Elastin in Lung Development and Disease Pathogenesis

Abstract

Elastin is expressed in most tissues that require elastic recoil. The protein first appeared coincident with the closed circulatory system, and was critical for the evolutionary success of the vertebrate lineage. Elastin is expressed by multiple cell types in the lung, including mesothelial cells in the pleura, smooth muscle cells in airways and blood vessels, endothelial cells, and interstitial fibroblasts. This highly crosslinked protein associates with fibrillin-containing microfibrils to form the elastic fiber, which is the physiological structure that functions in the extracellular matrix. Elastic fibers can be woven into many different shapes depending on the mechanical needs of the tissue. In large pulmonary vessels, for example, elastin forms continuous sheets, or lamellae, that separate smooth muscle layers. Outside of the vasculature, elastic fibers form an extensive fiber network that originates in the central bronchi and inserts into the distal airspaces and visceral pleura. The fibrous cables form a looping system that encircle the alveolar ducts and terminal air spaces and ensures that applied force is transmitted equally to all parts of the lung. Normal lung function depends on proper secretion and assembly of elastin, and either inhibition of elastin fiber assembly or degradation of existing elastin results in lung dysfunction and disease.

1. INTRODUCTION

As animals moved from an aquatic to a terrestrial habitat, the functional design of the respiratory system changed to accommodate appropriate ventilation and oxygen exchange. Invertebrates evolved versatile organs, including gills, skin, book lungs, and tracheae systems, for air breathing [1]. In contrast, the method of ventilation used in respiration by many lower land-dwelling vertebrates is via a pulse pump. Air movement in the pulse pump is driven by muscle within the respiratory structure itself or by movement of the structures in the mouth in what is called buccal pumping. This method of ventilation is inefficient, but is nonetheless used by all air-breathing amphibians and, to a varying extent, by reptile species [2].

In mammals, we see remarkable changes in lung design and function. To meet the higher metabolic demands of terrestrial mammals, the lung is subdivided into long narrow airways and progressively smaller air spaces, rendering the pulse pump inefficient. In its place evolved the aspiration pump, where the lungs are located within the pump (the chest cavity) so air is sucked in, or aspirated, by low pressure created around the lungs [2]. The two important evolutionary innovations that made aspiration ventilation possible were the muscular diaphragm and the extracellular matrix (ECM) protein elastin. Elastin imparts elasticity to tissues and its presence allows the lung to function as an elastic bag. In aspiration pumping, the potential energy created by contraction of the diaphragm during inhalation is stored in the elastic tissues of the lung, and is released when the lung recoils during exhalation. It is therefore no surprise that damage to lung elastic fibers can be detrimental to lung mechanics and overall respiratory efficiency.

Three elastic fiber systems are present and develop independently in the lung. They include: (a) the pleura intersegmental and interlobular connective tissue; (b) the blood vascular system; and (c) the bronchi and respiratory units [3]. All three continue to develop postnatally up to the young adult period when elastin production diminishes [4, 5]. This review will discuss these three elastin-containing networks and focus on the importance of elastin to lung architecture and function. How mutations in the elastin gene and degradation of elastin protein lead to pulmonary disease will also be addressed.

