State of the Art The Connective Tissue of Lung' ALLAN J. HANCE and RONALD G. CRYSTAL

Contents Introduction Collagen Structure Type I [al (I)] 2a2 Type II [al (Il)]g Type III [al (III)]g Type IV [al (IV)]g Biosynthesis of Collagen Translation of Collagen Messenger Ribonucleic Acid Hydroxylation of Proline Hydroxylation of Lysine Glycosylation Procollagen and Helix Formation Secretion Fibril Formation and Crosslinking Degradation of Collagen Microorganism Collagenase Cathepsin Bl Vertebrate Collagenase Lung Collagen Catabolism Morphology of Lung Collagen Tracheal and Bronchial Cartilage Bronchovascular Framework Alveolar Interstitium Pleura Elastic Fibers Morphology Composition, Biosynthesis, and Crosslinks of Elastic Fibers Elastin Microfibrils Analysis of Elastic Fibers Degradation of Elastic Fibers Elastic Fibers in Lung From the Section on Pulmonary Biochemistry, National Heart and Lung Institute, Bethesda, Md. 20014. 1

The Proteoglycans General Characteristics Glycosaminoglycans in Lung Quantification of Connective Tissue in Nonnal Lung Collagen Elastic Fibers Proteoglycans Control of Connective Tissue Accumulation in Normal Lung and Experimental Models of Lung Growth Collagen Elastic Fibers Proteoglycans Connective Tissue and Lung Disease General Principles Emphysema Quantity of Elastic Fibers Quality of Elastic Fibers Quantity of Collagen Quality of Collagen Proteogl ycans Experimental Protease-Induced Emphysema General Characteristics of the Enzymes Specificity for Elastin Specificity for Collagen or Proteoglycans Endogenous Factors Repair Processes Summary Pulmonary Fibrosis Biochemical Measurements in Human Fibrotic Disease Experimental Animal Models of Pulmonary Fibrosis In Vitro Tissue Culture Models of Fibrosis Relationship of Lung Connective Tissue to Lung Function Obstacles Models

AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 112, 1975

657

658

HANCE AND CRYSTAL

Experimental Evidence Morphologic Studies Quantity of Connective Tissue Components Effects of Decreased Cross! inks Effects of Increased Crosslinks Enzymatic Destruction of Specific Components Gencraliza tions Future Approaches Approaches to the Understanding and Treatment of Lung Disorders Involving Connective Tissue Explant Culture Systems Dispersed Lung Cells and Tissue Culture Models Introduction

In the past decade, there has been an explosion of interest in the connective tissue of the body. vVe know now that the 3 general categories of connective tissue, collagen, elastic fibers, and proteoglycans, are all heterogeneous, with multiple types and substructures. In addition, numerous enzyme systems have been discovered that are critical for preparing each connective tissue component for its structural function. Connective tissue is vital to the structure and function of every tissue; it gives blood vessels viscoelastic properties; skin and tendon, toughness; bone, a matrix to calcify. The lung, of course, is no exception. Connective tissue comprises approximately 25 per cent of the adult human lung and is an intimate part of all lung structures. The lung is the one organ of the body in which we can estimate the contribution of connective tissue to mechanical behavior in vivo and in which abnormalities in connective tissue very quickly appear as abnormalities in function. The purpose of this review is multifold: (1) to define the components of connective tissue in general terms and, specifically, as related to lung; (2) to describe the mechanisms controlling the presence of connective tissue in lung; (3) to summarize the current concepts of how connective tissue influences lung structure and function in both health and disease; (4) to .discuss some possible future directions for the study of lung connective tissue and approaches to the therapy of lung disease. Collagen

Because of the abundance and wide distribution of collagen, its biochemistry has received considerable attention, and it is the best characterized of the connective tissue proteins. In the adult human lung, collagen represents 15 to 20 per cent of the total tissue mass and 60 to 70 per cent of the total connective tissue mass (1, 2).

To provide a general background for understanding the function of collagen in the lung. this section will briefly review the molecular structure of collagen and will emphasize aspects of collagen metabolism that are important for understanding the control of lung collagen homeostasis. Where applicable, we will detail what is known about the composition, localization, biosynthesis, and degradation of lung collagen. In later sections, we will describe the alterations in quality, quantity, and localization of lung collagen in development and disease. Finally, these concepts will be combined with information on other lung connective tissue components to try to summarize the relationships of connective tissue to lung function and how these relationships can be modified to normalize the disordered connective tissue in certain lung diseases. Structure Collagen is an important structural component throughout the lung, including the tracheobronchial tree, vascular tree, parenchyma, and pleura. The fundamental unit of collagen is the tropocollagen molecule, which, by its capacity to copolymerize with other tropocollagen molecules and to interact with a variety of other connective tissue elements, is capable of forming structures such as cartilage, basement membrane, and various fibrils that form the pleura and the skeleton of the lung parenchyma. Tropocollagen is a rod-shaped molecule (1.5 X 300 nm) composed of 3 a chains in a righthanded helical arrangement (3). The righthanded "triple helical" tropocollagen is, in turn, wound in a left-handed "super coil" during fibril formation (4). Tropocollagen is heterogeneous; the 4 types found in the body are distinguishable by the a chains composing them. Type I tropocollagen contains two al (I) chains and one a2 chain, and thus has the structure [al (I)ha2. Each of the other tropocollagens, Type II, Type III, and Type IV, is composed of 3 identical a chains of the appropriate type; the structures are [al (II)Ja. [al (III)]a, and [al (IV)]a. respectively. The 5 known a chains, al (I), a2, al (II), al (III), and al (IV), have different primary amino acid sequences and thus are the products of different structural genes (5, 6). Lung contains all 4 tropocollagens, and thus is the most heterogeneous of all tissues studied with regard to collagen composition (I). Although the 4 tropocollagens are clearly distinguishable by their a chain composition, they

