Elastin, Arterial Mechanics, and Cardiovascular Disease

Am J Physiol Heart Circ Physiol 315: H189–H205, 2018. First published April 6, 2018; doi:10.1152/ajpheart.00087.2018.

REVIEW Extracellular Matrix in Cardiovascular Pathophysiology

Elastin, arterial mechanics, and cardiovascular disease

Austin J. Cocciolone,1 Jie Z. Hawes,2 Marius C. Staiculescu,2 Elizabeth O. Johnson,2 Monzur Murshed,3 and Jessica E. Wagenseil2 1Department of Biomedical Engineering, Washington University, St. Louis, Missouri; 2Department of Mechanical Engineering and Materials Science, Washington University, St. Louis, Missouri; and 3Faculty of Dentistry, Department of Medicine, and Shriners Hospital for Children, McGill University, Montreal, Quebec, Canada Submitted 2 February 2018; accepted in ﬁnal form 6 April 2018

Cocciolone AJ, Hawes JZ, Staiculescu MC, Johnson EO, Murshed M, Wagenseil JE. Elastin, arterial mechanics, and cardiovascular disease. Am J Physiol Heart Circ Physiol 315: H189–H205, 2018. First published April 6, 2018; doi:10.1152/ajpheart.00087.2018.—Large, elastic arteries are composed of cells and a specialized extracellular matrix that provides reversible elasticity and strength. Elastin is the matrix protein responsible for this reversible elasticity that reduces the workload on the heart and dampens pulsatile flow in distal arteries. Here, we summarize the elastin protein biochemistry, self-association behavior, cross-linking process, and multistep elastic fiber assembly that provide large arteries with their unique mechanical properties. We present measures of passive arterial mechanics that depend on elastic fiber amounts and integrity such as the Windkessel effect, structural and material stiffness, and energy storage. We discuss supravalvular aortic stenosis and autosomal dominant cutis laxa-1, which are genetic disorders caused by mutations in the elastin gene. We present mouse models of supravalvular aortic stenosis, autosomal dominant cutis laxa-1, and graded elastin amounts that have been invaluable for understanding the role of elastin in arterial mechanics and cardiovascular disease. We summarize acquired diseases associated with elastic fiber defects, including hypertension and arterial stiffness, diabetes, obesity, atherosclerosis, calcification, and aneurysms and dissections. We mention animal models that have helped delineate the role of elastic fiber defects in these acquired diseases. We briefly summarize challenges and recent advances in generating functional elastic fibers in tissue-engineered arteries. We conclude with suggestions for future research and opportunities for therapeutic intervention in genetic and acquired elastinopathies. aorta; compliance; elasticity; extracellular matrix; stiffness

INTRODUCTION elastic fibers also have important chemical signaling properties. Elastin is an extracellular matrix (ECM) protein with a unique biochemical structure that provides entropic elasticity, Elastin is highly hydrophobic, extensively cross-linked, and allowing the large arteries to reversibly expand and relax with is assembled into elastic fibers in a dynamic process involving every cardiac cycle. Elastin is the main component of elastic cells, cell surface receptors, and numerous elastic fiber associ- fibers that provide the large artery elasticity necessary for ated proteins. Mutations in the elastin gene cause supravalvular proper cardiovascular function in vertebrate animals. Insuffi- aortic stenosis (SVAS), associated with elastin insufficiency, ciency of elastin or disorganization, improper assembly, frag- and autosomal dominant cutis laxa-1 (ADCL1), associated mentation, and biochemical modifications of elastic fibers with tissue-specific defects in elastic fiber assembly. SVAS change the passive mechanical behavior of the large arteries mutations appear throughout the gene, but ADCL1 mutations and affect cardiovascular mechanics at multiple length scales. are clustered near the last few exons, suggesting a role for that Both genetic and acquired cardiovascular diseases are associ- region in tissue-specific elastic fiber assembly. Since elastin ated with elastin and elastic fiber defects and the resulting synthesis and elastic fiber assembly are primarily restricted to changes in arterial mechanics. Tropoelastin, the soluble pre- development and maturation, it is critical to understand the cursor to elastin, and the degradation products of fragmented assembly mechanisms for designing interventions in elastinopathies and to recreate elastic fiber structure and function in tissue-engineered arteries. Acquired diseases are generally as- Address for reprint requests and other correspondence: J. E. Wagenseil, sociated with elastic fiber degradation or biochemical modifi- Dept. of Mechanical Engineering and Materials Science, Washington Univ., One Brookings Dr., CB 1185, St. Louis, MO 63130 (e-mail: cation. This degradation initiates a destructive arterial remod- [email protected]). eling cascade that is difficult to stop and may be impossible to

http://www.ajpheart.org 0363-6135/18 Copyright © 2018 the American Physiological Society H189 Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (128.252.020.193) on September 19, 2018. Copyright © 2018 American Physiological Society. All rights reserved. H190 ELASTIN, ARTERIAL MECHANICS, AND CARDIOVASCULAR DISEASE reverse. As modern medicine has extended life expectancy 163). The narrow timeframe of elastin expression and synthesis beyond elastin’s 70-yr half-life, it is imperative to understand is unusual for an ECM component, as generally ECM produc- how degradation and modification of elastic fibers within the tion is dynamic throughout the organism’s lifespan. Because of arterial wall contribute to the pathogenesis of acquired dis- elastin’s resilience and a half-life of nearly 70 yr, the limited eases. time period of expression and synthesis is sufficient for the In this review, we summarize the biochemistry of elastin and protein to last a lifetime in most species (151, 165). elastic fiber assembly process. We discuss measures of passive arterial mechanics that are affected by elastin amounts, elastic Regulation of Tropoelastin Expression fiber integrity, and cross-linking. When elastin amounts are Immunohistochemistry studies in the developing chicken reduced or elastic fiber integrity is compromised, the vascular lung (71) and aorta (68) have shown that tropoelastin expres- wall cells compensate by producing other ECM proteins and sion correlates with smooth muscle differentiation markers and remodeling arterial geometry that can further alter the mechan- suggest that transcription of elastin is developmentally regu- ical behavior. Additionally, circulating elastin fragments pro- lated. Tropoelastin mRNA levels are decreased in adult com- duced during elastic fiber degradation can activate cytokine pared with neonatal rat lungs, but tropoelastin pre-mRNA and inflammatory signaling pathways that further degrade levels are similar, suggesting that posttranscriptional regulation elastic fibers in a positive feedback cycle. We summarize the turns off tropoelastin expression in adulthood (176). An ele- results of human and animal studies that have advanced our ment in the open reading frame specifically binds a cytosolic understanding of genetic elastinopathies and the cardiovascular protein that destabilizes tropoelastin mRNA. Binding affinity effects of varying elastin gene dosage. We address acquired increases in adult compared with neonatal lung fibroblast cells, cardiovascular diseases that are associated with elastic fiber mediating rapid tropoelastin mRNA decay and decreasing degradation or modification. We briefly discuss advances in overall mRNA levels. Binding affinity is decreased by trans- generating elastic fibers within tissue-engineered arteries and forming growth factor-␤ (TGF- ␤ ) (212), which stimulates conclude with suggestions for future research. 1 1 tropoelastin expression in human fetal lung fibroblasts by ELASTIN stabilizing its mRNA (89). Active Smads and de novo tropoelastin expression are required for TGF-␤1-mediated tro- Elastin Characterization poelastin mRNA stabilization in cell culture (90). TGF-␤1 immunoreactivity is present at the sites of arterial elastin Elastin is an elastic protein polymer that is highly resilient expression as detected by in situ hybridization, supporting a due to extensive posttranslational cross-linking of the mono- role for TGF-␤ regulation of tropoelastin expression in vivo meric precursor tropoelastin. Reviews focusing on tropoelastin 1 (159). TGF-␤1 may additionally regulate tropoelastin expres- and/or elastin have been written by Vrhovski and Weiss (191) sion through interactions with the promoter region in a tissue- and Kozel et al. (87). Due to the numerous cross-links at lysine specific manner (109). IGF-1, vitamin D, and IL-1␤ also residues and having a composition of nearly 75% hydrophobic modulate tropoelastin expression at either the promoter level or residues, elastin is extremely hydrophobic (191). The precursor through increased mRNA stability (191). Cytokine regulation tropoelastin is a 70-kDa protein transcribed from a single of tropoelastin expression has been reviewed by Sproul and elastin gene in the mammalian genome (51, 128, 158). The Argraves (171). human elastin gene is 45 kb, located on chromosome 7, and contains 34 exons of which each exon encodes either a hydro- Coacervation and Cross-Linking phobic or cross-linking domain in an alternating pattern (Fig. 1) (9, 138, 180, 206). Elastin is only present in vertebrate In the arterial wall, tropoelastin is primarily produced from animals, and there is extensive homology among human, chick, vascular smooth muscle cells (VSMCs) in the media (46, 68), bovine, and rat species at the nucleic and amino acid levels although it can also be produced by endothelial cells (ECs) in (191). During an organism’s lifespan, elastin gene expression the intima (19, 113) and fibroblasts in the adventitia (152, 187). and protein synthesis occur within a narrow timeframe initiat- After secretion to the extracellular space, tropoelastin simulta- ing late in embryonic development and terminating in adoles- neously undergoes coacervation and cross-linking. Cross-link- cence with essentially no de novo elastin production through- ing is facilitated by enzymes from the lysyl oxidase (LOX) out adult life of the animal (15, 26, 34, 119, 133, 142, 145, family, in particular LOX and LOX-like-1 (LOXL-1). The role of LOX in elastic fiber assembly has been reviewed by Lucero and Kagan (105). Coacervation is a reversible and thermody- namically driven self-assembly of tropoelastin into globular aggregates that occurs spontaneously at physiological temper- atures and is a consequence of the numerous hydrophobic regions in tropoelastin (22, 23, 134, 183, 186, 190). LOX activity appears to depend on coacervation, as LOX demon- strates higher affinity for insoluble, coacervated tropoelastin and is less effective at cross-linking tropoelastin at tempera- Fig. 1. Domain structure of the human elastin gene. Alternating hydrophobic tures below the threshold for spontaneous coacervation (75, and cross-linking domains are generally transcribed by individual exons. The 120). While accepted that coacervation is a critical step to locations of 64 unique polymorphisms (Human Gene Mutation Database, www.hgmd.cf.ac.uk) associated with either supravalvular aortic stenosis cross-linking, it is unclear whether this is due to induced (SVAS; red, 49 mutations not including large deletions) or autosomal domi- conformational changes in tropoelastin or to tropoelastin se- nant cutis laxa-1 (ADCL1; blue, 15 mutations) are indicated. questering (80, 120, 191).

Fig. 2. Structure of the elastin network in arterial elastic laminae. En face images of elastin in a wild-type mouse ascending aorta obtained using nonlinear ﬂuorescence microscopy are shown (166). A dense, fenes- trated (arrows) elastin network can be observed in elastic laminae near the inner media (left). The network appears more ﬁbrous toward the outer media (right). Circumfer- ential direction is horizontal; axial direction is vertical. Scale bars ϭ 20 ␮m.

Arterial Stiffness and Passive Mechanical Behavior Arterial stiffness is a structural mechanical property that depends on the material properties and geometry of the arterial wall. Common measurements of stiffness include Peterson’s modulus, distensibility, and PWV (50) and almost always refer to deformation and loading in the circumferential direction. We will call these measurements “structural stiffness” values. Pe- Fig. 3. Organization of the elastic laminae in large and small arteries. Cross- terson’s modulus and distensibility can be measured in vivo or sections of a mouse descending aorta (A) and mesenteric artery (B) stained for in vitro, while PWV is usually measured in vivo. PWV can be elastin (red) and cell nuclei (blue) are shown (93). The descending aorta has determined simply and reproducibly in the clinic by recording multiple layers of elastic laminae. The mesenteric artery has an internal elastic lamina at the luminal surface and a thin external elastic lamina at the the time it takes for a blood pressure pulse wave to travel a adventitial surface. L, lumen. Scale bars ϭ 20 ␮m. specified distance in the arterial tree (usually from the carotid to the femoral artery) (124). PWV is a measure of structural stiffness because the value is related to Young’s modulus of the outer wall cells that are too far from the arterial lumen for arterial wall (a material property), wall thickness, arterial diffusion of oxygen and nutrients across the wall. diameter, and density of blood according to the Moens- Korteweg equation (50). The derivation of the Moens- The Windkessel Effect Korteweg equation assumes that the aorta is a perfect cylinder Elastin is only present in vertebrate animals with a closed with no tapering or branches, made up of a linear elastic, circulatory system and pulsatile pressure and flow (155). The isotropic material that experiences small deformations during amount of elastin is highest in the large, elastic arteries closest the cardiac cycle, none of which are precisely true. While PWV to the heart and decreases as one moves distally in the cardio- is a useful measure of structural arterial stiffness, the assump- vascular system (16). Elastic fibers provide reversible elasticity tions accompanying its use must be kept in mind. to the large, elastic arteries. This allows the aorta to deform Arterial wall material properties are nonlinear and vary with elastically under an applied hemodynamic load, with no per- the direction of deformation and loading (i.e., circumferential manent deformation and no energy dissipation when the load is vs. axial); hence, they cannot be quantified by a single mea- removed. Stephen Hales in 1733 recognized the importance of surement, such as Young’s modulus in the Moens-Korteweg arterial elasticity in cardiovascular function and Otto Frank in equation. However, estimates of arterial material properties can 1895 formalized Hales’ ideas into a mathematical model of the be obtained by linearizing the mechanical behavior within a Windkessel effect (132). A Windkessel is an air chamber that given deformation or loading range in a specified direction to was used in fire engines during the 18th century to convert provide an incremental Young’s modulus for comparison pulsatile pumping into a constant flow of water. In the cardio- across samples (50). Material stiffness values can also be vascular system, the Windkessel effect dampens the pulsatile obtained by fitting constitutive models to detailed in vitro flow from the left ventricle (LV), so that the distal vasculature mechanical testing data and calculating derivatives of the receives almost constant perfusion. Blood ejected from the LV conjugate stress and strain measurements (41). Fitting consti- during systole distends the aortic wall and during diastole the tutive models allows comparison of material behavior and aorta reversibly returns to its original configuration. The strain stored strain energy for any loading condition in any direction energy stored during systolic deformation is returned as work but requires multiple experimental protocols for each sample to further pump blood downstream during diastole. If the and is dependent on the suitability of the chosen constitutive Windkessel effect is compromised, for example, by increased equation. arterial stiffness in aged or diseased arteries, the microvascu- The passive mechanical behavior of large, elastic arteries is lature of downstream organs, especially in the brain and determined mostly by the amount and organization of elastic kidney, can be damaged (125). Furthermore, the Windkessel fibers and collagen fibers in the wall. VSMCs contribute to effect reduces cardiac afterload and perfuses the coronary arterial mechanics through active contraction or relaxation and arteries during diastole; hence, cardiac function can deteriorate by producing the surrounding ECM proteins, but they do not when the Windkessel effect is compromised (126). contribute substantially to the passive mechanical behavior The Windkessel effect can be compromised through genetic (38). Although there is only one elastin protein, there are or acquired elastinopathies that alter elasticity of the arterial multiple collagen proteins. The most abundant arterial colla- wall. Elasticity can be altered by increasing wall stiffness or gens are types I and III, with lesser amounts of types IV, V, and increasing energy dissipation so that less stored strain energy is VI. The amounts and transmural organization of collagen types available to do work on circulating blood. Increased wall depend on location in the cardiovascular tree (64). Elastic stiffness also increases the pulse wave velocity (PWV) of arteries show nonlinear behavior with maximum distensibililty