2. ELASTIN

Unlike many matrix proteins that are phylogenetically ancient, elastin appeared relatively late in evolution and is present in all species from sharks to humans, but absent in lower chordates and invertebrates [6–9]. Its arrival coincides with the appearance of the closed circulatory system and the elastic lung, where elastic recoil is critical for appropriate tissue function. Elastin is encoded by a single gene whose transcript undergoes extensive alternative splicing [10–13]. It is secreted as a 70 kDa protein, called tropoelastin, that crosslinks with other tropoelastin molecules to form an insoluble, polymeric protein (Figure 1). It is this highly cross-linked form of elastin that functions as an elastomer [14, 15]. Lysyl oxidase, a copper-dependent enzyme, initiates cross-link formation by catalyzing the oxidative deamination of specific lysyl residues on the elastin precursor molecule to from α-aminoadipic δ-semialdehyde (trivial name allysine) [16]. There are two major bifunctional cross-links in elastin: dehydrolysinonorleucine, formed through the condensation of one residue of allysine and one of lysine, and allysine aldol formed through the association of two allysine residues [17–19]. These two cross-links can condense with each other, or with other intermediates, to form the tetrafunctional cross-links desmosine or isodesmosine (Figure 1) [20, 21]. Other cross-links that have been identi ed include a trifunctional cross-link, dehydromerodesmosine [22, 23], a cyclopentenosine trifunctional cross-link formed through the condensation of three allysine residues [24], and desmopyridine and isodesmopyridine found in trace amounts that form through the condensation of ammonia and allysine [25]. There is also evidence that desmosine/isodesmosine cross-links can be oxidized by reactive oxygen species resulting in dihydrooxopyridine forms [26].

Figure 1.

Elastin protein has a repeating domain structure consisting of lysine-containing sequences that populate crosslinking sites and hydrophobic sequences that contribute to elastic recoil (top). The ε-amino group of lysine residues in alanine-rich (KA) or proline-rich (KP) sequences are oxidized by the enzyme lysyl oxidase resulting in bifunctional and tetrafunctional crosslinks (see text). The electron micrograph of an elastic fiber shows crosslinked elastin (dark amorphous material) associated with a bed of microfibrils. In addition to facilitating elastin assembly, microfibrils have numerous signaling and mechanical roles indicated by the red arrows. Elastin also provides information to cells when fragments interact with signaling receptors on cells (green arrow).

The extensive cross-linking found in elastin is important for the protein’s insolubility and contributes to its longevity. Shapiro et al.[27] estimated the life span of elastin using aspartic acid racemization and ¹⁴C turnover to be ~80 years in humans. Studies using sensitive immunological techniques to measure elastin peptides in the blood or desmosine cross-links excreted in the urine suggest that less than 1% of the total body elastin pool turns over in a year [28]. Elastin expression in most tissues, including the lung, occurs over a narrow window of development, beginning in mid-gestation and continuing at high levels through the postnatal period [4, 29–31]. Both protein and gene array analysis show that there is minimal elastin synthesis in any tissue in the adult animal [32–35]. This explains why damage to elastic fibers during the adult period is so detrimental and why the elastin protein must have a long half-life.

Another factor contributing to the longevity of mature elastin is its relative resistance to proteolysis. Because there are few lysine or arginine residues in the fully cross-linked protein, and few amino acids with large aromatic side chains, elastin is not degraded by trypsin- or chymotrypsin-like proteases. Proteases that do degrade elastin are those that prefer amino acids with small hydrophobic side chains, such as alanine, valine, glycine, and leucine. These proteases, generally termed elastases [36], are predominantly serine proteases, although cathepsins will degrade elastin under appropriate circumstances [37, 38]. Elastases are produced by interstitial and inflammatory cells, and some of the most potent elastases are produced by bacteria [39–42]. Several matrix metalloproteinases (MMPs) secreted by mammalian cells also have elastolytic activity. These include MMP-2, -3, -7, -9, -10, and -12 [43, 44]. While elastin is clearly a substrate for the elastase class of protease, elastases are not specific for elastin. In fact, elastases—particularly the serine elastases like porcine pancreatic elastase and neutrophil elastase—are relatively nonspecific and have a broad range of substrates, including many, if not most, extracellular matrix proteins [36]. It is important to note that most of these proteases degrade microfibrillar components as well as elastin.