THE CONNECTIVE TISSUE OF LUNG

do share certain structural features. Every a chain has a molecular weight (MW) of 95,000 to 100,000 daltons; each is composed of approximately 1,050 amino acids (3). More than 1,000 of these amino acids occur in repetitive triplets of the form, (glycine-X-Y)n· Although the amino acids represented by X and Y are variable, there is a high content of alanine, proline, lysine, hydroxyproline, and hydroxylysine (5). The latter 2 amino acids are especially characteristic of collagen; their formation will be discussed in the section on biosynthesis. Although elastin (5), the Clq component of complement (7), and perhaps other noncollagen structural proteins also contain hydroxyproline (8), the content of hydroxyproline in collagen is considerably higher. Hydroxylysine is even more characteristic of collagen; it is not present in elastin, although there are small amounts in Clq (7). The a chains are also unique proteins in that they have low tyrosine and methionine contents, and no tryptophan (5). At the N-and C-terminal ends of each a chain, there are regions of approximately 20 amino acids that do not have the glycine-X-Y sequence (3). These regions, known as "teleopeptides," do not take part in helix formation, but they do have an important role in crosslink formation both within and between tropocollagen molecules (4) (see below). Not all of these common features have been studied in tropocollagen isolated from lung, but from what is known, there is nothing to suggest that there will be tissue-specific differences. Interestingly, although all tropocollagens share these characteristics, the structure of each type of tropocollagen can vary beyond that attributable to the differences in primary amino acid sequence. These differences will be discussed below in relation to each tropocollagen type; they include the degree of hydroxylation of lysyl and prolyl residues, the number and type of carbohydrates attached to the tropocollagen, and the number, type, and location of crosslinks that are formed during and after fibril formation. It is probably the uniqueness of each tropocollagen that allows it to perform its structural function in a different way, depending on the requirements of a given tissue. Type I ([al (l)ha2). Type I collagen is the most ubiquitous collagen in the body, being the most abundant type in skin, bone, tendon, and a number of other tissues (9-ll ). Large amounts of Type I collagen are also present in lung; pre-

659

sumably, Type I collagen constitutes a significant proportion of the collagen in the large bronchi and blood vessels. In addition, Type I collagen is also synthesized by lung parenchyma, indicating a role in support of the interstitium (12-14). In vitro, Type I collagen readily forms fibrils that have high tensile strength. These fibrils are tightly packed, leaving little room for ground substance (15). Because of the ubiquity of Type I collagen, it is not surprising that more than one cell type is capable of its synthesis. Because the interstitial cell, fibroblast, and pericyte have the same general morphologic features in lung, the general term "mesenchymal cell" will be used to describe them all. A very large proportion of adult lung cells are mesenchymal (16). The lung mesenchymal cell is capable (in tissue culture) of synthesizing Type I collagen (Hance, A. ]., Bradley, K., and Crystal, R. G.: J Clin Invest, in press); and in addition, a cell isolated from cat lung of apparent parenchymal epithelial origin has been shown to synthesize this collagen type (17, 18). In other tissues, a wide variety of nonmesenchymal cells, including cells of epithelial and neural origin, have been shown to be capable of collagen synthesis (1 9-21 ). Therefore, it is not unlikely that a number of other nonmesenchymal cell types in lung will also be shown to be capable of collagen synthesis. The current assessment of lung cells capable of collagen synthesis is given in table 1. Type II [a! (II)Js. Type II collagen has been identified only in cartilagenous tissue (6). This is also true in lung, where Type II has been found only in trachea and bronchi; it is the major collagen synthesized by these structures maintained in explant culture (13) (table 1). Type II collagen does not form prominent fibrils within these tissues, but rather is found in association with large amounts of proteoglycan (22) (see below for a discussion of these compounds). These proteoglycan-collagen complexes are maintained by strong ionic interactions that may be fostered by the presence of a considerable amount of carbohydrate attached to Type II collagen (23). The cell responsible for the synthesis of Type II collagen in lung has not been identified. In other tissues, chondroblasts are the only cells that synthesize Type II collagen in adult tissues (24), and chrondroblasts in tracheal and bronchial cartilage are probably responsible for its synthesis in lung.

Chondroblast

Probable No

? ?

Probabletttt ?

Probabletttt, • • • • • Probable***··"'

?

Probable* "'"'

cell

Smooth muscle

? ?

Probable*** Probable"'"'"'

?

Probable

No ?

Yes

Yes Probable' • •

? ?

? ?

?

No

No

No

No

Probable* •"'

Yes

?

Probable* • •

?

No-

Yes

Glycosamino-

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?

?

Yes

? ?

? ?

?

?

? ?

Yes

? Probable' • •

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Probable***

glycan DegradaElastase tive Enzymes

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Probable' • •

?

Yes Probable' • •

?

?

Probable* • • Yes

?

Probable • • •

Alveolar Types I and II

Mesenchymal"'"' * * Macrophage

Probable

?

?

Collagenase

Yestt

Proteoglycan Synthesis

Endothelial

Yes ?

Synthesis' •

Elastic Fiber

? ?

Type IV

Yes Probablettt

Type Ill

No ?

Type II

Collagen Synthesist

Yes Probablett t

Type I

• • • • *Probably chondroitin sulfates and keratan sulfate.

• References to the literature are given in the text. tTypa I ~ Type I tropocollagen, ate. • *In most cases, only the elastin component has been demonstrated to be synthesized. ttHyaluronic acid, chondroitin sulfates, dermatan sulfate, heparin, and heparan sulfate. • • *Shown in cells derived from tissue other than lung. It is probable that lung cells behave similarly. from lung will t t fEpithelial cells derived from fetal cat lung and maintained in culture make Types I and Ill; it is unknown whether alveolar Type II cells isolated directly make collagen. • • • '"Mesenchymal" cell is a general term including the fibroblast, the interstitital cell, and the pericyte. These cells are indistinguishable morphologically. ttttThe tracheobronchial tree contains cells (smooth muscle and mesenchymal) that probably synthesize elastic fibers and proteoglycans.

Tracheobronchial tree

Blood vessels

Parenchyma

Structure

Specific Cell Type

TABLE 1

RELATIONSHIP OF LUNG CELLS TO LUNG CONNECTIVE TISSUE SYNTHESIS AND DEGRADATION*

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THE CONNECTIVE TISSUE OF LUNG

Type III [al (III)h The structure of Type III collagen differs from that of Type I and Type II in that Type III has a higher hydroxyproline content and contains cysteine (25). Type III is more difficult to solubilize from adult tissues than Type I, although digestion of tissues with pepsin (which digests the teleopeptide, but not the helical, regions of tropocollagen), markedly increases its recovery (25, 26). Type III collagen constitutes a major percentage of fetal, but not adult, skin and has been found to be a constituent of a number of other tissues, including lung (11, 25-27). So far, it has been found only in tissues that also contain Type I collagen and that also support epithelium or endothelium. The localization of Type III collagen to specific structures in lung has not, as yet, been accomplished. Lung mesenchymal cells grown in tissue culture are capable of synthesizing Type III collagen in addition to Type I (Hance, A. J., Bradley, K., and Crystal, R. G.: J Clin Invest, in press). Interestingly, preliminary work indicates that certain cultured lung cells of epithelial origin also synthesize both Type I and Type III collagen (17, 18) (table I). Type IV [al (IV)] 3 • Type IV, or basement membrane, collagen has been difficult to isolate from lung, because it is closely associated with a number of proteoglycans and other glycoproteins through ionic and disulfide bonds (28). Type IV collagen investigated in other tissues appears to have a number of distinctive characteristics (29-31). It has a high content of cy~~ teine and hydroxylysine and contains a large amount of carbohydrate, including mannose and hexosamine, neither of which is found in other collagens. In addition, the unusual amino acid 3-hydroxyproline is found in abundance in Type IV collagen (31); 4-hydroxyproline is common in other collagen types. Type I contains a single 3-hydroxyproline residue (32), making it the only collagen other than Type IV to have this amino acid. Collagen isolated from rabbit lung does contain small amounts of 3-hydroxyproline, but its source has not been identified (12). Collagenous protein has been extracted from lung parenchyma using techniques that solubilize basement membrane in other tissues (29), but amino acid analysis suggested considerable contamination with noncollagen proteins. Notably, this "alveolar basement membrane" collagen did not contain significant 3-hydroxyproline, raising the possibility that alveolar Type IV collagen is different from the better-studied re-

