle relaxation [114]. is reduced and energy dissipation is increased in Interestingly, these mutations lead to reduced SMC aortas with elastic fiber defects. These energetic contractility, presumably affecting the coordinated alterations increase the work required by the heart to response of the aortic wall to mechanical forces. It is circulate blood and may contribute to secondary of note that SMCs isolated from postnatal day 7 cardiovascular diseases associated with elastic fiber Fbn1mgR/mgR aortas have increased SMC markers, defects. reduced elastin expression and decreased prolifera- Elasticity is an essential biomechanical property of tion, showing premature SMC differentiation [115]. the aorta and is provided by elastic fibers and Marfan iPS-derived SMCs originated from the neural enhanced by elastin-contractile units. Whereas insol- crest recapitulate the Marfan vascular phenotype, uble polymerized elastin confers dynamic elasticity exhibiting reduced FBN1 deposition, increased TGFβ, and recoil to the aortic wall, elastin extensions aid in and upregulation of SMC contractile proteins, but connecting elastic lamella to integrins and transmit decreased carbachol-induced cell contractility and mechanical forces through focal adhesions, thereby intracellular calcium retention [116]. Fbln4 mutant converting the physical stimuli into biochemical SMCs also show reduced SMC contractile markers signaling and regulating actin contractile filaments. [24]. Taken together, these observations underscore We hypothesize that a homeostatic stress state, the importance of the elastin-contractile unit for proper determined by external forces from ECM connections maintenance of SMC differentiation and coordinated and internal forces from contractile filaments, is contraction in the aortic wall. necessary for maintenance of the SMC phenotype. In the case of genetic defects to elastic fiber proteins, microfibrils may still be in place, but elastin extensions Concluding remarks and future directions are disrupted, interfering directly with the SMC homeostatic stress state or with continuity of the Studies in knockout mice have been instrumental force sensing apparatus. We also have preliminary in defining critical players in elastic fiber assembly data indicating that force promotes micro-remodeling and identifying molecules that work together to of the elastin contractile unit. Therefore, defects in the regulate elastin coacervation, cross-linking, deposi- mechanical environment (such as changes in the tion onto microfibrils, and interactions with SMCs. universal elastic modulus) or the cellular interpretation Additional in vivo and in vitro studies are needed to of the mechanical environment (such as defects in the fully understand the role(s) of the over 30 different elastin-contractile unit) lead to maladaptive remodel- molecules implicated in elastic fiber synthesis so that ing associated with disease. Disruption of the physical we may someday be able to encourage elastic fiber and functional connecting elastin-contractile units repair or de novo synthesis in the case of defects or weakens the aortic wall and exacerbates pathological damage caused by disease or aging. It is important conditions due to alterations in SMC phenotype. to remember that elastic fiber synthesis occurs in a Ineffective regeneration of elastic fibers hampers the dynamic mechanical environment, with the aortic control of these conditions and urges us to have a wall distending and recoiling with each heartbeat, so more comprehensive understanding of the regulation

Please cite this article as: H. Yanagisawa and J. Wagenseil, Elastic fibers and biomechanics of the aorta: Insights from mouse studies, Matrix Biology, https://doi.org/10.1016/j.matbio.2019.03.001 Elastic fibers and biomechanics of the aorta 9 of elastic fibers and organization of elastin-contractile expressed human elastin polypeptides as a model for units in vivo. investigations of structure and self-assembly of elastin, Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 357 (2002) 185–189. [12] H.A. Lucero, H.M. Kagan, Lysyl oxidase: an oxidative enzyme and effector of cell function, Cell. Mol. Life Sci. 63 Acknowledgment (2006) 2304–2316. [13] H.E. Barker, T.R. Cox, J.T. Erler, The rationale for targeting the The work in Yanagisawa Laboratory and Wagenseil LOX family in cancer, Nat. Rev. Cancer 12 (2012) 540–552. Laboratory was supported in part by grants from the [14] X. Zhang, Q. Wang, J. Wu, J. Wang, Y. Shi, M. 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Please cite this article as: H. Yanagisawa and J. Wagenseil, Elastic fibers and biomechanics of the aorta: Insights from mouse studies, Matrix Biology, https://doi.org/10.1016/j.matbio.2019.03.001