Fig. 4. Effects of elastin amounts on arterial mechanical behavior. AϪD: representative pressure-outer diameter (A and B) and circumferential Cauchy stress-stretch ratio (C and D) curves (197) for the ascending aorta (ASC; A and C) and left common carotid (LCC; B and D) from wild-type (WT; 100% elastin), Elnϩ/Ϫ (50– 60% elastin), and hBAC-mNull (30–40% elastin) mice. Decreased elastin amounts shift the pressure-diameter behavior for both artery locations, but the difference between Elnϩ/Ϫ and hBAC-mNull pressure-diameter behavior for the LCC is negligible. For both artery locations, the stress-stretch behavior is similar between WT and Elnϩ/Ϫ mice but becomes more nonlinear at lower stretch ratios for hBAC-mNull mice. The data highlight the importance of examining structural (pressure-diameter) and material (stress-stretch) mechanical behavior as well as differences between artery locations in the structural response to reduced elastin amounts. The results suggest that mouse arteries can remodel to maintain WT material behavior when elastin amounts are 50–60% of normal but cannot fully remodel and adapt the material behavior when elastin amounts are 30–40% of normal.

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00087.2018 • www.ajpheart.org Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (128.252.020.193) on September 19, 2018. Copyright © 2018 American Physiological Society. All rights reserved. H194 ELASTIN, ARTERIAL MECHANICS, AND CARDIOVASCULAR DISEASE an important consideration in diseases with highly frag- receptor, which is involved in melanoma chemotaxis and mented elastic fibers. expression of MMP-2 and MMP-3 (141). A review summariz- ing the effects of elastin degradation and EDPs in cardiovas- ELASTIN AS A SIGNALING MOLECULE cular aging and disease has been written by Duca et al. (35). Tropoelastin The Elastin Receptor Complex While we have focused so far on the mechanical effects of Many of the chemical signaling effects of tropoelastin and/or structural changes in elastic fibers, elastin and its degradation EDPs are thought to be mediated by the elastin receptor products have chemical signaling effects as well. For example, complex (ERC) (111). The ERC is composed of two mem- soluble tropoelastin can regulate the cell phenotype. The addi- brane-bound subunits, protective protein/cathepsin A (PPCA) tion of tropoelastin to the culture media of VSMCs from and neuraminidase-1 (Neu-1), plus elastin-binding protein ElnϪ/Ϫ mice inhibits proliferation (76) and increases stiffness (EBP). EBP (also known as S-Gal) is a splice variant of as measured by atomic force microscopy (37). Karnik et al. lysosomal ␤-galactosidase that retains the ability to bind galac- (77) showed that a specific domain within tropoelastin, tosugars but has lost enzymatic activity. In addition, the splice VGVAPG, induces actin polymerization in VSMCs through a variant has a 32-peptide sequence capable of binding tropoelas- G protein-coupled pathway. The VGVAPG peptide is a potent tin (59). In elastogenesis, EBP is thought to be responsible for chemoattractant for VSMCs (77), fibroblasts, and monocytes shuttling tropoelastin to the cell surface, and antagonistic (162). Sites have been identified at the COOH terminus and interaction between EBP and galactose or lactose may release central region of tropoelastin that mediate binding to ␣v␤3- and tropoelastin to the extracellular space (58). Neu-1 appears to ␣v␤5-integrins, which are involved in numerous signaling control signal transduction by ERC through the catalytic con- pathways (96, 149). A nonintegrin-mediated interaction be- version of ganglioside to lactosylceramide (153). Downstream tween tropoelastin and the cell membrane has also been iden- signaling of the ERC diverges depending on cell type but tified through the binding of fractionated tropoelastin to hep- reconvenes at the activation of ERK1/2 (35). arin sulfated proteoglycans (14). Tropoelastin expression is ␤ detectable in the adult human aorta at decreasing levels with TGF- Sequestration aging (44), implying that reduced tropoelastin as well as TGF-␤ is secreted from cells in the form of a large latent degradation and modification of existing elastic fibers may complex (LLC) that includes a latency-associated peptide modulate age-related changes in the arterial wall. (LAP) and a latent TGF-␤-binding protein (LTBP). LTBP localizes latent TGF-␤ to the ECM. LTBP1 and LTBP3 bind Elastin-Derived Peptides well to all three TGF-␤ isoforms (147). LTBP1 has specific Elastic fibers can be degraded and fragmented by mechani- interactions with fibrillin-1 microfibrils (129). Although direct cal fatigue, calcification, glycation, lipid peroxidation, and interactions of LTBP3 with fibrillin-1 have not been shown, LTBP3 protein is reduced in the aorta of fibrillin-1 knockout protease digestion (35). The resulting elastin-derived peptides Ϫ/Ϫ (EDPs) are bioactive and retain the VGVAPG peptide se- (Fbln1 ) mice (213). In the early stages of elastic fiber quence found in tropoelastin, rendering similar signaling ca- assembly, microfibrils are abundant and there is little elastin. pability. EDP levels in the blood are increased in humans with Throughout late embryonic development and early maturation, obliterative arteriosclerosis of the legs, type IIb hyperlipid- elastin synthesis increases and eventually obscures the micro- emia, hypertension, diabetes, and ischemic heart disease (45). fibrils (196). It is possible that elastin accumulation indirectly ␤ Whether the increased levels of EDPs are a cause or conse- regulates TGF- availability by modulating LLC binding to ␤ quence of the disease is unknown. Recent studies in mice have microfibrils. TGF- 1 immunoreactivity and tropoelastin ex- suggested that while elastic fiber fragmentation may be sec- pression colocalize in the developing rat aorta and decrease ondary to the genetic or acquired disease, circulating EDPs with age (159). This has been interpreted as evidence of ␤ contribute to further disease progression. Chronic doses of TGF- 1-regulated expression of tropoelastin in vivo, but it EDPs, as well as VGVAPG peptides, increase the size of could also be interpreted as tropoelastin-regulated availability ␤ ␤ atherosclerotic plaques at the aortic root in two different of TGF- 1 as elastin accumulates on microfibrils. TGF- 1 atherosclerosis susceptible mouse models (47). EDPs are ca- signaling is altered when arterial elastic fibers are not assem- pable of binding the lactose-insensitive receptor found on the bled properly and through proteolytic cleavage of elastic fiber surface of macrophages, implicating a role for EDPs in mac- associated proteins, indicating bidirectional effects between ␤ rophage migration in inflammation and atherosclerosis pro- TGF- 1 activity and elastic fiber integrity (61). Elastinopathies gression (107). A single dose of EDPs, as well as VGVAPG that alter elastin amounts or elastic fiber integrity may then ␤ peptides, causes hyperglycemia in mice (162). Rat aortic affect TGF- activity (185). VSMCs incubated with EDPs show increased expression of CARDIOVASCULAR DISEASE typical bone proteins, which may contribute to arterial calcification (167). An antibody to VGVAPG blocks EDP signaling Several genetic and acquired cardiovascular diseases are to inflammatory cells and protects against elastase-induced associated with elastin and elastic fiber defects. The defects emphysema in mice (63). VGVAPG antibody also reduces include altered amounts of elastin and improper assembly, aortic elastic fiber fragmentation, macrophage infiltration, and fragmentation, and modification of elastic fibers. Additional TGF-␤1 activity in a mouse model of aortic aneurysm due to reviews on cardiovascular diseases associated with elastin or reduced fibrillin-1 levels (54). Finally, VGVAPG sequence- elastic fiber defects have been written by Baldwin et al. (7) and carrying EDPs have been shown to interact with the galectin-3 Milewicz et al. (115).

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00087.2018 • www.ajpheart.org Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (128.252.020.193) on September 19, 2018. Copyright © 2018 American Physiological Society. All rights reserved. ELASTIN, ARTERIAL MECHANICS, AND CARDIOVASCULAR DISEASE H195 Genetic Elastinopathies is loose, is hyperextensible, and has redundant folds. Patients may have cardiovascular pathologies including bicuspid aortic Supravalvular aortic stenosis. SVAS (OMIM no. 185500) is valve, dilation of the aorta or pulmonary artery, and pulmonary a rare (~1:20,000 births) congenital obstruction of the LV emphysema (55).The majority of reported ADCL1 cases have outflow tract. The defining defect is a lesion in the aorta at the a frame shift mutation within the last five exons of the ELN sinotubular junction (STJ) as a result of VSMC hypertrophy, gene (Fig. 1). The result of the frame shift is usually an increased collagen, and reduced/disorganized elastic fibers in elongation of tropoelastin protein that is believed to form larger the medial layer (29, 136). Often there are additional morpho- aggregates during coacervation and impair the ability of elastin logical defects in other elastic arteries, but the arteries affected to bind to the microfibrillar scaffold during elastic fiber assem- and severity of the defects are patient specific. There are three bly. Callewaert et al. (17) demonstrated impaired tropoelastin forms of SVAS: autosomal dominant familial, sporadic, and deposition onto microfibrils, increased TGF-␤ signaling, and Williams-Beuren syndrome (WBS; OMIM no. 194050) asso- indications of endoplasmic reticulum stress in cultured fibro- ciated. The genetic origin defines the form of SVAS. Famil- blasts from patients with ADCL1. In nearly 50% of ADCL1 ial and sporadic SVAS arise from loss-of-function mutations cases, aneurysms are present in the aortic root, aortic arch, or to the elastin gene that are either inherited or de novo, pulmonary artery. Aneurysm development is likely a combined respectively. A variety of mutations have been reported, result of reduced elastic fiber synthesis and increased suscep- including translocations, partial deletions, and point muta- tibility to elastic fiber degradation (36). tions (Fig. 1), often resulting in a truncated elastin protein. In WBS-associated SVAS, the entire elastin gene, along Mouse Models of Genetic Elastinopathies with 25–27 other genes, is encompassed in a large deletion on chromosome 7 (140). In all cases, one functional elastin Targeted deletion of the Eln gene in mice was first reported gene remains and sporadic mutations are more common than by Li et al. in 1998 (98). ElnϪ/Ϫ mice represent an extreme inherited mutations (5). elastinopathy and provide insights into the role of elastin in SVAS can present as one of two different types. Type I arterial mechanics, VSMC phenotype modulation, and cardio- (discrete) is defined by a localized narrowing at the STJ. Type vascular disease. The same year, Li et al. showed that Elnϩ/Ϫ II (diffuse) shows uniform narrowing beginning at the STJ and mice demonstrate some of the cardiovascular phenotypes ob- extending as far as the aortic arch or even the descending aorta served in patients with SVAS, such as increased arterial struc- (97). The ratio of the type I to type II incidence is ~7:3 (202). tural stiffness and an increased number of arterial lamellar Due to the rarity of SVAS, data correlating type I or II with units but do not have the local aortic lesions observed in SVAS specific elastin mutations are not available (30). In addition to (99). A mouse model of WBS with a large deletion that the primary STJ lesion, vascular defects occur in other arteries. includes the Eln gene (100) has a similar cardiovascular phe- Often the aortic valve leaflets are hypertrophic, resulting in notype to Elnϩ/Ϫ mice (49). Elnϩ/Ϫ mice have been used to partial fusion of the leaflets to the STJ (118). Thirty-nine study cardiovascular effects of reduced elastin amounts and the percent of patients with SVAS have bicuspid aortic valve (30). associated increases in arterial structural stiffness. Pulmonary artery stenosis is present in 83% of patients with Due to the insertion of a short exon after exon 4 in the mouse SVAS (112, 202). Lesions in the right ventricular outflow tract and the loss of two exons (34, 35) in the human during prim- are also common (30). Coronary arteries can exhibit fibrodys- ate evolution, the mouse Eln gene has 37 exons, whereas the plasia and may have stenotic lesions (160). Because coronaries human ELN gene has 34 exons (178). The human ELN gene are muscular arteries, it is unclear whether the lesions are a also shows unique alternative splicing patterns (40). To create direct result of the elastinopathy or are a secondary pathology. a mouse model to study ELN mutations in human disease, Hypertension is common in patients with WBS even at early Hirano et al. expressed ELN gene in a bacterial artificial ages (139) and may be linked to increased arterial structural chromosome (BAC) in ElnϪ/Ϫ mice (60). These hBAC-mNull stiffness due to reduced elastin amounts. Kozel et al. (85) mice only express human elastin and have total elastin amounts measured PWV in 77 patients with WBS and found an increase at 30–40% of normal (60). hBAC-mNull mice also show versus healthy control subjects. However, the increased PWV VSMC hypertrophy and aortic luminal narrowing representa- was independent of whether or not the patient was hypertensive tive of SVAS lesions (72). Sugitani et al. engineered a single (85). While hypertension and increases in arterial structural base deletion associated with ADCL1 within the ELN gene in stiffness generally develop coincidentally in acquired cardio- a BAC and expressed it in mice (175). The mutant elastin was vascular disease, their dissociation in WBS may indicate that incorporated into elastic fibers in the mouse skin and lung and reduced elastin amounts are a common causal factor. Patients compromised tissue function. The mutant elastin was incorpo- with SVAS have an increased risk of sudden cardiac death, rated at low levels into the mouse aorta and did not affect tissue with patients with WBS having the highest risk (202). The function. The results show tissue specific differences in elastic most common cause of sudden cardiac death is myocardial fiber assembly and highlight the utility of “humanized” mice ischemia, likely resulting from coronary obstruction and LV for studying human disease (175). hypertrophy (11). Ischemia could also be caused by reduced Effects of graded elastin amounts in mice. ElnϪ/Ϫ, Elnϩ/Ϫ, coronary perfusion during diastole due to a compromised and hBAC-mNull mice allow investigation of how graded Windkessel effect. Compromised arterial Windkessel function amounts of elastin affect cardiovascular function. They are would also increase cardiac afterload and could contribute to relevant for determining threshold amounts of elastin necessary LV hypertrophy. for restoring cardiovascular function in genetic and acquired Autosomal dominant cutis laxa-1. ADCL1 (OMIM no. elastinopathies, as well as in tissue-engineered arteries. Each 123700) is a rare connective tissue disorder in which the skin mouse model is discussed in additional detail below. Compar-