3. THE ELASTIC FIBER

3.1 Elastic fiber assembly

Elastin is a component of a complex fiber structure consisting of, in addition to elastin, fibrillin-containing microfibrils (see electron micrograph in Figure 1). Microfibrils are 10–15 nm fibrils found throughout the ECM that facilitate tropoelastin polymerization and serve as molecular bridges connecting elastic fibers to adjacent cells [45–47]. The association of elastin with microfibrils occurs extracellularly and gene knockout studies confirm that microfibrils are required for elastin polymerization and fiber formation [48]. The process by which tropoelastin monomers are secreted and how they associate with microfibrils is still not completely understood. Numerous studies now support a stepwise model for elastic fiber assembly that involves a number of molecules that assist in the assembly process [49–51]. Tropoelastin is synthesized on membrane-bound polysomes, transported through the Golgi apparatus and packaged into secretory vesicles. At this point, elastin secretion may differ from other ECM proteins by trafficking through an acidic compartment (perhaps a sorting endosome) [50, 52]. There is also evidence that tropoelastin is secreted as a complex with a 67 kDa molecular chaperone that targets the tropoelastin molecule to assembly sites on the cell surface [53]. Electron microscopy and dynamic imaging studies show that tropoelastin is assembled into small globular aggregates in the secretory pathway or on the cell surface that begin the initial stages of cross-linking (a process called microassembly) [54–57]. Cross-linking is not required for tropoelastin’s interaction with microfibrils, however [58, 59]

3.2 Microfibril structure and function

The protein fibrillin provides the major structural component of the microfibril, and several associated proteins interact with fibrillin to modify microfibril function [60–62]. The ancestral fibrillin arose early in evolution (Cnidaria) [63, 64] and has remained relatively unchanged except for late gene duplication events that gave rise to fibrillin-2 &-3 [65]. Fibrillin monomers can self-assemble into individual microfibrils that then associate to form large microfibril bundles [66]. Interestingly, physiological studies show that microfibrillar bundles provide limited elasticity to invertebrate tissues that lack elastin [67–69], suggesting that microfibrils were among the earliest elastic structures in the ECM.

In addition to serving as a scaffold for elastin assembly, microfibrils are important modulators of growth factor localization and function [70]. Fibrillin can interact directly with bone morphogenic proteins (BMPs) as well as with latent forms of TGFβ [71, 72]. All three TGFβs are secreted as an inactive latent dimer bound to a member of the latent TGFβ-binding protein (LTBP) family [73]. This large latent complex (TGFβ-LLC) binds covalently to fibrillin molecules in microfibrils [74], thereby creating a latent growth factor reserve in the ECM that can be activated and mobilized, when needed, through interactions with proteases, αvβ6 or αvβ8 integrins, or other factors [73]. Mutations in fibrillin are proposed to compromise its ability to retain the TGFβ-LLC complex in the matrix, resulting in inappropriate growth factor release, activation, and increased TGFβ activity. Numerous studies suggest that elevated TGFβ activity is the pathogenic mechanism leading to emphysema and aortic aneurysms in diseases such as Marfan syndrome, which has been linked to mutations in Fbn1 [75–77]. However, other studies suggest that elevated TGFβ levels are protective against tissue damage and act to suppress disease progression [78–81].

With the arrival of chordates and vertebrates came several new fibrillin-associated proteins, including fibulins-4 & -5, MAGP-1 & -2, and, as discussed above, elastin [82]. Fibrillins and MAGPs are likely the only constitutive components of microfibrils in vertebrates [82–84]. Fibulins, elastin and LTBPs can associate with microfibrils, but are not integral components and are not found on all microfibrils. None of the microfibril-associated proteins are required for fibrillin assembly. Instead, throughout evolution, we see changes occur to microfibril function brought about by the appearance of the proteins that partner with fibrillin to bring new and specialized functional properties. The ability to tailor microfibril activity by changing associated proteins provides a dynamic mechanism for meeting specific needs, such as altering growth factor signaling or controlling protein assembly.