661

nal and lens capsule basement membrane collagen. The cell type(s) responsible for synthesis of basement membrane collagen in lung are currently unknown (table 1). It is interesting to speculate that lung epithelial and endothelial cells synthesize their own basement membrane, analogous to synthesis of basement membrane by chick lens epithelial cells (33).

Biosynthesis of Collagen Collagen is synthesized in a precursor form termed protropocollagen, which is composed of 3 precursor proa chains. Between the synthesis of component proa chains and the formation of the mature collagen fibril, a complex series of events must take place, including (J) hydroxylation of prolyl and lysyl residues; (2) glycosylation; (J) alignment of component proa chains and triple helix formation; (4) secretion; (5) selected proteolytic digestion at both the ends of the protropocollagen molecule; (6) fibril formation; and (7) crosslinking (figures 1 and 2). Each of these steps, and their importance in determining the nature of the final collagen product, will be discussed briefly_ Translation of collagen messenger ribonucleic acid (mRNA). Nothing is known concerning the transcription of collagen mRNA or the mechanism of its transcriptional control. Collagen mRNA is known to be translated on ribosomes found to the membranes of the rough endoplasmic reticulum (34-37), as is typical for proteins destined for secretion (38) (figure I). The large collagen mRNA (MW > 1.6 X 106 daltons) (39) is capable of accommodating many ribosomes, and therefore, the polysomes directing its synthesis are large (37, 40). Translation of collagen mRNA has been studied in a cell-free system derived from lung; the requirements for translation are similar to those of other proteins (37, 41). Although all somatic cells have the same complement of genetic material, the differences manifested by specific cell types presumably are secondary to the restriction of different genes in diffe~ent cells. Thus, all cells do not have collagen mRNA available to be translated, and those that do may have different collagen mRNAs coding for individual collagen types. For example, it is assumed, but not proved, that the mesenchymal cell probably has al (I), a2, and al (III) mRNA; the chrondoblast, al (II) mRNA; the alveolar macrophage, which does not synthesize collagen (17), no collagen mRNA. The translation product of collagen mRNA is

662

HANCE AND CRYSTAL

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Fig. I. Schematic representation of the biosynthesis of collagen. (a) Transcription of collagen structural genes results in synthesis of collagen messenger ribonucleic acid (mRNA). Each a chain type is translated from a distinct mRNA. It is assumed, but not proved, that each differentiated cell transcribes only the collagen mRN As specific for the collagen type characteristic for that cell. DNA deoxyribonucleic acid. (b) Collagen mRNAs are transported from the nucleus to the ribosomes lining the rough endoplasmic reticulum. As the precursor proa chains are synthesized, they move into the cisternae of the endoplasmic reticulum, where (c) some prolyl and lysyl residues are enzymatically hydroxylatcd (-OH). Proa chains contain an N-terminal noncollagen peptide (D-), a middle collagen a chain ( ), and a C-terminal noncollagen peptide ( ---1:1 ). (d) Three proa chains are aligned into protropocollagen (probably, in part, through disulfide bonds in the C-terminal noncollagen peptide), and the middle a chains coil into a triple helix. (e) Before secretion, some hydroxylysyl residues are glycosylated by enzymatic addition of the monosaccharide, galactose (GAL), or the disaccharide, glucosylgalactose (GLU-GAL-). Glycosylation takes place before proa chains are released from the ribosome, but may continue after helix formation. (f) The completed tropocollagen is secreted from the cell.

=

not the a chain, but rather, a large precursor (MW: 150,000 daltons) called the proa chain (42) (figure I). Translation time for an intact proa chain is probably 7 to 8 min in vivo (43). The proa chain contains 3 regions: an N-terminal noncollagenous region (MW: 20,000 daltons); a middle a chain region (MW: 95,000 daltons), and a C-terminal noncollagenous region (MW: 35,000 daltons). The amino acid sequences of the N- and C-terminal noncollagenous regions are very different from those of the a chain region in that they do not contain the repeating glycine-X-Y sequences, and tryptophan is present in the C-terminal region (42, 44-47). The importance of these N- and C-terminal regions and the conversion of the proa chain to its final a chain form will be discussed later. Before this conversion takes place, there arc 3 important

alterations to the middle a chain "collagenous" region of the proa chain: (I) conversion of selected prolyl residues to hydroxyproline; (2) conversion of selected lysyl residues to hydroxylysine; (3) glycosylation of some hydroxylysyl residues (figure 1). Hydroxylation of proline. As the proa chain is being synthesized on the collagen mRNA, certain prolyl residues are enzymatically converted to hydroxyproline by prolyl hydroxylase (15). This enzyme is bound to the membranes lining the cisternae of the rough endoplasmic reticulum (48-50) and is dependent on the cofactors Fe++, atmospheric 0 2 , a-ketogluterate, and ascorbic acid (or a substitute reducing agent) for activity (51, 52). The enzyme hydroxylatcs in the 4position only prolyl residues that arc in the sequence, glycine-X-proline-glycine (42). Forma-