Fig. 5. Effects of elastin amounts on arterial wall structure and composition. AϪI: Histological sections (93) of the left common carotid artery from wild-type (WT) mice with 100% elastin (A, D, and G), Elnϩ/Ϫ mice with 50–60% elastin (B, E, and H), and hBAC-mNull mice with 30–40% elastin (C, F, and I). A–C: Verhoeff-Van Gieson (VVG)-stained sections show black elastic laminae, brown muscle tissue, and pink collagen. The elastic laminae are thinner, more numerous, and have more diffuse staining as elastin amounts decrease. DϪF: picro- sirius red (PSR)-stained sections show collagen ﬁ- bers in red and other material in yellow. In WT arteries, red collagen clearly outlines the yellow elastic laminae. In Elnϩ/Ϫ and hBAC-mNull arteries, the collagen staining overlaps with the elastic laminae. GϪI: hematoxylin and eosin (H&E)- stained sections show cell nuclei in purple and other material in pink. Scale bars ϭ 20 ␮m.

isons of arterial mechanics, wall structure, and morphology for similar aortic material stiffness to WT mice in the high pres- WT mice with 100% of normal elastin amounts, Elnϩ/Ϫ mice sure range (82). VSMCs from ElnϪ/Ϫ mice have higher pro- with 50–60% of normal elastin amounts, and hBAC-mNull liferation rates (76) and lower material stiffness values (37), mice with 30–40% of normal elastin amounts are shown in and these phenotypes can be partially reversed by the addition Figs. 4, 5, and 6, respectively. of tropoelastin to the cell culture medium. Treatment with the Ϫ Ϫ 0% ELASTIN. Eln / mice die within a few days of birth due mechanistic target of rapamycin (mTOR) inhibitor rapamycin to VSMC overproliferation in the aortic wall leading to luminal decreases VSMC overproliferation in ElnϪ/Ϫ mice but does not occlusion that is similar to the aortic root lesion observed in extend their lifespan (101). ␤3-Integrin expression is upregu- Ϫ/Ϫ Ϫ/Ϫ SVAS (98). Eln mice have normal blood pressure and lated in the Eln aorta, and genetically reducing ␤3-integrin aortic structural stiffness at embryonic day 18 (192) but high dosage extends the lifespan of ElnϪ/Ϫ mice by ~2 days (116). blood pressure and increased aortic structural stiffness at birth Pharmacological inhibition of ␤3-integrin with cilengitide re- (193). Aortas from ElnϪ/Ϫ mice have decreased aortic material duces VSMC overproliferation in the ElnϪ/Ϫ aorta and may be stiffness compared with WT mice in the low pressure range but a novel treatment to prevent SVAS (116).

Fig. 6. Effects of elastin amounts on arterial morphology. Yellow latex (172) was injected for contrast into the ascending aorta, major branches, and descending thoracic aorta in wild-type (WT; 100% elastin), Elnϩ/Ϫ (50–60% elastin), and hBAC- mNull (30–40% elastin) mice. With decreasing elastin amounts, the aortic diameter decreases and length increases. There are also changes in branch- ing morphology with reduced elastin amounts. Scale bars ϭ 1 mm.

ϩ Ϫ 50–60% ELASTIN. Eln / mice live a normal lifespan but have media, Jiao et al. (72) showed that deficient circumferential hypertension, increased arterial structural stiffness (Fig. 4), and growth that limits outward expansion of the aorta, combined an increased number of lamellar units in the arterial wall (Fig. with normal VSMC proliferation, is a mechanism for luminal 5) (39, 99). Treatment with the mTOR inhibitor rapamycin narrowing in hBAC-mNull mice. Inhibition of mTOR with prevents the formation of increased lamellar units in Elnϩ/Ϫ rapalogs does not prevent luminal narrowing in hBAC-mNull mice (101). Elnϩ/Ϫ arteries show reduced in vivo axial stretch, mice but does prevent the observed collagen fibrosis and residual torsion (197), and increased length (Fig. 6). Increased arterial structural stiffening (73). hBAC-mNull mice have a arterial structural stiffness precedes increases in blood pressure longer aorta than WT mice (Fig. 6), and the axial growth may compared with WT mice during maturation, indicating that it be a compensation mechanism for the deficient circumferential may be a causative factor in the development of hypertension growth (67). in Elnϩ/Ϫ mice (94). Elnϩ/Ϫ mice have indications of diastolic dysfunction, which may be linked to compromised arterial Acquired Cardiovascular Diseases Windkessel function and decreased cardiac perfusion during diastole (95). Elnϩ/Ϫ mice have impaired glucose metabolism Due to the limited timeframe of elastin synthesis, elastic (28) and altered susceptibility to atherosclerotic plaque accu- fibers that are damaged or degraded with aging or disease are mulation (108, 174). Genetic modifiers of the Elnϩ/Ϫ cardio- generally not repaired. Elastic fibers are then replaced by vascular phenotype have been identified by outcrossing Elnϩ/Ϫ collagens and proteoglycans that stiffen the arterial wall and mice. Several genes, including Ren1, Ncf1, and Nos1, were lead to hypertension and damage to downstream organs. The identified that may synergize with elastin insufficiency to ratio of elastin to collagen in the human aorta decreases 30–40% predispose an individual to hypertension and increased arterial with age from the second to the ninth decade of life (62). In aging structural stiffness (86). Treatment of Elnϩ/Ϫ mice with anti- primates, it was recently observed that the elastin-to-collagen ratio hypertensive medications improves pulse pressure and subse- changes with age, but disarray of the remaining elastic and quently physiologic structural stiffness due to the shift in collagen fibers may be an even more important contributor to operating pressures but does not change material stiffness of increased arterial stiffness with aging (211). Many acquired dis- the arteries (56). Treatment of Elnϩ/Ϫ mice with minoxidil, a eases represent accelerated or exacerbated versions of the arterial KATP channel opener and vasodilator, also improves physio- remodeling observed with aging. The cardiovascular diseases logic structural stiffness (84). Elnϩ/Ϫ mice have reduced sus- discussed in this review, with a focus on elastic fibers and arterial ceptibility to vascular calcification (Fig. 7) (79). Mice with mechanics, include hypertension and arterial stiffening, diabetes, reduced elastin due to a large genetic deletion that mimics the obesity, atherosclerosis, calcification, and aneurysms and dissec- heterozygous deletion observed in WBS (100) have hyperten- tions. sion and increased arterial stiffness but do not have the in- Hypertension and arterial stiffening. Hypertension is fre- creased number of lamellar units observed in Elnϩ/Ϫ mice (49). quently observed in conjunction with diseases associated with Ϫ Ϫ 30–40% ELASTIN. Eln / mice can be rescued by genetic defects in elastin or elastic fibers (4). While hypertension is insertion of a BAC expressing the human elastin gene (60). typically associated with aging, the coincidental occurrence of These hBAC-mNull mice have 35% of normal elastin amounts hypertension in infants with SVAS indicates that age-related in the ascending aorta, have extreme hypertension, and have factors are not required for SVAS-associated hypertension (85, increased arterial structural and material stiffness (Fig. 4) (60). 139) and that elastin insufficiency likely plays a key role. Increased arterial structural stiffness has been confirmed in Modulation of TGF-␤ availability by emilin-1, an elastic fiber- vivo using ultrasound imaging and may be caused by addi- associated protein, determines systolic blood pressure in mice, tional collagen amounts in the aortic media (Fig. 5) and supporting a role for elastic fiber integrity and TGF-␤ signaling increased wall thickness (72). While lesion development in in hypertension (209). TGF-␤ is an important regulator of SVAS is attributed to VSMC overproliferation in the aortic ECM remodeling in the arterial wall (57). Inflammatory signaling stimulated by EDPs from elastic fiber degradation may also play a role in hypertension. Reduced amounts of elastin or compromised elastic fiber integrity and the subsequent alterations in ECM composition and arterial geometry lead to increased material and/or structural arterial stiffness. While hypertension has long been associated with changes in resistance of the small, muscular arteries, it is now appreciated that increased stiffness of the large arteries can contribute to hypertension. The role of large artery stiffness in hypertension has been reviewed by Greenwald (52), Wagenseil and Mecham (194), and Mitchell (117), among others. Targeting ECM remodeling and the resulting changes in arterial stiffness represent a challenging but potentially effective treatment for hypertension (137). Fig. 7. Effects of elastin amounts on medial calcification caused by matrix Gla Spontaneous hypertensive rats (SHRs) are an animal model Ϫ Ϫ protein (MGP) deficiency. Mgp / mouse arteries show extensive mineral for essential hypertension developed through selective breed- deposition (black) along the elastic laminae (A), which is markedly reduced (arrows) in MgpϪ/ϪElnϩ/Ϫ mice (B). Thin plastic sections of the thoracic ing (127). In young and adult SHRs, systolic blood pressure is aortae from 2-wk-old mice were stained with von Kossa and van Gieson (79). increased over Wistar-Kyoto (WKY) rats, but material stiff- Scale bars ϭ 20 ␮m. ness of the thoracic aorta is not different (110), indicating that

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00087.2018 • www.ajpheart.org Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (128.252.020.193) on September 19, 2018. Copyright © 2018 American Physiological Society. All rights reserved. H198 ELASTIN, ARTERIAL MECHANICS, AND CARDIOVASCULAR DISEASE increased stiffness of the large arteries does not precede hy- In a rat diabetic model, glycation increases over time in arterial pertension in SHRs. SHRs have reduced elastin amounts, samples as diabetes develops but is unchanged in control rats smaller arterial diameter, and increased structural stiffness of (182). Diabetic rats also have reduced elastin protein levels the mesenteric resistance arteries compared with WKY rats, in aortic extracts as well as an increase in elastolytic activity suggesting that increased resistance of the distal arteries con- (91). A decreased elastin-to-collagen mRNA ratio and in- tributes to hypertension development in SHRs (13). With creased structural stiffness of the aorta are observed in the aging, aortic material stiffness dramatically increases in SHRs leptin receptor-deficient mouse model for type 2 diabetes compared with WKY rats (110). The increased stiffness has (db/db). Interestingly, the same study (78) found opposite been attributed to endothelial dysfunction (154), increases in trends in the elastin-to-collagen mRNA ratio and structural advanced glycation end products (6), and decreases in the stiffness for the db/db coronary artery. Human patients with elastin-to-collagen ratio (20) in the aged SHR compared with diabetes show a similar decrease in elastin-to-collagen pro- WKY aorta. These results imply that the SHR is not an tein ratios in aortic tissues (179) and an increase in circu- appropriate model to investigate large artery stiffness in the lating CatS levels (103). There are increased EDP levels in development of hypertension but is an appropriate model to children with diabetes (122), indicating that degradation of investigate the mechanisms of ECM remodeling and increased elastic fibers may occur in diabetes that contributes to arterial arterial stiffness in aging coupled with hypertension. stiffening and inflammatory signaling. EDPs may also contrib- Genetic factors associated with selective breeding of inbred ute to insulin resistance and the onset of diabetes with aging rat strains are linked to changes in amount and organization of (12). These results suggest that high glucose associated with the aortic ECM. Behmoaras et al. (10) found that aortic, blood diabetes may physically stiffen elastic fibers as well as make pressure, elastin, collagen and cytokine amounts, and elastic them more susceptible to degradation, altering passive arterial laminae structure are different among seven inbred rat strains. mechanics and initiating a chemical signaling cascade through Of the seven strains, the Brown-Norway rat (BNR) has the EDPs. lowest aortic elastin amount and lowest elastin-to-collagen Obesity. Obesity is a risk factor for increased cardiovascu- ratio, leading to increased structural stiffness of the aorta lar-related mortality, but it is typically associated with addi- compared with young LOU control rats (130). BNRs are tional cardiovascular risk factors including hypertension and normotensive, suggesting that they are a good model for diabetes. Rider et al. (144) observed an increase in aortic PWV investigating interactions between elastin and arterial stiffness in obese patients absent other cardiovascular risk factors, that are independent of changes in blood pressure. Treatment providing evidence for a direct correlation between obesity and of cultured BNR VSMCs with Kϩ channel openers (minoxidil aortic structural stiffness. Elastin is decreased in adipose tissue or diazoxide) increases tropoelastin transcription and mRNA of obese patients (170); however, investigations on elastin and stability. Treatment of BNRs with minoxidil or diazoxide elastic fiber changes in large arteries from obese patients have increases aortic elastic fiber content (169). In contrast, treat- yet to be performed. TGF-␤1 levels positively correlate with ment of Elnϩ/Ϫ mice with minoxidil increases arterial size and body mass index in humans (184) and are upregulated in stimulates differential expression of 127 matrix or matrix- adipose tissue from obese humans (2) and mice (157). Cathep- associated genes but does not show an elastin specific effect sin elastases, CatK, CatL, and CatS have increased activity (84). Therapeutic options for increasing elastic fiber content with increased body mass index in humans and in the leptin- may depend on the primary cause of the elastinopathy. deficient (ob/ob) mouse model of obesity (92). In ob/ob mice, Diabetes. In diabetes, the body does not produce or respond aortic stiffening was observed by an increase in PWV, an to insulin appropriately, leading to increased blood glucose increase in abdominal aorta structural stiffness in vitro, and an levels. In vitro studies have examined the effects of increased increase in thoracic aorta material stiffness as measured by glucose on VSMCs, isolated arterial elastin, and arterial me- atomic force microscopy. Elastic fiber remodeling in the ob/ob chanical behavior. High-glucose media, with the addition of aorta was evidenced by a decrease in LOX protein levels and EDPs and TGF-␤1, increase expression of osteogenic genes in activity, a decrease in desmosine cross-links, an increase in cultured VSMCs, and may lead to arterial calcification associ- elastase activity, and a higher degree of fragmentation in the ated with diabetes (168). Incubation of isolated arterial elastin elastic lamellae (21). The observed increase in aortic stiffness in high-glucose solution increases the storage and loss modu- with obesity may arise from elastic fiber remodeling through lus, indicating an increase in material stiffness and viscous LOX downregulation and an upregulation of elastolytic activ- energy loss (102). After 48 h of incubation in a high-glucose ity. solution, Zou and Zhang (214) found an increase in the mate- Atherosclerosis. Both the synthesis of tropoelastin and deg- rial stiffness of isolated arterial elastin in the longitudinal radation of elastic fibers are increased in human atherosclerotic direction but not in the circumferential direction. With incu- lesions (1). This may be due to the production of tropoelastin bation times up to 28 days, Wang et al. (199) found an (88), metalloproteinases (121), and cathepsins (92) by macro- increased material stiffness in both longitudinal and circum- phages involved in atherosclerosis. Elastic fiber degradation ferential directions as well as increased hysteresis (or energy allows infiltration of lipids and immune cells into the aortic loss). The mechanical changes in isolated arterial elastin have wall for plaque formation and rupture in mice (188). EDPs been attributed to the dehydrating effects of glycation (102), resulting from elastic fiber fragmentation further enhance but it is unclear how these results translate in vivo for an intact atherogenesis (47). TGF-␤1 activity, which may be modulated arterial wall with ECs acting as an impermeable barrier. As- by elastic fiber degradation, has a wide range of effects in suming that the glucose can reach the medial layers, increased atherosclerosis (181). Elastin insufficiency and the associated glucose may lead to glycation of elastic fibers that alters increase in arterial structural stiffness do not increase athero- stiffness and protease susceptibility. sclerotic plaque deposition in mice (108, 174). Together, these