4. ELASTIN AND LUNG DEVELOPMENT

Elastin is widely distributed in compartments of the mammalian lung including pleura, septa, large vessels, and elastic cartilage. The highest concentration of elastin is in the respiratory parenchyma where it comprises 20–30% of the crude connective tissue dry weight [85–87]. The elastin content of pulmonary blood vessels is 7–16% and that of airways is 3–5%. Numerous cell types produce elastin in the lung, including mesothelial cells [88, 89], airway epithelial cells [90, 91], vascular and airway smooth muscle cells [92, 93], endothelial cells [94, 95], and interstitial and lipid-laden fibroblasts [96–98].

4.1 The Lung Pleura: Elastin production by pleural mesothelial cells

There have been many studies of elastin expression in late fetal and postnatal stages of lung development [99–101], but expression of elastic fiber proteins in early lung development is less well understood. Among the earliest cells to make elastin are the pleural cells that line the lung surface (Figure 2). The pleural mesothelial cell is mesenchymal in origin but exhibits characteristics typical of epithelial cells, such as tight junctions, epithelial cytokeratins, and abundant microvilli on the apical surface. Pleural cells are attached basally to a basement membrane, beneath which is an extracellular matrix layer containing elastic fibers organized into an “elastic lamina” and dense bundles of fibrillar collagen. Ultrastructural analysis suggests that the collagen and elastin in the connective tissue layer are produced by the mesothelial cell, although resident fibroblasts may also contribute. Indeed, mesothelial cells have been shown to produce both collagen and elastin under tissue culture conditions [88, 89]. There is evidence suggesting that a population of progenitor-like mesothelial cells can differentiate into multiple cell types and represent a common lineage to fibroblasts, smooth muscle cells, and vasculature [102, 103]. Lung mesothelial cells also regulate epithelial airway branching and organ size through a FGF9 signaling pathway [104].

Figure 2.

In situ hybridization showing elastin gene expression in a ~7-week bovine embryonic lung. Tissue sections were hybridized with a ³⁵S-labeled riboprobe specific for elastin mRNA and exposed to photographic emulsion. Developed slides were counterstained with hematoxylin-eosin, and white silver grains over areas of hybridization were visualized with dark-field microscopy. At this stage in development, tropoelastin showed highest expression in the visceral pleura (arrowhead) with detectable expression in vessels associated with large branching airways in the parenchyma. The image on the right is a reversal of dark-field only, where the hybridization signal shows up black.

The visceral pleura contributes to the mechanical properties of the lung and is in mechanical linkage with the lung parenchyma. This structural arrangement helps distribute mechanical stresses within the lung that facilitate patency of the alveoli and respiratory bronchioles. Without an elastic covering, the vertebrate lung would not be able to expand during inhalation or have the elastic recoil required for exhalation. The visceral pleura of the human and large animals tends to be thick, whereas the pleura in smaller animals, including the mouse, tends to be thin. The variable thickness is due to the submesothelial layer containing the connective tissue components, blood vessels, and lymphatics. The blood supply of the visceral pleura is considered to be of pulmonary origin in small mammals (such as mice) and of bronchial origin in larger mammals (including humans), although difficulties in assigning vascular origins make this generalization somewhat debatable (reviewed in [105]). In addition to contributing to lung elasticity, the pleural connective tissue helps prevent leakage of air from alveoli near the lung surface. Alterations in this barrier function may explain why inherited disorders of elastic fiber proteins, such as mutations in fibrillin associated with Marfan syndrome, permit the development of pneumothorax [77].

4.2 Expression of elastic fiber components in the pulmonary vasculature and bronchi

Among the earliest cells to synthesize elastin in the lung are the vascular cells associated with airways that invade the lung mesenchyme during early stages of lung development. The lungs begin as a ventral outpouching on the foregut. By successive dichotomous divisions, the airway tree increases in complexity as it invades the surrounding mesenchyme. Differentiation of the endodermally-derived epithelial tubules is strongly influenced by the surrounding mesoderm [104, 106, 107]. Development of pulmonary arteries and veins before birth is closely related to that of the bronchial tree [100] and the pre-acinar vascular branching pattern (proximal to the respiratory region of the lung) is present by the 20^th week of fetal life in the human lung [108]. The intra-acinar arteries, in contrast, form during late fetal life and about half are formed after birth as alveolar ducts and alveoli form.