663

THE CONNECTIVE TISSUE OF LUNG

tion of the collagen triple helix limits further action of the enzyme (53). A separate enzyme may be reguired to form 3-hydroxyproline (found only in al (I) and al (IV) chains), which can be formed from prolyl residues only in the sequence, glycine-proline-Y-glycine. Under normal circumstances, most prolyl hydroxylation occurs while the proa chain is still a nascent chain (before translation has been completed), although it is possible to hydroxylate prolyl residues in completed chains (54); however, not all prolyl residues in the appropriate sequence need be hydroxylated, and differences have been described in the hydroxylation of the same prolyl residue in different tissues from the same species (5, 55). In addition, the

degree of hydroxylation varies greatly among the various collagen a chain types; al (IV) is hydroxylated most completely (ratio of hydroxyproline to proline 1.5), followed by Type III (1.2) (25), Type II (0.9) (23), and Type I (0. 7 to 0.8) (5). Prolyl hydroxylase activity is easily detected in lung tissue; in one study, the level of activity (per mg of tissue protein) was higher in lung than any other tissue studied (50). The degree of hydroxylation of Type I collagen in lung is similar to that in other tissues (12). The hydroxylation of individual prolyl residues or degree of hydroxylation of other lung collagen types has not been examined. Hydroxylation of prolyl residues appears to

=

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FIBRIL FORMATION AND CROSSLINKING

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Fig. 2. Extracellular steps in collagen fibril formation. Newly secreted protropocollagen (upper right) consists of 3 coiled collagen a chains plus N-terminal ( c::J'-) and C-terminal ( ~ ) noncollagen peptides. Proteolytic action of procollagen peptidase cleaves the N-terminal noncollagen peptides, producing the intermediate p-tropocollagen (upper left). Further proteolytic activity removes the C-terminal noncollagen peptides, leaving tropocollagen (center left), composed of a helical region of 3 a chains with short, nonhelical teleopeptides ( and -II ) at either end. Tropocollagens then polymerize to form the collagen fibril (lower left), which is reinforced by crosslinks (heavy lines). The circle at lower right illustrates the steps in formation of the crosslink, hydroxylysinonorleudne. First, lysyl oxidase converts lysine to the aldehyde, allysine. Then, the allysine residue and a hydroxylysine in an adjacent chain interact to form the final crosslink. In the presence of ,B·aminopropionitrile, lysyl oxidase is inhibited, and crosslinking does not occur.

·cr--

664

HANCE AND CRYSTAL

be important for forming and stabilizing the collagen triple helix, although it may serve other functions (42). Hydroxylation can be inhibited severely by removing necessary cofactors or by using proline analogues that are incorporated into a chains, but cannot be hydroxylated. In several systems, underhydroxylation has been shown to result in delayed triple helix formation and inhibition of secretion (42, 56-59). These underhydroxylated proa chains may be more susceptible to intracellular degradation. This could be a vulnerable step in collagen biosynthesis, where therapeutic intervention might slow the process of pathologic collagen accumulation. Whether or not hydroxylation of prolyl residues by prolyl hydroxylase is an important control mechanism in normal collagen synthesis remains unknown. Tissue culture experiments have shown that the concentration of prolyl hydroxylase need not correlate with the rate of collagen synthesis (60, 61). In some cases, however, prolyl hydroxylase activity does correlate with increased collagen formation; activity in silicotic rat lung increased to 250 per cent more than the control value, and the increased activity preceded histologically apparent collagen accumulation (62). Hydroxylation of lysine. In many respects, the hydroxylation of lysyl residues is analogous to prolyl hydroxylation; lysyl residues are hydroxylated as translation proceeds, and only residues in the glycine-X-lysine-glycine sequence are hydroxylated. The necessary enzyme, lysyl hydroxylase, requires the same cofactors and is membrane-bound, and triple' helix formation inhibits further lysyl hydroxylation (42, 63-65). Lysyl hydroxylase activity has been detected in chick lung; in the 15-day-old chick embryo, the concentration was lower than that found in skin and bone, but similar to that in heart and kidney (66). Hydroxylation of lysine appears to be important in several respects. Lysyl and hydroxylysyl residues are the sites of crosslink formation between tropocollagens. Whether or not a lysyl residue involved in crosslink formation is hydroxylated changes the nature and strength of the crosslink formed (5, 67). Hydroxylysyl residues are also important sites at which carbohydrate is attached to tropocollagen (42). Although the precise function of these carbohydrate moieties is unknown, a number of functions have been postulated (see below). As with hydroxylation of proline, all lysyl res-

idues in the correct sequence need not be hydroxylated. Changes in the degree of hydroxylation of a single type of collagen in different tissues have been described. In addition, the degree of hydroxylation can change with age (6870). Type I collagen isolated from 4-month-old rabbit lung has twice the hydroxylysine content as newborn rabbit lung Type I; the sum of lysyl and hydroxylysyl residues remains constant (12). How this change modifies subsequent glycosylation, crosslink formation, or otherwise affects collagen structure is unknown, as are the mechanisms by which lysyl hydroxylase activity is regulated. The degree of hydroxylation is also different in the different collagen types; the lysyl residues are more fully hydroxylated in Type II and Type IV collagen than in Type I and Type III (5, 23, 25, 29). Glycosylation. During biosynthesis of collagen, certain hydroxylysyl residues in the a chain portion of the proa chain are altered by the addition of either the monosaccharide, galactose, or the disaccharide, glucosylgalactose (5). These saccharide units are added sequentially. First, the enzyme, uridine diphosphate (UDP)-galactose:galactosyl transferase, links galactose to collagen through the hydroxyl group of hydroxylysine. Subsequently, some, but not all, of the galactosyl residues are glucosylated by a separate enzyme, UDP-glucose: glucosyl transferase (71, 72). The site of glycosylation is uncertain, although hydroxylysine-linked mono- and disaccharides have recently been identified in nascent proa chains still attached to the ribosome (73). The fact that glycosylation enzymes are capable of acting on native protropocollagen suggests that further glycosylation could take place later. Other types of carbohydrate are attached to Type IV collagen (31) and may be present in the nonhelical peptide regions of other procollagen types (74). The enzymes necessary for glycosylation have been identified in lung microsomes and can use native tropocollagen as a substrate (75). The level of activity was very high in 10-day-old rat lung (only cartilage contained more activity), and the relative activities of the 2 enzymes were nearly equal. The level of activity of both enzymes decreased with aging, which correlates with the rates of collagen synthesis in lung (see below). The function of glycosylation remains uncertain, although a number of possible functions have been suggested, including identifying the proa chain as a protein destined for secretion