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00087.2018 • www.ajpheart.org Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (128.252.020.193) on September 19, 2018. Copyright © 2018 American Physiological Society. All rights reserved. ELASTIN, ARTERIAL MECHANICS, AND CARDIOVASCULAR DISEASE H199 results suggest that elastic fiber degradation and the resulting models demonstrate aortic dilation and aneurysm formation but circulating peptides are key mechanisms linking elastin to do not progress to aortic rupture or dissection (161). Models atherosclerosis progression. that progress to rupture and dissection will be critical for Calcification. Elastic fiber degradation has been linked to translating the results of animal studies to treatment of human ectopic calcium phosphate mineral deposition (calcification) in disease. the arterial walls, particularly in the medial layer. As demonstrated by Basalyga et al. (8), periadventitial treatment of TISSUE ENGINEERING surgically exposed rat aortas with calcium chloride leads to Increasing elastin amounts in genetic elastinopathies or re- ectopic mineral deposition along the elastic laminae. Calcified pairing fragmented elastic fibers in acquired cardiovascular aortas of treated rats show the presence of degraded ECM diseases are challenging problems. Tissue engineering, how- proteins, including elastin. Later, Khavandgar et al. (79) ever, encounters the most challenging problem, recreating showed that medial calcification in mice lacking matrix Gla functional elastic fibers de novo. Elastogenesis has been called protein (MGP), a potent inhibitor of soft tissue calcification, is the “holy grail” of arterial tissue engineering, as the lack of elastin dependent. The amount of deposited minerals in Ϫ Ϫ mature elastic fibers in tissue-engineered constructs results in Mgp / arteries scales with elastin amounts in the cardiovas- inferior mechanical properties compared with native arteries cular tree, with mineral deposition and elastin amounts de- (135). Long and Tranquillo (104) showed that the type of creasing from the thoracic to the abdominal aorta. Khavandgar Ϫ Ϫ ϩ Ϫ scaffold and cells chosen affects elastogenesis in tissue-engi- et al. (79) further showed that Mgp / Eln / mice have neered arteries, with increased elastin amounts secreted from markedly reduced arterial mineral deposition compared with Ϫ Ϫ neonatal compared with adult cells and on fibrin compared Mgp / mice (Fig. 7). This genetic experiment provides evi- with collagen scaffolds. They also used chemokines known to dence that elastin content, and not just elastic fiber degradation, increase steady-state elastin mRNA expression, but the result- is a critical determinant of arterial medial calcification. ing elastic fiber structure in their constructs does not resemble Currently, the mechanism of mineral accumulation by elas- arterial elastic laminae. Trying to replicate in vivo loading tin and how this process is inhibited by MGP are not well conditions, Kim et al. (81) showed that long-term application understood. Also, the role of elastin in intimal calcification of circumferential cyclic strain increases tropoelastin mRNA associated with atherosclerotic plaques is still unknown. Future and protein expression along with increasing the material mechanistic work focusing on these aspects of elastin biology stiffness and ultimate tensile strength of tissue-engineered may lead to new therapeutic approaches to treat vascular arteries, although the mechanical properties are still inferior to calcification, for which no treatment is currently available native aorta. Huang et al. (65) recently showed that application Aneurysms and dissections. Degraded and fragmented elas- of biaxial cyclic strain, more representative of the in vivo tic fibers in the arterial wall are a common histopathology in loading environment, produces tissue-engineered arteries with thoracic and abdominal aortic aneurysms (18, 114). It appears evidence of mature elastic fibers and improved mechanical that fragmented elastic fibers or improper elastic fiber deposi- properties compared with circumferentially strained constructs tion is critical to aneurysm formation rather than reduced or constructs grown without cyclic strain. A tissue-engineered elastin amounts. ADCL1, characterized by improper elastic graft implanted as a pulmonary artery replacement in 8-wk-old fiber deposition, is generally associated with aneurysms, lambs exposed to cytokines and mechanical loading present in whereas SVAS, characterized by elastin insufficiency, is gen- early maturation and growth showed abundant elastic fiber erally not associated with aneurysms. Additionally, genetic deposition and ~50% of the insoluble elastin levels of the mutations that affect proteins necessary for elastic fiber assem- native pulmonary artery when evaluated 42 wk after implan- bly, such as FBLN4 (66) and LOX (53), cause aneurysms. tation (177). Overall, these results demonstrate the importance Loss-of-function mutations in genes that affect TGF-␤ se- 1 of reproducing the biochemical and biomechanical environ- questration in the ECM, such as fibrillin-1, are associated with ment of developing arteries to encourage elastogenesis in tissue aberrant TGF-␤ activity and aortic aneurysms (31). Frag- 1 engineering. mented, degraded, or improperly assembled elastic fibers may be more susceptible to protease digestion, allowing aneurysmal SUMMARY AND FUTURE DIRECTIONS dilation over time and stimulating cytokine and inflammatory signaling. Homogenates of abdominal aortic aneurysms show We have summarized genetic and acquired cardiovascular high elastinolytic activity (18). Degraded or improperly assem- diseases linked to elastin and elastic fiber defects with a focus bled elastic fibers allow inflammatory cell infiltration and on large artery mechanics. Elastin and elastic fibers play a affect TGF-␤1 sequestration, and the released EDPs can en- critical mechanical role at multiple length scales from pulse hance macrophage activation (24) and TGF-␤1 activity (54). wave reflections in the cardiovascular system, to local material Researchers have capitalized on the relationship among properties of the arterial wall, to interactions of tropoelastin elastic fiber degradation, inflammatory cell activation, and with VSMCs. Elastin is a unique protein due to its limited time chemokine signaling to generate a variety of chemically stim- period of synthesis, long half-life, multistep assembly process, ulated models of abdominal aortic aneurysms and dissections and high degree of cross-linking that provides mechanical in animals (3, 25, 106). The models include intraluminal or elasticity to the large arteries. Acquired damage to the elastic periadventitial application of elastase, periadventitial incuba- fibers is especially problematic due to the inability of the tion with calcium chloride, and subcutaneous infusion of an- organism to generate de novo elastic fibers postadolescence. giotensin II. Several of the models have been extended to Despite the increasing therapeutic options for controlling dis- create thoracic aortic aneurysms and dissections (69, 74). It is eases such as hypertension, obesity, and diabetes, elastic fiber important to note that many of the chemically induced animal damage is currently irreparable, meaning that the cardiovascu-

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00087.2018 • www.ajpheart.org Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (128.252.020.193) on September 19, 2018. Copyright © 2018 American Physiological Society. All rights reserved. H200 ELASTIN, ARTERIAL MECHANICS, AND CARDIOVASCULAR DISEASE lar risks associated with arterial stiffness may persist even 2. Alessi MC, Bastelica D, Morange P, Berthet B, Leduc I, Verdier M, when other disease aspects are controlled. The process of Geel O, Juhan-Vague I. Plasminogen activator inhibitor 1, transforming growth factor-beta1, and BMI are closely associated in human adipose elastic fiber assembly, especially inducing assembly in vitro or tissue during morbid obesity. Diabetes 49: 1374–1380, 2000. doi:10. in vivo, is an active and important area of investigation for 2337/diabetes.49.8.1374. treating elastinopathies and the associated changes in cardio- 3. Anidjar S, Salzmann JL, Gentric D, Lagneau P, Camilleri JP, Michel vascular mechanics, as well as advancing tissue engineering. JB. Elastase-induced experimental aneurysms in rats. Circulation 82: We would like to highlight the differential effects of reduced 973–981, 1990. doi:10.1161/01.CIR.82.3.973. 4. Arribas SM, Hinek A, González MC. Elastic fibres and vascular elastin amounts compared with improper assembly or degra- structure in hypertension. Pharmacol Ther 111: 771–791, 2006. doi:10. dation of elastic fibers. Reduced elastin amounts lead to altered 1016/j.pharmthera.2005.12.003. arterial wall structure, narrowed arterial lumen, increased ar- 5. Ashkenas J. Williams syndrome starts making sense. Am J Hum Genet terial structural stiffness, and hypertension in humans and 59: 756–761, 1996. mice. In mice, the structural, mechanical, and functional phe- 6. Atanasova M, Dimitrova A, Ruseva B, Stoyanova A, Georgieva M, Konova E. Quantification of elastin, collagen, and advanced glycation notypes scale with elastin amounts. For the most part, mice end products as a function of age and hypertension. In: Senescence, appear to adapt reasonably well to 50–60% elastin, with more edited by Nagata T. Rijeka, Croatia: In Tech, 2012, p. 519Ϫ530. severe phenotypes appearing when elastin amounts drop to 7. Baldwin AK, Simpson A, Steer R, Cain SA, Kielty CM. Elastic fibres 30–40% of normal. The results of the mouse studies suggest in health and disease. Expert Rev Mol Med 15: e8, 2013. doi:10.1017/ erm.2013.9. that a threshold amount of elastin may provide near-normal 8. Basalyga DM, Simionescu DT, Xiong W, Baxter BT, Starcher BC, cardiovascular function in genetic diseases associated with Vyavahare NR. Elastin degradation and calcification in an abdominal elastin insufficiency. Hence, therapies that moderately increase aorta injury model: role of matrix metalloproteinases. Circulation 110: elastin amounts, rather than completely restoring them to 3480–3487, 2004. doi:10.1161/01.CIR.0000148367.08413.E9. normal levels, may be sufficient for improved cardiovascular 9. Bashir MM, Indik Z, Yeh H, Ornstein-Goldstein N, Rosenbloom JC, Abrams W, Fazio M, Uitto J, Rosenbloom J. Characterization of the outcomes. complete human elastin gene. Delineation of unusual features in the Improperly assembled or degraded elastic fibers alter the 5=-flanking region. J Biol Chem 264: 8887–8891, 1989. passive mechanical properties of the arterial wall. The conse- 10. Behmoaras J, Osborne-Pellegrin M, Gauguier D, Jacob MP. Char- quences extend far beyond physical changes to the elastic acteristics of the aortic elastic network and related phenotypes in seven Ϫ fibers to the initiation of several arterial remodeling events inbred rat strains. Am J Physiol Heart Circ Physiol 288: H769 H777, 2005. doi:10.1152/ajpheart.00544.2004. including VSMC phenotype modulation, ECM deposition, in- 11. Bird LM, Billman GF, Lacro RV, Spicer RL, Jariwala LK, Hoyme flammatory cell infiltration, and the release of EDPs. EDPs HE, Zamora-Salinas R, Morris C, Viskochil D, Frikke MJ, Jones stimulate cytokine signaling and activate inflammatory cells. MC. Sudden death in Williams syndrome: report of ten cases. J Pediatr The inflammatory cells secrete proteases that contribute to 129: 926–931, 1996. doi:10.1016/S0022-3476(96)70042-2. 12. Blaise S, Romier B, Kawecki C, Ghirardi M, Rabenoelina F, Baud S, additional elastic fiber degradation in a positive feedback Duca L, Maurice P, Heinz A, Schmelzer CE, Tarpin M, Martiny L, cycle. It is unclear whether the arterial remodeling signaling Garbar C, Dauchez M, Debelle L, Durlach V. Elastin-derived peptides cascade is similar for genetic diseases with improperly assem- are new regulators of insulin resistance development in mice. Diabetes bled elastic fibers, such as ADCL1, and acquired diseases with 62: 3807–3816, 2013. doi:10.2337/db13-0508. elastic fiber degradation, such as diabetes or abdominal aortic 13. Briones AM, González JM, Somoza B, Giraldo J, Daly CJ, Vila E, González MC, McGrath JC, Arribas SM. Role of elastin in sponta- aneurysms. However, elastic fiber fragmentation, inflammatory neously hypertensive rat small mesenteric artery remodelling. J Physiol signaling, and arterial stiffening are common factors in many 552: 185–195, 2003. doi:10.1113/jphysiol.2003.046904. of the cardiovascular diseases discussed in this review. Better 14. Broekelmann TJ, Kozel BA, Ishibashi H, Werneck CC, Keeley FW, understanding of the biochemical and biomechanical signaling Zhang L, Mecham RP. Tropoelastin interacts with cell-surface glycos- associated with improperly assembled or degraded elastic fi- aminoglycans via its COOH-terminal domain. J Biol Chem 280: 40939– 40947, 2005. doi:10.1074/jbc.M507309200. bers may allow therapeutic interventions that halt or slow down 15. Burnett W, Finnigan-Bunick A, Yoon K, Rosenbloom J. Analysis of the arterial remodeling cascade and prevent disease progres- elastin gene expression in the developing chick aorta using cloned elastin sion. cDNA. J Biol Chem 257: 1569–1572, 1982. 16. Burton AC. Relation of structure to function of the tissues of the wall of blood vessels. Physiol Rev 24: 619Ϫ642, 1954. GRANTS 17. Callewaert B, Renard M, Hucthagowder V, Albrecht B, Hausser I, This work was partially supported by National Heart, Lung, and Blood Blair E, Dias C, Albino A, Wachi H, Sato F, Mecham RP, Loeys B, Institute Grants HL-115560 and HL-105314. Coucke PJ, De Paepe A, Urban Z. New insights into the pathogenesis of autosomal-dominant cutis laxa with report of five ELN mutations. Hum Mutat 32: 445–455, 2011. doi:10.1002/humu.21462. DISCLOSURES 18. Campa JS, Greenhalgh RM, Powell JT. Elastin degradation in abdom- No conflicts of interest, financial or otherwise, are declared by the authors. inal aortic aneurysms. Atherosclerosis 65: 13–21, 1987. doi:10.1016/ 0021-9150(87)90003-7. AUTHOR CONTRIBUTIONS 19. Cantor JO, Keller S, Parshley MS, Darnule TV, Darnule AT, Cer- reta JM, Turino GM, Mandl I. Synthesis of crosslinked elastin by an J.Z.H., M.C.S., and E.O.J. performed experiments. A.J.C., J.Z.H., M.C.S., endothelial cell culture. Biochem Biophys Res Commun 95: 1381–1386, E.O.J., M.M., and J.E.W. prepared figures; A.J.C., M.M., and J.E.W. drafted 1980. doi:10.1016/S0006-291X(80)80050-7. manuscript; A.J.C. and J.E.W. edited and revised manuscript; A.J.C., J.Z.H., 20. Chamiot-Clerc P, Renaud JF, Safar ME. Pulse pressure, aortic reac- M.C.S., E.O.J., M.M., and J.E.W. approved final version of manuscript. tivity, and endothelium dysfunction in old hypertensive rats. Hyperten- sion 37: 313–321, 2001. doi:10.1161/01.HYP.37.2.313. REFERENCES 21. Chen JY, Tsai PJ, Tai HC, Tsai RL, Chang YT, Wang MC, Chiou YW, Yeh ML, Tang MJ, Lam CF, Shiesh SC, Li YH, Tsai WC, Chou 1. Akima T, Nakanishi K, Suzuki K, Katayama M, Ohsuzu F, Kawai T. CH, Lin LJ, Wu HL, Tsai YS. Increased aortic stiffness and attenuated Soluble elastin decreases in the progress of atheroma formation in human lysyl oxidase activity in obesity. Arterioscler Thromb Vasc Biol 33: aorta. Circ J 73: 2154–2162, 2009. 839–846, 2013. doi:10.1161/ATVBAHA.112.300036.