There is a double arterial and double venous supply in the adult lung, with the pulmonary arteries supplying the respiratory units. The bronchial vessels, which supply oxygenated blood from the systemic arteries, supply the airway walls and lung hilum, including pleura and large blood vessel walls. Precursors of the major bronchial vessels initiate by an angiogenic process in which endothelial cells comprising the vestiges of the brachial arches and segmental arteries migrate into the mesenchyme surrounding the early lung buds. In peripheral regions of the developing lung, small vessels form directly through a vasculogenic process in undifferentiated mesenchyme surrounding the growing airway buds. These vessels form a vascular plexus that links up with larger vessels that sprouted and migrated from more differentiated arteries and veins in proximal regions of the lung. Branching of the airways and arteries is contemporaneous, although branching of the airways occurs in advance of the arteries. Pulmonary veins develop later than the arteries and tend to run in the mesenchyme that demarcates the boundary between segments and subsegments of the lung [100].

In developing systemic arteries, expression of the elastin gene occurs coincident with the accumulation and condensation of mesenchymal cells around endothelial tubes and after expression of smooth muscle cell markers [109–112]. When expression of elastin and other elastic fiber proteins was examined in developing lung, unique and divergent expression patterns were found, suggesting that the function of elastic fiber proteins is more complex than initially anticipated.

Figure 3 shows ~10 week bovine lung hybridized with in situ probes that detect mRNA for elastin, fibrillin-1 and fibrillin-2. At this stage of development (pseudoglandular period), hilar regions of the lung show large, well-differentiated airways, arteries and veins that branch within the enclosing mesenchyme. Regions of the lung around the large airways (lower left region in each figure) are more differentiated than areas where branching is still occurring (upper right region).

Figure 3.

In situ hybridization of ~10-week bovine embryonic lung with RNA probes for elastin (A), fibrillin-1(B), and fibrillin-2 (C). Panel 3D is a composite of gene expression in the pulmonary artery indicated by the arrowhead in panels A–C. Samples were prepared as described in Figure 2. See text for interpretation and details.

4.2.1 Elastin

Elastin gene expression (Figure 3A) continues in the visceral pleura (see Figure 2) as well as in the airway smooth muscle layer around the large, well-developed airways in the hilar region of the lung. In blood vessels, tropoelastin expression was detected in endothelial cells in both large and small vessels. SMCs in the medial layer of larger arteries also expressed elastin, although at lower levels than endothelial cells. Little tropoelastin expression was detected in the adventitia except for the outermost rim of cells (Figure 3D). Interestingly, tropoelastin was the only probe of the three that clearly hybridized with the small, less-differentiated vessels in peripheral regions of the lung. Probes for tropoelastin gene expression did not hybridize with all cells in the medial layer of the wall (not shown), suggesting heterogeneity in cellular phenotypes, at least in terms of ECM production. Cellular diversity in terms of the ECM gene expression is consistent with our previous observations of elastin and collagen expression in vessels from adult animals [113] and with studies using smooth muscle cell cytoskeletal markers to identify multiple phenotypically distinct SMC populations in pulmonary arteries [114–116]. There was no tropoelastin expression by epithelial cells in any region of the lung at this stage of development, or in the esophagus (ES). High levels of expression were found in the descending aorta (DA), especially by endothelial cells.

4.2.2 Fibrillin-1

Fibrillin-1 was expressed in the loose connective tissue that suspends the hilar region of the lung and, to a lesser extent, in the peripheral mesenchyme separating the branching segments (Figure 3B). Expression was also evident in well-differentiated blood vessels with high expression throughout the wall (Figure 3D). There was no detectable expression in the condensed mesenchyme around the branching epithelium or in the epithelium itself. In contrast to elastin, fibrillin-1 was not detected in the forming blood vessels in less-differentiated, peripheral regions of the lung. It is interesting to note that fibrillin-1 first appears in the large arteries at about the same time that it appears in the airways associated with those arteries, supporting co-differentiation of cells within arteries and airways.