THE CONNECTIVE TISSUE OF LUNG

(42), partially regulating crosslink formation (67, 76), and inducing collagen-mediated platelet aggregation (77). Glycosylated hydroxylysine residues do participate in crosslink formation, but how the rate of formation or stability of these crosslinks is modified remains unknown (67, 76). Differences in the degree of glycosylation have been found between tissues, and at different ages in the same tissue, analogous to differences in the hydroxylation of prolyl and lysyl residues (78, 79). Differences in glycosylation of different collagen types is also considerable. Hydroxylysyl residues in Type IV collagen are more than 80 per cent glycosylated, almost entirely with glucosylgalactose. Hydroxylysyl residues in Type II collagen are approximately 40 per cent glycosylated, with an equal distribution of monoand disaccharides (23). The degree of glycosylation of lung collagens has not been studied. Procollagen and helix formation. The importance of the N- and C-terminal noncollagenous "pro" regions of the proa chain is just beginning to be understood. Near the time of release the newly synthesized proa chains from the ribosome, but before helix formation, 3 proa chains of the appropriate type are aligned, probably through interaction of the noncollagenous regions. This association is further stabilized by disulfide bonds in the C-terminal region (42, 46, 4 7, 80-83). Subsequent triple helix formation of the middle a chain "collagenous" region results in the formation of protropocollagen, the form in which collagen is probably secreted from the cell (42). Other functions that have been suggested for the "pro" regions include (J) ensuring solubility of collagen during cellular and extracellular transport (80, 83, 84); (2) facilitating proper orientation of tropocollagen during fibrillogenesis (85); and (3) inhibiting premature crosslink formation (86). Secretion. Optimal secretion requires protropocollagen to be in the triple helical configuration (42) (figure 1). The mechanism by which the newly synthesized protropocollagen is secreted by the cell remains uncertain. A merocrine pattern of secretion, typical of many proteins, has been suggested; protropocollagen moves from the smooth endoplasmic reticulum to the Golgi apparatus, to Golgi-derived vacuoles, and eventually to the extracellular space (87, 88). Other patterns of secretion have been suggested, such as secretion of protropocollagen in vesicles that bypass the Golgi apparatus, or secretion by exocytosis of protropocollagen released from the endoplasmic reticulum into the cell cytoplasm

665

(6). The mechanism of secretion of collagen in lung cells has not been investigated. Fibril formation and cross/inking. The ultimate ability of collagen to serve its supportive function requires the formation of strong collagen fibrils, which, together with associated proteoglycans and noncollagen connective tissue glycoproteins, form the matrix for individual tissues. This requires cleavage of the "pro" region, polymerization of tropocollagen in a specific orientation, and stabilization of the fibril through intra- (between a chains within tropocollagen) and intermolecular (between tropocollagen molecules) crosslinks (figure 2). Before completion of extracellular fibrillogenesis, the N- and C-terminal "pro" regions are cleaved in a series of steps (protropocollagen -+ p-tropocollagen-+ tropocollagen) that requires at least 2 distinct proteolytic enzymes (46, 47). Under normal circumstances, the N-terminal peptide is cleaved first, and .subsequently, the disulfide-linked C-terminal peptide is removed. All 5 types of a chain are first synthesized in precursor form. The rate at which theN- and C-terminal "pro" regions are cleaved may differ for the different types of collagen, and in the case of basement membrane collagen, cleavage may never be complete (89). In the types of collagen in which it has been studied (Types I and II), the polymerization of tropocollagen molecules during fibrillogenesis occurs in a specific fashion, with a staggered arrangement, each tropocollagen overlapping its neighbor by 67 nm or a multiple of that distance (3). Crosslinking is not necessary for fibril formation, but without crosslinking, the fibrils lack high tensile strength. Crosslinks are derived through lysyl or hydroxylysyl residues on neighboring chains, and their formation begins with the oxidation of the «/~/////h,

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COLLAGEN FRAGMENTS

CELL

Fig. 3. Schematic representation of collagen degradation. (1) Intracellular degradation can occur before triple helix formation. This process does not require collagenase; numerous nonspecific proteases can hydrolyze collagen when it is in the nonhelical form. (2) Once the a chains are iri the triple helix, and under normal physiologic extracellular conditions, the a. chains can be degraded only by vertebrate collagenase or microorganism collagenase (presumabiy, the latter is not normally present) . (3) If the triple helix is denatured (e.g., underhydroxylated collagen will denature under physiologic conditions), the a chains can be attacked by nonspecific proteases. (4) The conversion, of protropocollagcn to soluble tropocollagen leaves 3 a chains in helical form; they can be degraded only by collagenase. (5) Proteolysis by vertebrate collagenase leaves tropocollagen in 2 helices, and N-terminal TCA fragment, and a smaller C-terminal TCB fragment. Under physiologic conditions, these fragments denature and can be attacked by nonspecific proteases. (6) If the tropocollagen is incorporated into the insoluble crosslinked fibril, proteolysis can occur with (7) collagenase, or (8) if the intermolecular crosslinks leave the postcollagenase pieces still in fibrillar form, the partially degraded fibril can be (9) phagocytized. In the resulting phagolysosome, enzymes such as cathepsin Bl can degrade the fibril at acid pH.

ase secretion from explants of rabbit lung parenchyma and cultured rabbit alveolar macrophages. As with the microorganism collagenases, these collagenases require Ca++, and are inhibited by EDTA, cysteine, and serum a-globulins (97101). A precursor form of the enzyme ("procollagenase" or "zymogen") has been described (107-109). Although vertebrate collagenase will attack denatured collagen, it is most active against the tropocollagen molecule, whether in solution or in the fibrillar form (97-101). This e nzyme has extraordinary specificity; it cleaves the tropocollagen at one site three-fourths of the distance from the N-terminus of the molecule, always between a glycine:! isoleucine (or leucine) bond (110). Whereas intact tropocollagen remains helical at 37°C, the 2 helical fragments resulting from vertebrate collage nase attack will denature at body temperature. Once collagen is

in a nonhelical form, it ca n be degraded nonsp ecifically by a large number of enzymes active at neutral pH (e.g., extracellular) or acid pH (e.g., within phagolysosomes) (101) (figure 3). Lung collagen catabolism. There are 2 known sources of vertebrate collagenase that can a ttack lung collagen, the polymorphonuclear leukocyte and the alveolar macrophage. '\Ve do no t know which cells are responsible for the production of the enzyme secreted by rabbit parenchyma, although by a nalogy to other tissues, it is probable tha t lung mesenchyma l cells synthesize and secrete collagenase (table 1). The fact that the ubiquitous serum a-globulins inhibit collagenases makes the study of these enzymes in lung very difficult, because the a.-globulins are present in parenchyma (Ill , 112). Although there is clearly a collagenase present