22. Clarke AW, Arnspang EC, Mithieux SM, Korkmaz E, Braet F, 42. Fonck E, Prod’hom G, Roy S, Augsburger L, Rüfenacht DA, Ster- Weiss AS. Tropoelastin massively associates during coacervation to giopulos N. Effect of elastin degradation on carotid wall mechanics as form quantized protein spheres. Biochemistry 45: 9989–9996, 2006. assessed by a constituent-based biomechanical model. Am J Physiol doi:10.1021/bi0610092. Heart Circ Physiol 292: H2754–H2763, 2007. doi:10.1152/ajpheart. 23. Cox BA, Starcher BC, Urry DW. Communication: coacervation of 01108.2006. tropoelastin results in fiber formation. J Biol Chem 249: 997–998, 1974. 43. Foster JA, Rubin L, Kagan HM, Franzblau C, Bruenger E, Sand- 24. Dale MA, Xiong W, Carson JS, Suh MK, Karpisek AD, Meisinger berg LB. Isolation and characterization of cross-linked peptides from TM, Casale GP, Baxter BT. Elastin-derived peptides promote abdom- elastin. J Biol Chem 249: 6191–6196, 1974. inal aortic aneurysm formation by modulating M1/M2 macrophage 44. Fritze O, Romero B, Schleicher M, Jacob MP, Oh DY, Starcher B, polarization. J Immunol 196: 4536–4543, 2016. doi:10.4049/jimmunol. Schenke-Layland K, Bujan J, Stock UA. Age-related changes in the 1502454. elastic tissue of the human aorta. J Vasc Res 49: 77–86, 2012. doi:10. 25. Daugherty A, Cassis LA. Mouse models of abdominal aortic aneu- 1159/000331278. rysms. Arterioscler Thromb Vasc Biol 24: 429–434, 2004. doi:10.1161/ 45. Fülöp T Jr, Wei SM, Robert L, Jacob MP. Determination of elastin 01.ATV.0000118013.72016.ea. peptides in normal and arteriosclerotic human sera by ELISA. Clin 26. Davidson JM, Smith K, Shibahara S, Tolstoshev P, Crystal RG. Physiol Biochem 8: 273–282, 1990. Regulation of elastin synthesis in developing sheep nuchal ligament by 46. Gadson PF Jr, Rossignol C, McCoy J, Rosenquist TH. Expression of elastin mRNA levels. J Biol Chem 257: 747–754, 1982. elastin, smooth muscle alpha-actin, and c-jun as a function of the 27. Davis EC. Smooth muscle cell to elastic lamina connections in devel- embryonic lineage of vascular smooth muscle cells. In Vitro Cell Dev oping mouse aorta. Role in aortic medial organization. Lab Invest 68: Biol Anim 29A: 773–781, 1993. doi:10.1007/BF02634344. 89–99, 1993. 47. Gayral S, Garnotel R, Castaing-Berthou A, Blaise S, Fougerat A, 28. DeMarsilis AJ, Walji TA, Maedeker JA, Stoka KV, Kozel BA, Berge E, Montheil A, Malet N, Wymann MP, Maurice P, Debelle L, Mecham RP, Wagenseil JE, Craft CS. Elastin Insufficiency Predis- Martiny L, Martinez LO, Pshezhetsky AV, Duca L, Laffargue M. poses Mice to Impaired Glucose Metabolism. J Mol Genet Med 8: 129, Elastin-derived peptides potentiate atherosclerosis through the immune ␥ 2014. doi:10.4172/1747-0862.1000129. Neu1-PI3K pathway. Cardiovasc Res 102: 118–127, 2014. doi:10. 29. Denie JJ, Verheugt AP. Supravalvular aortic stenosis. Circulation 18: 1093/cvr/cvt336. 902–908, 1958. doi:10.1161/01.CIR.18.5.902. 48. Gibbons CA, Shadwick RE. Functional similarities in the mechanical 30. Deo SV, Burkhart HM, Dearani JA, Schaff HV. Supravalvar aortic design of the aorta in lower vertebrates and mammals. Experientia 45: stenosis: current surgical approaches and outcomes. Expert Rev Cardio- 1083–1088, 1989. doi:10.1007/BF01950164. vasc Ther 11: 879–890, 2013. doi:10.1586/14779072.2013.811967. 49. Goergen CJ, Li HH, Francke U, Taylor CA. Induced chromosome 31. Dietz HC, Loeys B, Carta L, Ramirez F. Recent progress towards a deletion in a Williams-Beuren syndrome mouse model causes cardiovas- molecular understanding of Marfan syndrome. Am J Med Genet C Semin cular abnormalities. J Vasc Res 48: 119–129, 2011. doi:10.1159/ Med Genet 139C: 4–9, 2005. doi:10.1002/ajmg.c.30068. 000316808. 50. Gosling RG, Budge MM. Terminology for describing the elastic behav- 32. Dingemans KP, Teeling P, Lagendijk JH, Becker AE. Extracellular ior of arteries. Hypertension 41: 1180–1182, 2003. doi:10.1161/01.HYP. matrix of the human aortic media: an ultrastructural histochemical and 0000072271.36866.2A. immunohistochemical study of the adult aortic media. Anat Rec 258: 1–14, 51. Gray WR, Sandberg LB, Foster JA. Molecular model for elastin 2000. doi:10.1002/(SICI)1097-0185(20000101)258:1Ͻ1::AID-AR1Ͼ3.0. structure and function. Nature 246: 461–466, 1973. doi:10.1038/ CO;2-7. 246461a0. 33. Dobrin PB, Canfield TR. Elastase, collagenase, and the biaxial elastic 52. Greenwald SE. Ageing of the conduit arteries. J Pathol 211: 157–172, properties of dog carotid artery. Am J Physiol Heart Circ Physiol 247: 2007. doi:10.1002/path.2101. H124–H131, 1984. doi:10.1152/ajpheart.1984.247.1.H124. 53. Guo DC, Regalado ES, Gong L, Duan X, Santos-Cortez RL, Arnaud Dubick MA, Rucker RB, Cross CE, Last JA. 34. Elastin metabolism in P, Ren Z, Cai B, Hostetler EM, Moran R, Liang D, Estrera A, Safi rodent lung. Biochim Biophys Acta 672: 303–306, 1981. doi:10.1016/ HJ, Leal SM, Bamshad MJ, Shendure J, Nickerson DA, Jondeau G, 0304-4165(81)90297-X. Boileau C, Milewicz DM; University of Washington Center for 35. Duca L, Blaise S, Romier B, Laffargue M, Gayral S, El Btaouri H, Mendelian Genomics. LOX mutations predispose to thoracic aortic Kawecki C, Guillot A, Martiny L, Debelle L, Maurice P. Matrix aneurysms and dissections. Circ Res 118: 928–934, 2016. doi:10.1161/ ageing and vascular impacts: focus on elastin fragmentation. Cardiovasc CIRCRESAHA.115.307130. Res 110: 298–308, 2016. doi:10.1093/cvr/cvw061. 54. Guo G, Muñoz-García B, Ott CE, Grünhagen J, Mousa SA, Pletsch- 36. Duz MB, Kirat E, Coucke PJ, Koparir E, Gezdirici A, Paepe A, acher A, von Kodolitsch Y, Knaus P, Robinson PN. Antagonism of Callewaert B, Seven M. A novel case of autosomal dominant cutis laxa GxxPG fragments ameliorates manifestations of aortic disease in Marfan in a consanguineous family: report and literature review. Clin Dysmor- syndrome mice. Hum Mol Genet 22: 433–443, 2013. doi:10.1093/hmg/ phol 26: 142–147, 2017. doi:10.1097/MCD.0000000000000179. dds439. 37. Espinosa MG, Gardner WS, Bennett L, Sather B, Yanagisawa H, 55. Hadj-Rabia S, Callewaert BL, Bourrat E, Kempers M, Plomp AS, Wagenseil JE. The effects of elastic fiber protein insufficiency and Layet V, Bartholdi D, Renard M, De Backer J, Malfait F, Vanakker treatment on the modulus of arterial smooth muscle cells. J Biomech Eng OM, Coucke PJ, De Paepe AM, Bodemer C. Twenty patients including 136: 021030, 2014. doi:10.1115/1.4026203. 7 probands with autosomal dominant cutis laxa confirm clinical and 38. Faury G, Maher GM, Li DY, Keating MT, Mecham RP, Boyle WA. molecular homogeneity. Orphanet J Rare Dis 8: 36, 2013. doi:10.1186/ Relation between outer and luminal diameter in cannulated arteries. Am 1750-1172-8-36. J Physiol Heart Circ Physiol 277: H1745–H1753, 1999. 56. Halabi CM, Broekelmann TJ, Knutsen RH, Ye L, Mecham RP, 39. Faury G, Pezet M, Knutsen RH, Boyle WA, Heximer SP, McLean Kozel BA. Chronic antihypertensive treatment improves pulse pressure SE, Minkes RK, Blumer KJ, Kovacs A, Kelly DP, Li DY, Starcher B, but not large artery mechanics in a mouse model of congenital vascular Mecham RP. Developmental adaptation of the mouse cardiovascular stiffness. Am J Physiol Heart Circ Physiol 309: H1008–H1016, 2015. system to elastin haploinsufficiency. J Clin Invest 112: 1419–1428, 2003. doi:10.1152/ajpheart.00288.2015. doi:10.1172/JCI19028. 57. Harvey A, Montezano AC, Lopes RA, Rios F, Touyz RM. Vascular 40. Fazio MJ, Olsen DR, Kauh EA, Baldwin CT, Indik Z, Ornstein- fibrosis in aging and hypertension: molecular mechanisms and clinical Goldstein N, Yeh H, Rosenbloom J, Uitto J. Cloning of full-length implications. Can J Cardiol 32: 659–668, 2016. doi:10.1016/j.cjca.2016. elastin cDNAs from a human skin fibroblast recombinant cDNA library: 02.070. further elucidation of alternative splicing utilizing exon-specific oligo- 58. Hinek A, Pshezhetsky AV, von Itzstein M, Starcher B. Lysosomal nucleotides. J Invest Dermatol 91: 458–464, 1988. doi:10.1111/1523- sialidase (neuraminidase-1) is targeted to the cell surface in a multipro- 1747.ep12476591. tein complex that facilitates elastic fiber assembly. J Biol Chem 281: 41. Ferruzzi J, Bersi MR, Humphrey JD. Biomechanical phenotyping of 3698–3710, 2006. doi:10.1074/jbc.M508736200. central arteries in health and disease: advantages of and methods for 59. Hinek A, Wrenn DS, Mecham RP, Barondes SH. The elastin receptor: murine models. Ann Biomed Eng 41: 1311–1330, 2013. doi:10.1007/ a galactoside-binding protein. Science 239: 1539–1541, 1988. doi:10. s10439-013-0799-1. 1126/science.2832941.