4.2.3 Fibrillin-2

Fibrillin-2 expression overlapped with elastin and fibrillin-1 in the bronchial smooth muscle of large airways but was the only protein of the three that was expressed by epithelial cells in the distal regions of the lung (Figure 3C). No signal was detected in the regions separating the branching segments, but low expression levels were evident in the condensed mesenchyme around the branching epithelium. Interestingly, fibrillin-2 expression was not detected in either large or small pulmonary vessels (Figure 3D).

4.3 Elastin in the respiratory units of the lung

The basic structure of the gas exchange alveolar-capillary units becomes established during the canalicular phase of lung development, roughly from 16 to 26 weeks in humans. Capillary sprouts from larger vessels interact with and fuse to the basal lamina of epithelial cells in what will become the alveolar ducts and alveoli. These capillaries attach to the epithelial basement membrane and form a network at the surface between neighboring air sacs. The mesenchymal cells within the interstitium of the acini differentiate to produce collagen and elastin fibers, with elastic fibers in the walls of the alveolar ducts, saccule, and alveoli confined essentially to areas immediately surrounding the mouths of the alveoli [3, 117, 118]. These elastic fibers are part of an elastic fiber network that forms an elastic “line element” [119], or cable-membrane architecture [117], that originates in the central bronchi and inserts into the distal airspaces and visceral pleura. The cables from a looping system that encircle the alveolar duct and terminal air spaces and ensures that applied forces will be transmitted equally to all parts of the lung [85, 120].

Numerous studies show that elastic fiber formation in the respiratory region of the lung is tightly linked with alveolar formation [99, 121]. As large amounts of elastin are added and alveolar walls thin in the early postnatal period, lung recoil increases and stress relaxation becomes rapid [122]. In humans, alveolarization begins in the late fetal period and continues during the first eight to ten years of life [121]. In contrast to humans, true alveoli do not exist in the mouse or rat lung at birth, but form between postnatal days 3–15 [122–124]. Within the first 2–3 days of mouse postnatal life narrow ridges appear in the walls of the primary saccule. These “secondary crests” always contain elastin fibers and rapidly elongate to divide the primary saccule into many smaller units of alveoli—a process that continues until about postnatal day 14 [123]. Apically situated elastic and collagen fibers appear to be an essential component of all secondary crests, and the initiation of crest formation is associated with the deposition of these fibers.

Electron microscopy of postnatal lungs identified two types of interstitial cells in the alveolar wall [96]. Cells that appear at the tip of developing septa are associated with the surrounding elastic fibers and have ultrastructural features characteristic of cells engaged in protein synthesis and secretion. Fibroblasts at the base of developing septa are markedly different, having fewer secretory organelles and extensive accumulations of intracellular lipid [125]. Recently, multiple lung fibroblast subsets have been identified (reviewed in [97, 126, 127]) using markers associated with the PDGF signaling pathway [118, 128, 129], FGF signaling [130, 131] and Gli1-expression [132, 133]. How each of these unique cell populations relate to extracellular matrix production, especially elastin, is under investigation.

The importance of elastin to alveolar formation was shown by the absence of alveoli in Pdgf-a^−/− mice that fail to produce elastin in the lung parenchyma [134]. Likewise, mice in which the elastin gene has been inactivated (Eln^−/−) show abnormal terminal airway branches and distal air sacs that are dilated with attenuated tissue septae. Additional studies have documented that disrupting elastin fiber assembly in neonatal mice by inhibiting the crosslinking enzyme, lysyl oxidase, through copper-deficiency or treatment with lathrogens such as β–aminopropionitrile (BAPN) resulted in impaired alveolar septal formation [135, 136]. In contrast to PDGF-A-null mice where ablation of alveolar elastin synthesis is not associated with an aberrant phenotype before P4 (this is true for lathrogen treatment as well), inactivation of the elastin gene in Eln^−/− mice results in lungs that have large air-filled cavities at birth. These differences suggest that disruption of elastin expression in regions other than the alveoli contribute to the pulmonary phenotype when elastin production is ablated in the embryo [137].