668

HANCE AND CRYSTAL

in the azurophilic granules of the human polymorphonuclear leukocyte, certain aspects of this enzyme suggest that it may be different from other vertebrate collagenases (101, 104, 105, 109). It is not inhibited by serum a-globulins, and in purified form it will not attack collagen fibrils unless an "assistor enzyme," also from the granulocyte, is added; however, a pure collagenase isolated from human leukemic granulocytes can be inhibited by arantitrypsin and a 2 -macroglobulin (II3). The role the leukocyte collagenase plays in lung collagen destruction is not clear at this time. Although collagenase is required to initiate the degradation of helical collagen, there are noncollagenase enzymes that degrade nonhelical collagen. Collagen is in this form in 2 circumstances: (1) newly synthesized proa chains before normal helix formation; (2) when postsynthetic modifications (e.g., proline hydroxylation) have not been completed, the proa or a chains may be unable to maintain a helical configuration. This type of collagen degradation, including at least some intracellular degradation, has been demonstrated in in vitro systems, including parenchymal explants of lung (114). Although proteolysis of newly synthesized collagen (e.g., before fibril formation) appears, at first glance, to be a wasteful mechanism to control net collagen secretion (i.e., it expends energy to synthesize and then to degrade a significant amount of that which is synthesized), it is a well-described characteristic of both mammalian and bacterial cells (!!5). It may be useful in several ways: (1) it allows the cell to remove abnormal proteins arising by mutations, errors in synthesis, denaturation, or modification (e.g., underhydroxylation of collagen secondary to ascorbic acid deficiency); (2) the ability to "turn over" newly synthesized proteins continuously gives the cell an additional rapid means of controlling the quantity of that protein. Regardless of its role in control of net collagen synthesis, reduction of collagen overproduction could be approached therapeutically by increasing intracellular degradation. In summary, if lung collagen is not degraded early by noncollagense mechanisms, it may be degraded in the helical (soluble or fibrillar) form by macrophage, leukocyte, or (possibly) parenchymal collagenase. A cleavage by any of these enzymes could result in 2 subsequent mechanisms to complete proteolysis (figure 3): (1) denaturation and attack by nonspecific proteases in the extracellular milieu; (2) phago-

cytosis and intracellular degradation (116) in the phagolysosome by an enzyme analogous to liver cathepsin Bl. Both are potential sites for therapeutic intervention in human lung disorders involving collagen.

i\!Iorphology of Lung Collagen Approximately 30 to 40 per cent of collagen in the lung is in the large airways, large blood vessels, and septae dividing major lung segments (16). The remainder is present in the various subcompartments of parenchyma, including alveolar interstitium, basement membrane, small blood vessels, and transitional airways. Although differences exist in the morphologic organization of collagen in each location, certain anatomic generalizations are possible. The terminology used to describe collagen morphologically is confusing·. vVe will use the term "fibers" to refer to structures seen with the light microscope; "fibrils," for structures with crossbanding; "nonbanded fibrils," for fibrils < 40 nm in diameter. The term "microfibrils," sometimes used to describe the smallest collagen fibrils, should be avoided because of confusion with the term "microfibrils" or "microfibrillar component" used to describe a specific substructure of the elastic fiber. "Reticulin" is a light microscopic term that has no clear electron microscopic counterpart, although "reticular filaments" are described in electron micrographs (116). Collagen fibers are distinguishable by light microscopy with a number of stains (118); none of these methods is specific for one collagen type. In the lung, collagen fibers are seen in all structures, although they are not prominent in the interstitium. Electron microscopic views of collagen fibers show that the fibers consist of a large number of densely packed fibrils. The fibrils have prominent cross striations occurring with a regular periodicity of 68 nm, with finer banding in between (3, 15). The major 68 nm bands result from the staggered packing of tropocollagen during fibrillogenesis. As a result, the N-terminal end of one tropocollagen does not closely abut the C-terminal end of the adjacent tropocollagen; instead, a space is left in the fibril that stains intensely as the major band. The minor cross striations result from the alignment of polar amino acids in tropocollagens that overlap in the fibril. Fibrils have a wide spectrum (40 to 200 nm) of diameters and can exist together or alone. So-called "reticulin" or "reticulin fibers" were

THE CONNECTIVE TISSUE OF LUNG

first defined by a silver methionine stain; reticulin stains intensely black, whereas collagen fibers stain yellow (ll9). Reticulin is widely distributed in lung. It is probably a complex structure composed of several classes of connective tissue; their exact compositions are unknown. It has been suggested that they contain collagen, proteoglycans, lipid, and noncollagen protein (119-120). The resistance of reticulin to trypsin and its sensitivity to collagenase suggest that the collagenous portion (of unknown type) is an important constituent (100). Tracheal and bronchial cartilage. A distinctive feature of the larger airways is the presence of cartilagenous support within the walls. In the trachea and first-order bronchi, cartilage is present as irregular plates that do not encircle the airway. Once the airways penetrate the parenchyma, cartilage is present as irregular rings that are part of the bronchial walls down to the ninth-order branches (121). The appearance of bronchial cartilage has not received detailed study, but it appears similar to hyaline cartilage found elsewhere in the body. The cartilage contains collagen (Type II) in the form of fine, nonbanded fibrils associated with an amorphous proteoglycan matrix. The perichondrium contains, in part, Type I collagen that appears as banded fibrils. These fibrils fuse with the continuous bronchovascular framework, firmly attaclling cartilage within the walls of the airway (121). The structural importance of cartilage is underscored by the consequences of congenital absence of cartilage, a finding frequently confined to individual lung segments. Without the added support given by cartilage, airway closure produces tension emphysema in the affected segments (122). Bronchovascular framework. Large collagen fibers traverse the lung in association with the subdividing bronchial and vascular trees. These fibers are present in both longitudinal and circular arrays, both of which branch and fuse with each other. The fibers supporting the smaller branches of the airways and vasculature become progressively smaller in diameter. Reticulin is a more prominent feature of the bronchovascular parenchyma (121). Within the parenchyma, collagen fibrils are arranged in a helical fashion as they encircle respiratory bronchioles and alveolar ducts. Finally, the fibrils circle the alveolar openings and spread out as a thin net within the individual walls of the polygonal alveoli (123). Alveolar interstitium. At the level of the al-