60. Hirano E, Knutsen RH, Sugitani H, Ciliberto CH, Mecham RP. pattern of remodeling associated with decreased vessel stiffness. Basic Functional rescue of elastin insufficiency in mice by the human elastin Res Cardiol 106: 1123–1134, 2011. doi:10.1007/s00395-011-0201-0. gene: implications for mouse models of human disease. Circ Res 101: 79. Khavandgar Z, Roman H, Li J, Lee S, Vali H, Brinckmann J, Davis 523–531, 2007. doi:10.1161/CIRCRESAHA.107.153510. EC, Murshed M. Elastin haploinsufficiency impedes the progression of 61. Horiguchi M, Ota M, Rifkin DB. Matrix control of transforming arterial calcification in MGP-deficient mice. J Bone Miner Res 29: growth factor-␤ function. J Biochem 152: 321–329, 2012. doi:10.1093/ 327–337, 2014. doi:10.1002/jbmr.2039. jb/mvs089. 80. Kielty CM, Sherratt MJ, Shuttleworth CA. Elastic fibres. J Cell Sci 62. Hosoda Y, Kawano K, Yamasawa F, Ishii T, Shibata T, Inayama S. 115: 2817–2828, 2002. Age-dependent changes of collagen and elastin content in human aorta 81. Kim BS, Nikolovski J, Bonadio J, Mooney DJ. Cyclic mechanical and pulmonary artery. Angiology 35: 615–621, 1984. doi:10.1177/ strain regulates the development of engineered smooth muscle tissue. Nat 000331978403501001. Biotechnol 17: 979–983, 1999. doi:10.1038/13671. 63. Houghton AM, Quintero PA, Perkins DL, Kobayashi DK, Kelley 82. Kim J, Staiculescu MC, Cocciolone AJ, Yanagisawa H, Mecham RP, DG, Marconcini LA, Mecham RP, Senior RM, Shapiro SD. Elastin Wagenseil JE. Crosslinked elastic fibers are necessary for low energy fragments drive disease progression in a murine model of emphysema. J loss in the ascending aorta. J Biomech 61: 199–207, 2017. doi:10.1016/ Clin Invest 116: 753–759, 2006. doi:10.1172/JCI25617. j.jbiomech.2017.07.011. 64. Howard PS, Macarak EJ. Localization of collagen types in regional 83. Kim YM, Haghighat L, Spiekerkoetter E, Sawada H, Alvira CM, segments of the fetal bovine aorta. Lab Invest 61: 548–555, 1989. Wang L, Acharya S, Rodriguez-Colon G, Orton A, Zhao M, Rabin- 65. Huang AH, Balestrini JL, Udelsman BV, Zhou KC, Zhao L, Ferruzzi ovitch M. Neutrophil elastase is produced by pulmonary artery smooth J, Starcher BC, Levene MJ, Humphrey JD, Niklason LE. Biaxial muscle cells and is linked to neointimal lesions. Am J Pathol 179: stretch improves elastic fiber maturation, collagen arrangement, and 1560–1572, 2011. doi:10.1016/j.ajpath.2011.05.051. mechanical properties in engineered arteries. Tissue Eng Part C Methods 84. Knutsen R, Beeman SC, Broekelmann TJ, Liu D, Tsang KM, Kovacs 22: 524–533, 2016. doi:10.1089/ten.tec.2015.0309. A, Ye L, Danback J, Watson A, Wardlaw A, Wagenseil J, Garbow 66. Hucthagowder V, Sausgruber N, Kim KH, Angle B, Marmorstein JR, Shoykhet M, Kozel BA. Minoxidil improves vascular compliance, LY, Urban Z. Fibulin-4: a novel gene for an autosomal recessive cutis restores cerebral blood flow and alters extracellular matrix gene expres- laxa syndrome. Am J Hum Genet 78: 1075–1080, 2006. doi:10.1086/ sion in a model of chronic vascular stiffness. Am J Physiol Heart Circ 504304. Physiol. doi:10.1152/ajpheart.00683.2017. 67. Humphrey JD, Eberth JF, Dye WW, Gleason RL. Fundamental role 85. Kozel BA, Danback JR, Waxler JL, Knutsen RH, de Las Fuentes L, of axial stress in compensatory adaptations by arteries. J Biomech 42: Reusz GS, Kis E, Bhatt AB, Pober BR. Williams syndrome predisposes 1–8, 2009. doi:10.1016/j.jbiomech.2008.11.011. to vascular stiffness modified by antihypertensive use and copy number 68. Hungerford JE, Owens GK, Argraves WS, Little CD. Development of changes in NCF1. Hypertension 63: 74–79, 2014. doi:10.1161/ the aortic vessel wall as defined by vascular smooth muscle and extra- HYPERTENSIONAHA.113.02087. cellular matrix markers. Dev Biol 178: 375–392, 1996. doi:10.1006/dbio. 86. Kozel BA, Knutsen RH, Ye L, Ciliberto CH, Broekelmann TJ, 1996.0225. Mecham RP. Genetic modifiers of cardiovascular phenotype caused by 69. Ikonomidis JS, Gibson WC, Gardner J, Sweterlitsch S, Thompson elastin haploinsufficiency act by extrinsic noncomplementation. J Biol RP, Mukherjee R, Spinale FG. A murine model of thoracic aortic Chem 286: 44926–44936, 2011. doi:10.1074/jbc.M111.274779. aneurysms. J Surg Res 115: 157–163, 2003. doi:10.1016/S0022- 87. Kozel BA, Mecham RP, Rosenbloom J. Elastin. In: The Extracellular 4804(03)00193-8. Matrix: an Overview, edited by Mecham RP. Berlin, Germany: Spring- 70. Jain D, Dietz HC, Oswald GL, Maleszewski JJ, Halushka MK. Verlag, 2011, p. 267–301. Causes and histopathology of ascending aortic disease in children and 88. Krettek A, Sukhova GK, Libby P. Elastogenesis in human arterial young adults. Cardiovasc Pathol 20: 15–25, 2011. doi:10.1016/j.carpath. disease: a role for macrophages in disordered elastin synthesis. Arterio- 2009.09.008. scler Thromb Vasc Biol 23: 582–587, 2003. doi:10.1161/01.ATV. 71. James MF, Rich CB, Trinkaus-Randall V, Rosenbloom J, Foster JA. Elastogenesis in the developing chick lung is transcriptionally regulated. 0000064372.78561.A5. Dev Dyn 213: 170–181, 1998. doi:10.1002/(SICI)1097-0177(199810) 89. Kucich U, Rosenbloom JC, Abrams WR, Bashir MM, Rosenbloom J. 213:2Ͻ170::AID-AJA2Ͼ3.0.CO;2-D. Stabilization of elastin mRNA by TGF-beta: initial characterization of 72. Jiao Y, Li G, Korneva A, Caulk AW, Qin L, Bersi MR, Li Q, Li W, signaling pathway. Am J Respir Cell Mol Biol 17: 10–16, 1997. doi:10. Mecham RP, Humphrey JD, Tellides G. Deficient circumferential 1165/ajrcmb.17.1.2816. growth is the primary determinant of aortic obstruction attributable to 90. Kucich U, Rosenbloom JC, Abrams WR, Rosenbloom J. Transform- partial elastin deficiency. Arterioscler Thromb Vasc Biol 37: 930–941, ing growth factor-beta stabilizes elastin mRNA by a pathway requiring 2017. doi:10.1161/ATVBAHA.117.309079. active Smads, protein kinase C-delta, and p38. Am J Respir Cell Mol Biol 73. Jiao Y, Li G, Li Q, Ali R, Qin L, Li W, Qyang Y, Greif DM, Geirsson 26: 183–188, 2002. doi:10.1165/ajrcmb.26.2.4666. A, Humphrey JD, Tellides G. mTOR (mechanistic target of rapamycin) 91. Kwan CY, Wang RR, Beazley JS, Lee RM. Alterations of elastin and inhibition decreases mechanosignaling, collagen accumulation, and stiff- elastase-like activities in aortae of diabetic rats. Biochim Biophys Acta ening of the thoracic aorta in elastin-deficient mice. Arterioscler Thromb 967: 322–325, 1988. doi:10.1016/0304-4165(88)90027-X. Vasc Biol 37: 1657–1666, 2017. doi:10.1161/ATVBAHA.117.309653. 92. Lafarge JC, Naour N, Clément K, Guerre-Millo M. Cathepsins and 74. Johnston WF, Salmon M, Pope NH, Meher A, Su G, Stone ML, Lu cystatin C in atherosclerosis and obesity. Biochimie 92: 1580–1586, G, Owens GK, Upchurch GR Jr, Ailawadi G. Inhibition of interleu- 2010. doi:10.1016/j.biochi.2010.04.011. kin-1␤ decreases aneurysm formation and progression in a novel model 93. Le VP, Cheng JK, Kim J, Staiculescu MC, Ficker SW, Sheth SC, of thoracic aortic aneurysms. Circulation 130, Suppl 1: S51–S59, 2014. Bhayani SA, Mecham RP, Yanagisawa H, Wagenseil JE. Mechanical doi:10.1161/CIRCULATIONAHA.113.006800. factors direct mouse aortic remodelling during early maturation. J R Soc 75. Kagan HM, Vaccaro CA, Bronson RE, Tang SS, Brody JS. Ultra- Interface 12: 20141350, 2015. doi:10.1098/rsif.2014.1350. structural immunolocalization of lysyl oxidase in vascular connective 94. Le VP, Knutsen RH, Mecham RP, Wagenseil JE. Decreased aortic tissue. J Cell Biol 103: 1121–1128, 1986. doi:10.1083/jcb.103.3.1121. diameter and compliance precedes blood pressure increases in postnatal 76. Karnik SK, Brooke BS, Bayes-Genis A, Sorensen L, Wythe JD, development of elastin-insufficient mice. Am J Physiol Heart Circ Schwartz RS, Keating MT, Li DY. A critical role for elastin signaling Physiol 301: H221–H229, 2011. doi:10.1152/ajpheart.00119.2011. in vascular morphogenesis and disease. Development 130: 411–423, 95. Le VP, Wagenseil JE. Echocardiographic characterization of postnatal 2003. doi:10.1242/dev.00223. development in mice with reduced arterial elasticity. Cardiovasc Eng 77. Karnik SK, Wythe JD, Sorensen L, Brooke BS, Urness LD, Li DY. Technol 3: 424–438, 2012. doi:10.1007/s13239-012-0108-4. Elastin induces myofibrillogenesis via a specific domain, VGVAPG. 96. Lee P, Bax DV, Bilek MM, Weiss AS. A novel cell adhesion region in Matrix Biol 22: 409–425, 2003. doi:10.1016/S0945-053X(03)00076-3. tropoelastin mediates attachment to integrin ␣V␤5. J Biol Chem 289: 78. Katz PS, Trask AJ, Souza-Smith FM, Hutchinson KR, Galantowicz 1467–1477, 2014. doi:10.1074/jbc.M113.518381. ML, Lord KC, Stewart JA Jr, Cismowski MJ, Varner KJ, Lucchesi 97. Lev M. The pathologic anatomy of cardiac complexes associated with PA. Coronary arterioles in type 2 diabetic (db/db) mice undergo a distinct transposition of arterial trunks. Lab Invest 2: 296–311, 1953.