5. ELASTIN INTEGRITY AND LUNG DISEASE

5.1 Emphysema

5.1.1 Elastin degradation, inflammation, and emphysema

The discovery of an association between emphysema and α1- antitrypsin deficiency [138], in concert with the use of animal models that develop emphysema after intrapulmonary instillation of elastolytic enzymes [139, 140], lead to the recognition that destruction of elastin is central to disease pathogenesis (i.e., the elastase: anti-elastase hypothesis) [141]. The best-known source of proteases in the lung is inflammatory cells recruited to the airspaces by stimulators of inflammation, such as cigarette smoke (reviewed in [141, 142]). Analysis of the cell profile in alveoli and small airways of smokers shows an increase in all of the cell types implicated in chronic obstructive pulmonary disease (COPD), including macrophages, T lymphocytes, B lymphocytes, and neutrophils.

For many years, neutrophils were thought to be the cell type most responsible for tissue destruction in emphysema through the release of neutrophil elastase [143, 144]. Studies in knockout mice, however, found that the macrophage also plays a significant role in the pathophysiology of COPD and can account for many of the known features of the disease [145–147]. Both neutrophils and macrophages secrete potent elastases that degrade elastin and other matrix proteins [148]. Alveolar macrophages from patients with COPD secrete more inflammatory proteins and have a greater elastolytic activity at baseline than those from normal smokers and this is further increased by exposure to cigarette smoke [38]. Elastin fragments liberated during elastic fiber degradation recruit more inflammatory cells to the lung, leading to enhanced lung tissue destruction by placing more macrophages, and thus more elastin-degrading enzymes, within close proximity of the airspace [149–151].

The instillation of elastase into the air spaces of experimental animals produces lesions with the anatomic and physiologic features of emphysema [152]. After the initial destruction of elastin, connective tissue synthesis is stimulated and lung elastin content returns to normal. However, the resynthesized elastic fibers are disorganized and morphologically defective, and the normal elastic network is not restored [153]. The result is progressive airway loss and worsening lung function. Elastic fibers are among the most difficult matrix structures to repair because of their size, molecular complexity, and the requirement for numerous helper proteins to facilitate fiber assembly [154].

5.1.2 Elastin and disease susceptibility

Targeting elastic fiber genes with loss of function mutations illustrates the importance of elastin to lung development and supports the idea that loss of elastin is a major factor in emphysema and destructive lung diseases. As mentioned, elastin-deficient mice (Eln^−/−) die within 48h of birth with abnormal lung development. Interestingly, mice heterozygous for the elastin mutation (Eln^+/−) with elastin levels about half those of wild-type animals have normal lung morphology [137]. However, on exposure to cigarette smoke (representing chronic pulmonary injury), Eln^+/− mice develop more severe airspace enlargement than wild-type (WT) littermate control mice (Figure 4) [155]. These findings suggest that while lower than normal levels of elastin can support regular lung development, elastin insufficient mice are more prone to develop severe lung disease when exposed to injurious environmental stimuli. Thus, the quantity of elastin in the lung may be an important factor in determining susceptibility to lung injury. While 50% elastin can support normal lung development, mice with less than 50% normal elastin levels exhibit airway abnormalities [156]. For example, mice that express only 30% of normal elastin levels have enlarged thoracic cavities occupied by highly distended lungs that are larger than both WT and Eln^+/− lungs at identical inflation pressures [155]. Thus, when elastin levels drop below ~50% of normal, lung development is compromised.

Figure 4.

Histological sections of lungs from adult wild-type (Eln^+/+) and elastin haploinsufficient (Eln^+/−) mice. Eln^+/− animals exhibit normal alveolar structure (top panels) but develop worse emphysema than normal mice following cigarette smoke exposure (bottom panels).