669

veolar interstitium, collagen fibrils and elastic fibers may be found in close association; but frequently, they are individually grouped (figure 4). Considerable attention has been directed toward understanding the precise arrangement of collagen fibrils that allows maximal support without interference with the primary function of gas exchange. Within the interstitium, fibrils are present in a central "sheet" or "meshwork" along with cellular elements (macrophages, mesenchymal cells, and mast cells) and other connective tissue components (125-130). On each side of this central supporting layer is the extensive capillary network composed of capillaries that weave their way through the interstitium, appearing first on one side of the alveolar septum and then on the other. Thus, the capillaries seem to have a polarity, with 50 per cent or less of the capillary basement membrane embedded in the central fibrillar meshwork; the remaining capillary basement membrane fused with the alveolar basement membrane (126, 128) (figure 4). Thus, the pathway traversed by gases is reduced to a minimum; gases have only to penetrate the epithelial cell layer and the fused alveolar-capillary basement membrane to reach blood within the pulmonary circulation. In disease states, this orientation can be destroyed, but early thickening of the central interstitial sheet (e.g., in edema or fibrosis) need not lengthen the path for gas diffusion (130, 131). Basement membrane is a prominent structural feature of the alveolar interstitium, serving as the site of attachment of epithelial lining cells to the alveolar walls and surrounding the capillaries of the pulmonary vasculature. The composition of pulmonary basement membrane has received little study, but probably contains a specialized collagen type (Type IV) and associated proteoglycans and noncollagen glycoproteins. Usually, basement membrane appears amorphous, although small (< 10-nm) nonbanded fibrils have been described (116); the collagen composition of tllese nonbanded fibrils is unknown. Fibrils from the interstitial sheet merge with the alveolar basement membrane, especially in areas where capillaries are not closely applied to the alveolar basement membrane (figure 4). Pleura. The pleura also contains substantial amounts of connective tissue. The parietal pleura consists of a pleural cell layer, a thin layer of reticulin, and a dense, collagenous layer, but no elastic fibers (132). The visceral pleura also has a laminated construction. The outer cellular layer rests on a thin, connective tissue layer under

670

HANCE AND CRYSTAL

Fig. 4. Connective tissue in the adult rabbit lung parenchyma. T h e lung from a 1-year-old r a bbit was inflated to a pressure of 15 em H 2 0 with 3 per cent glutaraldehyde in 0.1 M phospha te buffer (p H 7 2. ). (a) Epithelial, capillary, and interst itial structures in the p arenchyma. Alveolar type II cell (TYPE II), capillary (CAP), basem ent membrane (B M), collagen (CO), and elastin (EL) are labeled. The basement m embra n e surrounding the capillary m erges with the collagen and elastin in the interstitium. Mesenchyma l cells are seen throughout the i nterstitium (uran yl acetate a nd lead citrate sta ins; original magnificatio n: X 7,500). (b) Higher-power view of collagen (CO) and ela stic fibers in the interstitium. Crossbanding of the collagen fibrils is ap parent. The

THE CONNECTIVE TISSUE OF LUNG

which lies the "chief layer." The latter contains both collagen and elastic fibers, which lie parallel to the lung surface, but which often turn abruptly, penetrating the parenchyma to join the fibers of the bronchovascular framework. The visceral pleura is therefore firmly attached to the underlying lung. Plaques of collagen have been described lying on the parietal pleura, especially in association with asbestos exposure (133). The collagen types composing the pleura are unknown.

671

(Weigert), orecem-1ron hematoxylin (Verhoef£), and aldehyde-fuchsin (Gomori) (118, 134). Some are not specific for elastic fibers (e.g., the Gomori-aldehyde fuchsin method stains both elastic fibers and mucopolysaccharides deep purple), whereas others do differentiate connective tissue elements (e.g., the Weigert method stains elastic fibers blue-black to black; collagen, pink to red, and other connective tissue components, yellow) (134). Other stains commonly used for light microscopy either do not stain elastic fibers (e.g., acidophilic dyes, including eosin, fast green, Elastic Fibers and orange G; basic dyes, including hematoxyThe presence of elastic fibers in the lung was · lin, methylene blue, and safranin) or variably demonstrated histologically more than 70 years stain elastic fibers (e.g., periodic acid-Schiff, ago. A knowledge of the general mechanical metachromic dyes, or the colloidal iron stain) properties of lung in vivo led to the natural as(134). The mechanisms of action of these stains signment of lung "elastic" properties to the pres- are, in general, not known. In lung, these stains ence of these fibers in lung parenchyma, airways, demonstrate elastic fibers in all structures, inblood vessels, and pleura. Although this associacluding visceral pleura (121). The elastic fibers tion seems obvious, the actual assignment of appear in the parenchyma as a continuum, comlung mechanical behavior to specific biochemplexly intertwined with collagen and ground ically defined components of the elastic fiber was substance elements (123). only begun in the past 10 years. There have been Early observations with the electron micro2 major problems to overcome: (1) until recentscope demonstrated that the elastic fibers were ly, the actual composition of elastic fibers was heterogeneous, with both amorphous and fibrilnot known; (2) the complex anatomic and biolar components being visualized (135). The conchemical interactions among the various comsensus is that the fibrillar constituents (termed ponents of connective tissue have prevented the "microfibrils" or "microfibrillar component") development of simple models of connective are each 10 to 12 nm in diameter, with a denser tissue determinants of lung mechanical properperiphery than center, giving a tubular appearties. This section will detail the current knowlance (135-138). The second component of the edge of elastic fiber morphology, composition, elastic fiber is amorphous to the limits of resolusynthesis, and degradation in general, and as tion of the electron microscope (figure 4). One specifically related to lung. In a later section, wt of the problems in studying these 2 components will combine these data with comparable inforis that they both cannot be well stained with the mation on other lung connective tissue composame method, and thus, electron micrographs nents in order to interpret studies relating lung usually demonstrate the microfibrillar compocomposition to lung mechanical properties in nent with "holes" in place of the amorphous normal and diseased lungs. component, or vice versa. Anionic stains (phosphotungstic acid and silver tetraphenylporphine Morphology solfonate) are used for the positively charged Classic morphologic stains used to define elas- amorphous component, whereas cationic stains tic fibers include resorcinol-iron-basic fuchsin (uranyl acetate, lead tartrate, or lead citrate) elastic fibers are composed of amorphous elastin (EL) and a microfibrillar component (MF) (uranyl acetate and lead citrate stains; original magnification: X 75,000). With this stain, the microfibrils are well seen, but the elastin is not (see text). (c) Interstitium stained with Verhoeff's hematoxylin and lead citrate (124). The elastin (EL) is densely stained; the microfibrils (MF) are seen on end as tubular structures. Collagen (CO), basement membrane (BM), and the cytoplasm of an interstitial cell are seen (IC). At the lower left corner, surrounding the elastic fiber, collagen fibrils are seen on end. Note that the elastic fiber is in close association with the interstitial cell and that the fiber is composed of central elastin and more peripheral microfibrils (original magnification: X 36,000). These electron micrographs were kindly done by V. Ferrans, section on Pathology, National Heart and Lung Institute.

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*The most prevalent nonpolar amino acids include glycine, alanine, valine, proline, leucine, and isoleucine. rPolar amino acids include aspartic acid, glutamic acid, lysine, arginine, and histidine. *. Desmosine, isodesmosine, and Iysinonorleucine.