98. Li DY, Brooke B, Davis EC, Mecham RP, Sorensen LK, Boak BB, 117. Mitchell GF. Arterial stiffness and hypertension: chicken or egg? Hy- Eichwald E, Keating MT. Elastin is an essential determinant of arterial pertension 64: 210–214, 2014. doi:10.1161/HYPERTENSIONAHA. morphogenesis. Nature 393: 276–280, 1998. doi:10.1038/30522. 114.03449. 99. Li DY, Faury G, Taylor DG, Davis EC, Boyle WA, Mecham RP, 118. Morrow AG, Waldhausen JA, Peters RL, Blood-Well RD, Braun- Stenzel P, Boak B, Keating MT. Novel arterial pathology in mice and wald E. Supravalvular aortic stenosis: clinical, hemodynamic and patho- humans hemizygous for elastin. J Clin Invest 102: 1783–1787, 1998. logic observations. Circulation 20: 1003–1010, 1959. doi:10.1161/01. doi:10.1172/JCI4487. CIR.20.6.1003. 100. Li HH, Roy M, Kuscuoglu U, Spencer CM, Halm B, Harrison KC, 119. Myers B, Dubick M, Last JA, Rucker RB. Elastin synthesis during Bayle JH, Splendore A, Ding F, Meltzer LA, Wright E, Paylor R, perinatal lung development in the rat. Biochim Biophys Acta 761: 17–22, Deisseroth K, Francke U. Induced chromosome deletions cause hyper- 1983. doi:10.1016/0304-4165(83)90357-4. sociability and other features of Williams-Beuren syndrome in mice. 120. Narayanan AS, Page RC, Kuzan F, Cooper CG. Elastin cross-linking EMBO Mol Med 1: 50–65, 2009. doi:10.1002/emmm.200900003. in vitro. Studies on factors influencing the formation of desmosines by 101. Li W, Li Q, Qin L, Ali R, Qyang Y, Tassabehji M, Pober BR, Sessa lysyl oxidase action on tropoelastin. Biochem J 173: 857–862, 1978. WC, Giordano FJ, Tellides G. Rapamycin inhibits smooth muscle cell doi:10.1042/bj1730857. proliferation and obstructive arteriopathy attributable to elastin defi- 121. Newby AC. Metalloproteinase production from macrophagesϪa perfect ciency. Arterioscler Thromb Vasc Biol 33: 1028–1035, 2013. doi:10. storm leading to atherosclerotic plaque rupture and myocardial infarc- 1161/ATVBAHA.112.300407. tion. Exp Physiol 101: 1327–1337, 2016. doi:10.1113/EP085567. 102. Lillie MA, Gosline JM. Swelling and viscoelastic properties of osmot- 122. Nicoloff G, Petrova C, Christova P, Nikolov A. Detection of free ically stressed elastin. Biopolymers 39: 641–652, 1996. doi:10.1002/ elastin-derived peptides among diabetic children. Atherosclerosis 192: (SICI)1097-0282(199611)39:5Ͻ641::AID-BIP3Ͼ3.0.CO;2-W. 342–347, 2007. doi:10.1016/j.atherosclerosis.2006.08.007. 103. Liu J, Ma L, Yang J, Ren A, Sun Z, Yan G, Sun J, Fu H, Xu W, Hu 123. O’Connell MK, Murthy S, Phan S, Xu C, Buchanan J, Spilker R, C, Shi GP. Increased serum cathepsin S in patients with atherosclerosis Dalman RL, Zarins CK, Denk W, Taylor CA. The three-dimensional and diabetes. Atherosclerosis 186: 411–419, 2006. doi:10.1016/j. micro- and nanostructure of the aortic medial lamellar unit measured atherosclerosis.2005.08.001. using 3D confocal and electron microscopy imaging. Matrix Biol 27: 104. Long JL, Tranquillo RT. Elastic fiber production in cardiovascular 171–181, 2008. doi:10.1016/j.matbio.2007.10.008. tissue-equivalents. Matrix Biol 22: 339–350, 2003. doi:10.1016/S0945- 124. O’Rourke MF, Hashimoto J. Mechanical factors in arterial aging: a 053X(03)00052-0. clinical perspective. J Am Coll Cardiol 50: 1–13, 2007. doi:10.1016/j. 105. Lucero HA, Kagan HM. Lysyl oxidase: an oxidative enzyme and jacc.2006.12.050. effector of cell function. Cell Mol Life Sci 63: 2304–2316, 2006. 125. O’Rourke MF, Safar ME. Relationship between aortic stiffening and doi:10.1007/s00018-006-6149-9. microvascular disease in brain and kidney: cause and logic of therapy. 106. Lysgaard Poulsen J, Stubbe J, Lindholt JS. Animal models used to Hypertension 46: 200–204, 2005. doi:10.1161/01.HYP.0000168052. explore abdominal aortic aneurysms: a systematic review. Eur J Vasc 00426.65. Endovasc Surg 52: 487–499, 2016. doi:10.1016/j.ejvs.2016.07.004. 126. Ohtsuka S, Kakihana M, Watanabe H, Sugishita Y. Chronically 107. Maeda I, Mizoiri N, Briones MP, Okamoto K. Induction of macro- decreased aortic distensibility causes deterioration of coronary perfusion phage migration through lactose-insensitive receptor by elastin-derived during increased left ventricular contraction. J Am Coll Cardiol 24: nonapeptides and their analog. J Pept Sci 13: 263–268, 2007. doi:10. 1406–1414, 1994. doi:10.1016/0735-1097(94)90127-9. 1002/psc.845. 127. Okamoto K. Spontaneous hypertension in rats. Int Rev Exp Pathol 7: 108. Maedeker JA, Stoka KV, Bhayani SA, Gardner WS, Bennett L, 227–270, 1969. Procknow JD, Staiculescu MC, Walji TA, Craft CS, Wagenseil JE. 128. Olliver L, Luvalle PA, Davidson JM, Rosenbloom J, Mathew CG, Hypertension and decreased aortic compliance due to reduced elastin Bester AJ, Boyd CD. The gene coding for tropoelastin is represented as amounts do not increase atherosclerotic plaque accumulation in Ldlr-/- a single copy sequence in the haploid sheep genome. Coll Relat Res 7: mice. Atherosclerosis 249: 22–29, 2016. doi:10.1016/j.atherosclerosis. 77–89, 1987. doi:10.1016/S0174-173X(87)80022-5. 2016.03.022. 129. Ono RN, Sengle G, Charbonneau NL, Carlberg V, Bächinger HP, 109. Marigo V, Volpin D, Bressan GM. Regulation of the human elastin Sasaki T, Lee-Arteaga S, Zilberberg L, Rifkin DB, Ramirez F, Chu promoter in chick embryo cells. Tissue-specific effect of TGF-beta. ML, Sakai LY. Latent transforming growth factor beta-binding proteins Biochim Biophys Acta 1172: 31–36, 1993. doi:10.1016/0167- and fibulins compete for fibrillin-1 and exhibit exquisite specificities in 4781(93)90265-F. binding sites. J Biol Chem 284: 16872–16881, 2009. doi:10.1074/jbc. 110. Marque V, Kieffer P, Atkinson J, Lartaud-Idjouadiene I. Elastic M809348200. properties and composition of the aortic wall in old spontaneously 130. Osborne-Pellegrin M, Labat C, Mercier N, Challande P, Lacolley P. hypertensive rats. Hypertension 34: 415–422, 1999. doi:10.1161/01. Changes in aortic stiffness related to elastic fiber network anomalies in HYP.34.3.415. the Brown Norway rat during maturation and aging. Am J Physiol Heart 111. Maurice P, Blaise S, Gayral S, Debelle L, Laffargue M, Hornebeck Circ Physiol 299: H144–H152, 2010. doi:10.1152/ajpheart.00040.2010. W, Duca L. Elastin fragmentation and atherosclerosis progression: the 131. Papke CL, Yanagisawa H. Fibulin-4 and fibulin-5 in elastogenesis and elastokine concept. Trends Cardiovasc Med 23: 211–221, 2013. doi:10. beyond: Insights from mouse and human studies. Matrix Biol 37: 142– 1016/j.tcm.2012.12.004. 149, 2014. doi:10.1016/j.matbio.2014.02.004. 112. McDonald AH, Gerlis LM, Somerville J. Familial arteriopathy with 132. Parker KH. A brief history of arterial wave mechanics. Med Biol Eng associated pulmonary and systemic arterial stenoses. Br Heart J 31: Comput 47: 111–118, 2009. doi:10.1007/s11517-009-0440-5. 375–385, 1969. doi:10.1136/hrt.31.3.375. 133. Parks WC, Secrist H, Wu LC, Mecham RP. Developmental regulation 113. Mecham RP, Madaras J, McDonald JA, Ryan U. Elastin production of tropoelastin isoforms. J Biol Chem 263: 4416–4423, 1988. by cultured calf pulmonary artery endothelial cells. J Cell Physiol 116: 134. Pasquali-Ronchetti I, Baccarani-Contri M. Elastic fiber during devel- 282–288, 1983. doi:10.1002/jcp.1041160304. opment and aging. Microsc Res Tech 38: 428–435, 1997. doi:10.1002/ 114. Milewicz DM, Guo DC, Van TF, Lafont AL, Papke CL, Inamoto S, (SICI)1097-0029(19970815)38:4Ͻ428::AID-JEMT10Ͼ3.0.CO;2-L. Kwartler CS, Pannu H. Genetic basis of thoracic aortic aneurysms and 135. Patel A, Fine B, Sandig M, Mequanint K. Elastin biosynthesis: The dissections: focus on smooth muscle cell contractile dysfunction. Annu missing link in tissue-engineered blood vessels. Cardiovasc Res 71: Rev Genomics Hum Genet 9: 283–302, 2008. doi:10.1146/annurev. 40–49, 2006. doi:10.1016/j.cardiores.2006.02.021. genom.8.080706.092303. 136. Perou ML. Congenital supravalvular aortic stenosis. A morphological 115. Milewicz DM, Urbán Z, Boyd C. Genetic disorders of the elastic fiber study with attempt at classification. Arch Pathol 71: 453–466, 1961. system. Matrix Biol 19: 471–480, 2000. doi:10.1016/S0945-053X(00) 137. Pierce GL. Mechanisms and subclinical consequences of aortic stiffness. 00099-8. Hypertension 70: 848–853, 2017. doi:10.1161/HYPERTENSIONAHA. 116. Misra A, Sheikh AQ, Kumar A, Luo J, Zhang J, Hinton RB, Smoot 117.08933. L, Kaplan P, Urban Z, Qyang Y, Tellides G, Greif DM. Integrin ␤3 138. Pierce RA, Deak SB, Stolle CA, Boyd CD. Heterogeneity of rat inhibition is a therapeutic strategy for supravalvular aortic stenosis. J Exp tropoelastin mRNA revealed by cDNA cloning. Biochemistry 29: 9677– Med 213: 451–463, 2016. doi:10.1084/jem.20150688. 9683, 1990. doi:10.1021/bi00493a024.

139. Pober BR, Johnson M, Urban Z. Mechanisms and treatment of car- Abdominal Aortic Aneurysm. Arterioscler Thromb Vasc Biol 37: 401– diovascular disease in Williams-Beuren syndrome. J Clin Invest 118: 410, 2017. doi:10.1161/ATVBAHA.116.308534. 1606–1615, 2008. doi:10.1172/JCI35309. 162. Senior RM, Griffin GL, Mecham RP, Wrenn DS, Prasad KU, Urry 140. Pober BR. Williams-Beuren syndrome. N Engl J Med 362: 239–252, DW. Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemot- 2010. doi:10.1056/NEJMra0903074. actic for fibroblasts and monocytes. J Cell Biol 99: 870–874, 1984. 141. Pocza P, Süli-Vargha H, Darvas Z, Falus A. Locally generated doi:10.1083/jcb.99.3.870. VGVAPG and VAPG elastin-derived peptides amplify melanoma inva- 163. Sephel GC, Buckley A, Davidson JM. Developmental initiation of sion via the galectin-3 receptor. Int J Cancer 122: 1972–1980, 2008. elastin gene expression by human fetal skin fibroblasts. J Invest Dermatol doi:10.1002/ijc.23296. 88: 732–735, 1987. doi:10.1111/1523-1747.ep12470403. 142. Pollock J, Baule VJ, Rich CB, Ginsburg CD, Curtiss SW, Foster JA. 164. Shadwick RE. Mechanical design in arteries. J Exp Biol 202: 3305– Chick tropoelastin isoforms. From the gene to the extracellular matrix. J 3313, 1999. Biol Chem 265: 3697–3702, 1990. 165. Shapiro SD, Endicott SK, Province MA, Pierce JA, Campbell EJ. 143. Reiser K, McCormick RJ, Rucker RB. Enzymatic and nonenzymatic Marked longevity of human lung parenchymal elastic fibers deduced cross-linking of collagen and elastin. FASEB J 6: 2439–2449, 1992. from prevalence of D-aspartate and nuclear weapons-related radiocarbon. doi:10.1096/fasebj.6.7.1348714. J Clin Invest 87: 1828–1834, 1991. doi:10.1172/JCI115204. 144. Rider OJ, Tayal U, Francis JM, Ali MK, Robinson MR, Byrne JP, 166. Shih CC, Oakley DM, Joens MS, Roth RA, Fitzpatrick JAJ. Nonlin- Clarke K, Neubauer S. The effect of obesity and weight loss on aortic ear optical imaging of extracellular matrix proteins. Methods Cell Biol pulse wave velocity as assessed by magnetic resonance imaging. Obesity 143: 57–78, 2018. doi:10.1016/bs.mcb.2017.08.004. (Silver Spring) 18: 2311–2316, 2010. doi:10.1038/oby.2010.64. 167. Simionescu A, Philips K, Vyavahare N. Elastin-derived peptides and 145. Ritz-Timme S, Laumeier I, Collins MJ. Aspartic acid racemization: TGF-beta1 induce osteogenic responses in smooth muscle cells. Biochem evidence for marked longevity of elastin in human skin. Br J Dermatol Biophys Res Commun 334: 524–532, 2005. doi:10.1016/j.bbrc.2005.06. 149: 951–959, 2003. doi:10.1111/j.1365-2133.2003.05618.x. 119. 146. Roach MR, Burton AC. The reason for the shape of the distensibility 168. Sinha A, Vyavahare NR. High-glucose levels and elastin degradation curves of arteries. Can J Biochem Physiol 35: 681–690, 1957. doi:10. products accelerate osteogenesis in vascular smooth muscle cells. Diab 1139/y57-080. Vasc Dis Res 10: 410–419, 2013. doi:10.1177/1479164113485101. 147. Robertson IB, Rifkin DB. Regulation of the Bioavailability of TGF-␤ 169. Slove S, Lannoy M, Behmoaras J, Pezet M, Sloboda N, Lacolley P, and TGF-␤-Related Proteins. Cold Spring Harb Perspect Biol 8: Escoubet B, Buján J, Jacob MP. Potassium channel openers increase a021907, 2016. doi:10.1101/cshperspect.a021907. aortic elastic fiber formation and reverse the genetically determined 148. Roccabianca S, Ateshian GA, Humphrey JD. Biomechanical roles of elastin deficit in the BN rat. Hypertension 62: 794–801, 2013. doi:10. medial pooling of glycosaminoglycans in thoracic aortic dissection. 1161/HYPERTENSIONAHA.113.01379. Biomech Model Mechanobiol 13: 13–25, 2014. doi:10.1007/s10237-013- 170. Spencer M, Unal R, Zhu B, Rasouli N, McGehee RE Jr, Peterson CA, 0482-3. Kern PA. Adipose tissue extracellular matrix and vascular abnormalities 149. Rodgers UR, Weiss AS. Integrin alpha v beta 3 binds a unique non-RGD in obesity and insulin resistance. J Clin Endocrinol Metab 96: E1990– site near the C-terminus of human tropoelastin. Biochimie 86: 173–178, E1998, 2011. doi:10.1210/jc.2011-1567. 2004. doi:10.1016/j.biochi.2004.03.002. 171. Sproul EP, Argraves WS. A cytokine axis regulates elastin formation 150. Roy CS. Elastic properties of the arterial wall. J Physiol 3: 125–159, and degradation. Matrix Biol 32: 86–94, 2013. doi:10.1016/j.matbio. 1881. doi:10.1113/jphysiol.1881.sp000088. 2012.11.004. 151. Rucker RB, Tinker D. Structure and metabolism of arterial elastin. Int 172. Staiculescu MC, Kim J, Mecham RP, Wagenseil J. Mechanical be- Rev Exp Pathol 17: 1–47, 1977. havior and matrisome gene expression in aneurysm-prone thoracic aorta 152. Ruckman JL, Luvalle PA, Hill KE, Giro MG, Davidson JM. Pheno- of newborn lysyl oxidase knockout mice. Am J Physiol Heart Circ typic stability and variation in cells of the porcine aorta: collagen and Physiol 313: H446ϪH456, 2017. doi:10.1152/ajpheart.00712.2016. elastin production. Matrix Biol 14: 135–145, 1994. doi:10.1016/0945- 173. Stoilov I, Starcher BC, Mecham RP, Broekelmann TJ. Measurement 053X(94)90003-5. of elastin, collagen, and total protein levels in tissues. Methods Cell Biol 153. Rusciani A, Duca L, Sartelet H, Chatron-Colliet A, Bobichon H, 143: 133–146, 2018. doi:10.1016/bs.mcb.2017.08.008. Ploton D, Le Naour R, Blaise S, Martiny L, Debelle L. Elastin peptides 174. Stoka KV, Maedeker JA, Bennett L, Bhayani SA, Gardner WS, signaling relies on neuraminidase-1-dependent lactosylceramide genera- Procknow JD, Cocciolone AJ, Walji TA, Craft CS, Wagenseil JE. tion. PLoS One 5: e14010, 2010. doi:10.1371/journal.pone.0014010. Effects of increased arterial stiffness on atherosclerotic plaque amounts. 154. Safar M, Chamiot-Clerc P, Dagher G, Renaud JF. Pulse pressure, J Biomech Eng 140: 051007, 2018. doi:10.1115/1.4039175. endothelium function, and arterial stiffness in spontaneously hyperten- 175. Sugitani H, Hirano E, Knutsen RH, Shifren A, Wagenseil JE, Cili- sive rats. Hypertension 38: 1416–1421, 2001. doi:10.1161/hy1201. berto C, Kozel BA, Urban Z, Davis EC, Broekelmann TJ, Mecham 096538. RP. Alternative splicing and tissue-specific elastin misassembly act as 155. Sage H, Gray WR. Studies on the evolution of elastinϪI. Phylogenetic biological modifiers of human elastin gene frameshift mutations associ- distribution. Comp Biochem Physiol B 64: 313–327, 1979. doi:10.1016/ ated with dominant cutis laxa. J Biol Chem 287: 22055–22067, 2012. 0305-0491(79)90277-3. doi:10.1074/jbc.M111.327940. 156. Sakai LY, Keene DR, Engvall E. Fibrillin, a new 350-kD glycoprotein, 176. Swee MH, Parks WC, Pierce RA. Developmental regulation of elastin is a component of extracellular microfibrils. J Cell Biol 103: 2499–2509, production. Expression of tropoelastin pre-mRNA persists after down- 1986. doi:10.1083/jcb.103.6.2499. regulation of steady-state mRNA levels. J Biol Chem 270: 14899–14906, 157. Samad F, Yamamoto K, Pandey M, Loskutoff DJ. Elevated expres- 1995. doi:10.1074/jbc.270.25.14899. sion of transforming growth factor-beta in adipose tissue from obese 177. Syedain Z, Reimer J, Lahti M, Berry J, Johnson S, Tranquillo RT. mice. Mol Med 3: 37–48, 1997. Tissue engineering of acellular vascular grafts capable of somatic 158. Sandberg LB, Weissman N, Gray WR. Structural features of tropoelas- growth in young lambs. Nat Commun 7: 12951, 2016. doi:10.1038/ tin related to the sites of cross-links in aortic elastin. Biochemistry 10: ncomms12951. 52–56, 1971. doi:10.1021/bi00777a008. 178. Szabó Z, Levi-Minzi SA, Christiano AM, Struminger C, Stoneking 159. Sauvage M, Hinglais N, Mandet C, Badier C, Deslandes F, Michel M, Batzer MA, Boyd CD. Sequential loss of two neighboring exons of JB, Jacob MP. Localization of elastin mRNA and TGF-beta1 in rat aorta the tropoelastin gene during primate evolution. J Mol Evol 49: 664–671, and caudal artery as a function of age. Cell Tissue Res 291: 305–314, 1999. doi:10.1007/PL00006587. 1998. doi:10.1007/s004410051000. 179. Tanno T, Yoshinaga K, Sato T. Alteration of elastin in aorta from 160. Schmidt RE, Gilbert EF, Amend TC, Chamberlain CR Jr, Lucas RV diabetics. Atherosclerosis 101: 129–134, 1993. doi:10.1016/0021- Jr. Generalized arterial fibromuscular dysplasia and myocardial infarc- 9150(93)90109-8. tion in familial supravalvular aortic stenosis syndrome. J Pediatr 74: 180. Tassabehji M, Metcalfe K, Donnai D, Hurst J, Reardon W, Burch M, 576–584, 1969. doi:10.1016/S0022-3476(69)80041-7. Read AP. Elastin: genomic structure and point mutations in patients with 161. Sénémaud J, Caligiuri G, Etienne H, Delbosc S, Michel JB, Coscas R. supravalvular aortic stenosis. Hum Mol Genet 6: 1029–1036, 1997. Translational Relevance and Recent Advances of Animal Models of doi:10.1093/hmg/6.7.1029.