5.2 Elastin Gain-of-Function Mutations and Autosomal Dominant Cutis Laxa

Elastin gain-of-function mutations that modify key assembly domains in elastin can also lead to lung disease by altering the ability of elastin to assemble into the functional polymer. Autosomal dominant cutis laxa (ADCL, OMIM #123700) is an inherited disease that produces frameshift mutations near the 3′ end of the gene. The result is missense sequence through a critical assembly domain in exon 36 [157, 158]. Several studies have documented severe COPD in individuals with ADCL, especially those who smoke [159, 160]. ADCL mutations alter the ability of the protein from the mutant allele to assemble properly. There is also the likely possibility that the mutant product acts in a dominant–negative fashion to alter the assembly of the protein from the normal allele. A humanized mouse model of ADCL expressing the human elastin gene with a common ADCL mutation showed that alternative splicing of down-stream exons generates multiple transcripts with different lengths of missense sequence and different stabilities [10]. Analysis of tissues expressing the human ADCL transgene found that the mutant protein is incorporated into elastic fibers in skin and lung with adverse effects, but low levels of incorporation were observed in the aorta, which explains why the vasculature is less affected in this disease as a consequence of this mutation [10]. These results suggest that the process of elastin assembly may be different in the aorta compared with lung and skin, which challenges the current thinking of a common fiber assembly mechanism in all tissues.

In addition to causality mutations like those in ADCL, the genetics of COPD susceptibility has been studied extensively [161–163]. A novel variant in the terminal exon of human elastin (c.2318 G A) resulting in an amino acid substitution of glycine 773 to aspartate (G773D), for example, was identified in a pedigree with severe early onset chronic obstructive pulmonary disease (COPD) [164]. Evidence of airflow limitation was most severe among those mutation carriers who smoked, suggesting a gene-by-environment interaction. These results suggest that the G773D variant confers structural and functional consequences relevant to the pathogenesis of COPD and that individual carriers of this polymorphism could be at increased risk for developing lung disease. Similarly, mutations in most of the known elastic fiber genes, including fibulin-4 [165, 166], fibulin-5 [167–169], fibrillin-1[77, 170], latent TGFβ binding proteins [171, 172], and lysyl oxidase-like 1[173, 174], lead to lung disease in humans and mice.

5.3 Elastin as a biomarker for disease

Because of elastin’s low turnover rate, elastin fragments detected in body fluids can be used diagnostically as markers for elastin-degrading diseases, such as emphysema (reviewed in [175]). The cross-linking amino acids desmosine and isodesmosine exist only in insoluble elastin and their presence in blood, urine or sputum can serve as a marker for elastic fiber degradation by elastases associated with inflammation. Desmosine and isodesmosine levels in sputum reflect the state of elastin degradation in the lung specifically [175]. Assays that can accurately measure elastin cross-links or elastin peptide fragments [176] hold great promise for determining disease activity, severity and responses to therapy.

6. CONCLUSIONS

The ECM is a complex integrated system of interacting molecules required for the normal functioning of the lung. Elastic fibers are a major component of this ECM and are essential for lung development and for ensuring that mechanical forces are transmitted equally to all parts of the lung. Understanding the critical role for elastin in lung development and disease will facilitate the discovery of therapies aimed at promoting pulmonary regeneration through alveolation and lung growth in an effort to improve the outcomes of patients afflicted with emphysema and other destructive lung diseases.

Highlights.

• The appearance of elastin in vertebrates was important for the advent of a closed circulatory system and the elastic lung.

• In the developing lung, elastin is expressed in the pleura, the blood vascular system, and in the bronchi and respiratory units.

• Elastin is required for normal lung development and elastic fiber production in the respiratory region of the lung is tightly linked with alveolar formation.

• Destruction of elastin, or abnormalities in elastic fiber assembly, are major factors in emphysema and destructive lung diseases.