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Molecular Weight (da/tons) Uranyl Acetate

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Composition, Biosynthesis, and Cross links of Elastic Fibers The extraordinary insolubilty of elastic fibers has been the major reason for the delay in their biochemical definition. The methods generally used to isolate elastic fibers consist of a series of harsh biochemical extraction techniques, with the elastic fibers defined as "the residue." Most of the biochemical definition of elastic fibers has been accomplished using bovine ligamentum nuchae, because of its high (approximately 70 to 80 per cent) elastic fiber content (140). When the 2 morphologic components of the fetal bovine ligamentum nuchae were "biochemically"

TABL.E 2

are used for the negatively charged microfibrillar component (135) (table 2). Studies of aortic elastic fibers have shown that portions of the elastic fibers stain with ruthenium red, suggesting a carbohydrate component (139). This is probably in the microfibrillar component of the fibers. Electron microscopic observation of tissues at various ages has shown that the ratio of microfibrillar to amorphous component appears to decrease with maturation (137). In the developing fetal bovine ligamentum nuchae, elastic fibers are almost entirely composed of the microfibrils. When the microfibrils first appear outside a cell, they remain in close intimacy, occupying infoldings of the cell surface. With advancing age, the microfibrils appear to be invested with the amorphous component within the preformed cylinder of the microfibrils. With maturation, almost the entire center of the fiber is amorphous, with the microfibrils moved peripherally (135, 137) (figure 4). The differential staining characteristics and changes in relative abundance during maturation led to the proposal that the microfibrillar and amorphous components were distinct macromolecules, although until these components were defined biochemically, it was believed that the microfibrils might be precursors of the amorphous component. Given the classic work of Ross and Bornstein (137), however, we now know that they are distinct, with no precursorproduct relationship. Elastic fibers have not been investigated thoroughly in lung, although numerous studies have demonstrated their presence. With Verhoeff's iron hematoxylin stain (124), both components can be seen in the alveolar interstitium of the adult rabbit lung, although the more peripheral microfibrils ·stain less intensely (figure 4).

Crosslinks" ...

HANCE AND CRYSTAL

COMPOSITION AND PROPERTIES OF THE MATURE ELASTIC FIBER AND ITS COMPONENTS

672

673

THE CONNECTIVE TISSUE OF LUNG

dissected (135-138), it was shown that the microfibrils were rich in polar amino acids (e.g., aspartic and glutamic acids), contained significant amounts of cysteine and methionine, and had no hydroxyproline or crosslinks (table 2). Quite distinctly, the amorphous component was rich in nonpolar amino acids (alanine, valine, leucine, and isoleucine), contained no cysteine or methionine, but did have small amounts of hydroxyproline and crosslinks (table 2). Neither the amorphous component nor the microfibrillar component contains hyclroxylysine, thus distinguishing them from collagen. The amino acid composition of the amorphous component was identical to that previously described for "elastin," a protein classically isolated from ligamentum nuchae as the residue not solubilized in the Lansing "hot alkali" procedure (141). These findings have been confirmed by other investigators, and current terminology holds that the elastic fiber is composed of both components, with "elastin" being the amorphous compo-

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nent. Although th ese biochemical studies are just beginning to be applied to lung, it is important to keep in mind that the "elastic fiber" is a variably heterogeneous mixture of elastin and microfibrils, whereas "elastin" is only one of the protein components of the elastic fiber. Elastin. The fact that elastin is an insoluble polymer severely hampered the development of the technology necessary to understand its composition and synthesis. The discoveries of a soluble elastin precursor (142-150), elastin crosslinks (5, 140, 151, 152), and the enzyme necessary to begin the process of crosslinking the precursor into insoluble elastin (90, 153) have resulted in a generally cohesive story of elastin biosynthesis and composition (figure 5). The precursor to elastin, tropoelastin, is synthesized on the rough endoplasmic reticulum of mesenchymal cells. Two cell types, the fibroblast and the smooth muscle cell, have now been been shown to participate in tropoelastin synthesis, although it is probable that other cells

BIOSYNTHESIS OF ELASTIC FIBERS

TROPOELASTIN CHAINS

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CRO SSLI N KED TR O PO ELASTIN CHAINS

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Fig. 5. Biosynthesis of the elastic fiber and elastin crosslinks. (A) A mesenchymal cell first synthesizes the microfibrils. These are elongated, beaded structures lying in semiparallel array. At some later point, the cell synthesizes tropoelastin chains. The enzyme, lysyl oxidase, converts selected lysyl residues in the tropoelastin to aldehydes that subsequently form crosslinks. The crosslinked amorphous component (elastin) invests the microfibrils to form the mature elastic fiber. It is not known whether the conversion of tropoelastin to elastin occurs before or after the association with the microfibrils. (B) An example of the formation of a dcsmosine crosslink bebetween 2 tropoelastin chains. The lysyl residues are surrounded by alanine residues. Although it has been proposed that as many as 4 tropoclastin chains could be held together by a single desmosinc crosslink, only the configuration shown here has, as yet, been demonstrated (140, 154-156).

674

HANCE AND CRYSTAL

have this capacity (135, 157, 158) (table 1). Tropoelastin has an MW of 40,000 to 70,000 daltons, depending on the method used in its isolation (145, 147, 149). The disparity in MW values found by different investigators suggests the possibility that tropoelastin is not the actual protein synthesized by the cell, but that a larger protein, protropoelastin, is the original form synthesized (analogous to proa chains of collagen). Such a precursor has not been found as yet. Tropoelastin is predominantly composed of amino acids that are nonpolar, so the molecule is not very soluble in water. Tropoelastin has not been completely sequenced, but it is known to have a primary structure very different from that of collagen, including repetitious sequences of the peptides glycine-glycine-valine-proline, proline-glycine-valine-glycine-valine, and prolineglycine-valine-glycine-valine-alanine ( 159). It is known that, like collagen, some of the prolyl residues in tropoelastin are hydroxylated, forming hydroxyproline. There is no methionine, cysteine, or hydroxylysine in tropoelastin, but there are significantly larger amounts of lysine in tropoelastin than in elastin (135) (table 2). The excess of lysine residues in tropoelastin compared to elastin is due to the conversion of some lysyl residues into covalent crosslinks that hold together the tropoelastin molecules (140). Although there are many intermediates in the formation of elastin crosslinks, the 3 rna jor final crosslinks ( desmosine, isodesmosine, and lysinonorleucine) have been identified ( 140, 151, 152) (figure 6). The crosslinking process begins

DESMOSINE

with a copper-dependent extracellular enzyme, lysyl oxidase, that catalyzes the oxidative deamination of the r'

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