181. Toma I, McCaffrey TA. Transforming growth factor-␤ and atheroscle- 198. Wan W, Yanagisawa H, Gleason RL Jr. Biomechanical and micro- rosis: interwoven atherogenic and atheroprotective aspects. Cell Tissue structural properties of common carotid arteries from fibulin-5 null mice. Res 347: 155–175, 2012. doi:10.1007/s00441-011-1189-3. Ann Biomed Eng 38: 3605–3617, 2010. doi:10.1007/s10439-010-0114-3. 182. Tomizawa H, Yamazaki M, Kunika K, Itakura M, Yamashita K. 199. Wang Y, Zeinali-Davarani S, Davis EC, Zhang Y. Effect of glucose on Association of elastin glycation and calcium deposit in diabetic rat aorta. the biomechanical function of arterial elastin. J Mech Behav Biomed Diabetes Res Clin Pract 19: 1–8, 1993. doi:10.1016/0168-8227(93) Mater 49: 244–254, 2015. doi:10.1016/j.jmbbm.2015.04.025. 90138-U. 200. Wang Z, Lakes RS, Golob M, Eickhoff JC, Chesler NC. Changes in 183. Toonkool P, Jensen SA, Maxwell AL, Weiss AS. Hydrophobic do- large pulmonary arterial viscoelasticity in chronic pulmonary hyperten- mains of human tropoelastin interact in a context-dependent manner. J sion. PLoS One 8: e78569, 2013. doi:10.1371/journal.pone.0078569. Biol Chem 276: 44575–44580, 2001. doi:10.1074/jbc.M107920200. 201. Wells SM, Langille BL, Lee JM, Adamson SL. Determinants of 184. Torun D, Ozelsancak R, Turan I, Micozkadioglu H, Sezer S, Oz- mechanical properties in the developing ovine thoracic aorta. Am J demir FN. The relationship between obesity and transforming growth Physiol Heart Circ Physiol 277: H1385–H1391, 1999. factor beta on renal damage in essential hypertension. Int Heart J 48: 202. Wessel A, Pankau R, Kececioglu D, Ruschewski W, Bürsch JH. Three 733–741, 2007. doi:10.1536/ihj.48.733. decades of follow-up of aortic and pulmonary vascular lesions in the 185. Urban Z, Davis EC. Cutis laxa: intersection of elastic fiber biogenesis, Williams-Beuren syndrome. Am J Med Genet 52: 297–301, 1994. doi: ␤ TGF signaling, the secretory pathway and metabolism. Matrix Biol 33: 10.1002/ajmg.1320520309. 16–22, 2014. doi:10.1016/j.matbio.2013.07.006. 203. Wolinsky H. Comparison of medial growth of human thoracic and 186. Urry DW. Entropic elastic processes in protein mechanisms. I. Elastic abdominal aortas. Circ Res 27: 531–538, 1970. doi:10.1161/01.RES.27. structure due to an inverse temperature transition and elasticity due to 4.531. internal chain dynamics. J Protein Chem 7: 1–34, 1988. doi:10.1007/ 204. Wolinsky H, Glagov S. A lamellar unit of aortic medial structure and BF01025411. function in mammals. Circ Res 20: 99–111, 1967. doi:10.1161/01.RES. 187. Ushiki T, Murakumo M. Scanning electron microscopic studies of 20.1.99. tissue elastin components exposed by a KOH-collagenase or simple KOH 205. Yanagisawa H, Davis EC. Unraveling the mechanism of elastic fiber digestion method. Arch Histol Cytol 54: 427–436, 1991. doi:10.1679/ assembly: The roles of short fibulins. Int J Biochem Cell Biol 42: aohc.54.427. 1084–1093, 2010. doi:10.1016/j.biocel.2010.03.009. 188. Van der Donckt C, Van Herck JL, Schrijvers DM, Vanhoutte G, 206. Yeh H, Anderson N, Ornstein-Goldstein N, Bashir MM, Rosenbloom Verhoye M, Blockx I, Van Der Linden A, Bauters D, Lijnen HR, JC, Abrams W, Indik Z, Yoon K, Parks W, Mecham R, Rosenbloom Sluimer JC, Roth L, Van Hove CE, Fransen P, Knaapen MW, J. Structure of the bovine elastin gene and S1 nuclease analysis of Hervent AS, De Keulenaer GW, Bult H, Martinet W, Herman AG, alternative splicing of elastin mRNA in the bovine nuchal ligament. De Meyer GR. Elastin fragmentation in atherosclerotic mice leads to Biochemistry 28: 2365–2370, 1989. doi:10.1021/bi00432a003. intraplaque neovascularization, plaque rupture, myocardial infarction, 207. Yu Q, Stamenkovic I. Cell surface-localized matrix metalloproteinase-9 stroke, and sudden death. Eur Heart J 36: 1049–1058, 2015. doi:10. proteolytically activates TGF-beta and promotes tumor invasion and 1093/eurheartj/ehu041. angiogenesis. Genes Dev 14: 163–176, 2000. 189. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of Yurchenco PD, O’Rear JJ. Curr Opin Cell Biol metalloproteinases: structure, function, and biochemistry. Circ Res 92: 208. Basal lamina assembly. 827–839, 2003. doi:10.1161/01.RES.0000070112.80711.3D. 6: 674–681, 1994. doi:10.1016/0955-0674(94)90093-0. 190. Volpin D, Urry DW, Cox BA, Gotte L. Optical diffraction of tropoelas- 209. Zacchigna L, Vecchione C, Notte A, Cordenonsi M, Dupont S, tin and alpha-elastin coacervates. Biochim Biophys Acta 439: 253–258, Maretto S, Cifelli G, Ferrari A, Maffei A, Fabbro C, Braghetta P, 1976. doi:10.1016/0005-2795(76)90181-1. Marino G, Selvetella G, Aretini A, Colonnese C, Bettarini U, Russo 191. Vrhovski B, Weiss AS. Biochemistry of tropoelastin. Eur J Biochem G, Soligo S, Adorno M, Bonaldo P, Volpin D, Piccolo S, Lembo G, 258: 1–18, 1998. doi:10.1046/j.1432-1327.1998.2580001.x. Bressan GM. Emilin1 links TGF-beta maturation to blood pressure 192. Wagenseil JE, Ciliberto CH, Knutsen RH, Levy MA, Kovacs A, homeostasis. Cell 124: 929–942, 2006. doi:10.1016/j.cell.2005.12.035. Mecham RP. The importance of elastin to aortic development in mice. 210. Zhang H, Apfelroth SD, Hu W, Davis EC, Sanguineti C, Bonadio J, Am J Physiol Heart Circ Physiol 299: H257–H264, 2010. doi:10.1152/ Mecham RP, Ramirez F. Structure and expression of fibrillin-2, a novel ajpheart.00194.2010. microfibrillar component preferentially located in elastic matrices. J Cell 193. Wagenseil JE, Ciliberto CH, Knutsen RH, Levy MA, Kovacs A, Biol 124: 855–863, 1994. doi:10.1083/jcb.124.5.855. Mecham RP. Reduced vessel elasticity alters cardiovascular structure 211. Zhang J, Zhao X, Vatner DE, McNulty T, Bishop S, Sun Z, Shen YT, and function in newborn mice. Circ Res 104: 1217–1224, 2009. doi:10. Chen L, Meininger GA, Vatner SF. Extracellular matrix disarray as a 1161/CIRCRESAHA.108.192054. mechanism for greater abdominal versus thoracic aortic stiffness with 194. Wagenseil JE, Mecham RP. Elastin in large artery stiffness and hyper- aging in primates. Arterioscler Thromb Vasc Biol 36: 700–706, 2016. tension. J Cardiovasc Transl Res 5: 264–273, 2012. doi:10.1007/s12265- doi:10.1161/ATVBAHA.115.306563. 012-9349-8. 212. Zhang M, Pierce RA, Wachi H, Mecham RP, Parks WC. An open 195. Wagenseil JE, Mecham RP. New insights into elastic fiber assembly. reading frame element mediates posttranscriptional regulation of tro- Birth Defects Res C Embryo Today 81: 229–240, 2007. doi:10.1002/ poelastin and responsiveness to transforming growth factor beta1. Mol bdrc.20111. Cell Biol 19: 7314–7326, 1999. doi:10.1128/MCB.19.11.7314. 196. Wagenseil JE, Mecham RP. Vascular extracellular matrix and arterial 213. Zilberberg L, Todorovic V, Dabovic B, Horiguchi M, Couroussé T, mechanics. Physiol Rev 89: 957–989, 2009. doi:10.1152/physrev.00041. Sakai LY, Rifkin DB. Specificity of latent TGF-␤ binding protein 2008. (LTBP) incorporation into matrix: role of fibrillins and fibronectin. J Cell 197. Wagenseil JE, Nerurkar NL, Knutsen RH, Okamoto RJ, Li DY, Physiol 227: 3828–3836, 2012. doi:10.1002/jcp.24094. Mecham RP. Effects of elastin haploinsufficiency on the mechanical 214. Zou Y, Zhang Y. The orthotropic viscoelastic behavior of aortic elastin. behavior of mouse arteries. Am J Physiol Heart Circ Physiol 289: Biomech Model Mechanobiol 10: 613–625, 2011. doi:10.1007/s10237- H1209–H1217, 2005. doi:10.1152/ajpheart.00046.2005. 010-0260-4.