JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 1
JANUARY 1991
BASIC SCIENCE REVIEW
COLLAGEN: A MULTIFUNCTIONAL FAMILY OF PROTEINS
triplets of gly-X-Y, where generally X represents proline and Y hydroxy-L-proline residues, producing a rigid structure as the individual a-chains assemble into a triple helix. What distinguishes the different forms of collagen are the extent and distribution of non-helical regions within the molecule which, in turn, determine the flexibility and biophysical features of the overall protein. Consequently, type I collagen, with no intervening globular regions, is rigid while type IX collagen, with two globular regions, would be expected to be flexible. Type IX collagen is also distinctive, having a glycosaminoglycan side chain, thereby making this collagen a large hybrid complex which, in turn, is covalently linked to type II collagen in cartilage.3 The different collagen types are synthesized from at least 26 genes located on six different chromoCollagen Biochemistry and Pharmacology somes. Many times, the genes which encode for aWhile many people conceptualize the biochemi- chains of one collagen type are present on different cal features of collagen using type I collagen as a chromosomes. For example, the gene for the al(I) prototype, this is only a partial reflection of the entire chain of type I collagen is present on chromosome 17, multi-gene family of proteins. Figure 1 provides a com- while the gene for a2(I) is on chromosome 7.4 These parison of the currently identified 13 types of collagen, different genes are used to produce mature mRNA demonstrating the diverse size and shape of these which is used to translate protein on the rough endomolecules. Each molecule is comprised of three en- plasmic reticulum (RER) of the cell. This initial protein chains, either identical or a heterocomplex of chains. product is termed "preprocollagen," having its leader The molecular weight of the triple helical domain of sequence (denoting a secreted protein) removed as each protein varies greatly. For example, the col- the protein moves through the wall of the RER. Each lagenous component of each type VI collagen ("short- collagen nascent chain contains globular domains at chain" collagen) a-chain is 52 to 62 kD1 or 40 percent of both the C-terminal and N-terminal, which are rethe total a-chain size of 140 kD. In contrast, type VII moved from selected types of collagen. The segments ("long-chain" collagen)2 has 90 percent of its total 170 that are removed are termed "propeptides." kD a-chain as a collagenous domain. It should be Within the cisternae, specific prolyl and lysyl resinoted that all of these proteins contain the hallmarks dues in the nascent protein are hydroxylated by their of collagen, namely a primary structure of repeating respective hydroxylases. These enzymes require fer-
Downloaded by: Universite de Sherbrooke. Copyrighted material.
William J. Lindblad and Arpad I. Kormos Our understanding of the biochemical structure and physiologic role of the collagen protein family has undergone significant advances during the past decade. It is now appreciated that these proteins are responsible for an impressive array of functions, in addition to the maintenance of tissue mechanical strength. Collagen imparts structure to basement membranes, provides information to cells to elicit a specific cellular phenotype, and provides crucial signals during embryogenesis for the appropriate migration and phenotypic expression of cells. This review will attempt to summarize a few aspects of the field of collagen biochemistry, to provide a glimpse at this rapidly expanding area of study.
Department of Pharmaceutical Sciences, Wayne State University, Detroit and The Wound Healing Center, Division of Plastic and Reconstructive Surgery, Medical College of Virginia, Richmond Reprint requests-. Dr. Lindblad, Dept. of Pharmaceutical Sciences, Wayne State University, 721 Shapero Hall, Detroit, MI 48202 Accepted for publication August 14, 1990 Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
37
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 1
JANUARY 1991 FUNCTION
hyali membr
/ •O 'O
Hyali
Eimi la r t o type I diafibe rs
e.g.
fibr
V
All t
XI
Hyali ne cartilage
eshwoi k
la r t o type III Unkn ow n (may fc rm co
nt membrane
fibrils
Oi
Anchoring fibrils
Unknown (fc
Hypertrophic car
Unkn
Descemet's membr
Unkn
Figure 1. Comparison of different collagen proteins, with each type drawn approximately to scale. Bars indicate triple helical segments and circles, globular regions. Closed segments remain in the mature collagen protein, while open portions indicate regions that are removed during post-translational modifications. The bar indicates lOOnm. (Reprinted in modified form with permission from Burgeson, RE: New collagens, new concepts. Ann Rev Cell Biol 4:551, 1988)
rous iron, ascorbate, molecular oxygen, and a-ketoglutarate as cofactors in the hydroxylation process, with the extent of hydroxylation dependent on the amount of time the non-helical peptides reside in the cistemae. 5 Following the hydroxylation of lysyl residues, sugar residues are added to the hydroxylysyl groups. This glycosylation process is catalyzed by two specific glucosyl and galactosyl transferases.5 Three appropriate nascent chains then spontaneously assemble into a triple helix, effectively stopping modifications of the protein by these enzymes. The stability of the collagen helix is determined by inter-chain hydrogen bonding resulting from the proper hydroxylation of prolyl residues. Consequently, any condition that interferes with this process can dramatically alter the thermal stability of the collagen molecule which, in turn, decreases its denaturation temperature and increases proteolytic breakdown. Scorbutic individuals manifest connective tissue defects partly due to this impairment in collagen hydroxylation. As a control mechanism, a portion of newly synthesized collagen is constantly degraded within the cell, prior to secretion.6 This presumably reflects the degradation of protein with structural defects, although the level can be altered as a means of control38 ling total secreted collagen.
The triple helical procollagen molecule is then secreted into the extracellular space surrounding the cell, where additional processing occurs. For the fibrillar forms of collagen, the propeptides at the C- and N-terminal of the collagen molecule are removed by specific endoproteinases. These endoproteinases, some forms as large as 500 kD,7 rapidly remove the C-terminal propeptides and the majority of the N-terminal propeptides before the individual collagen molecules begin to self-assemble into small-diameter fibers. Once the residual N-terminal propeptides are removed, these fibers can grow by merging with other small-diameter fibrils. At some point after the time of extrusion of the procollagen molecules from the cell, and before large aggregates are formed, lysyl and hydroxylysine residues in the collagen molecules are oxidatively deaminated by the enzyme lysyl oxidase. This deamination enables crosslinking between collagen molecules to occur spontaneously, leading to the formation of water-insoluble fibrils of collagen. Once collagen has assumed its native triple helical configuration, only one class of enzyme at physiologic pH and temperature is able to specifically cleave the molecules, namely, collagenase. Several different species of collagenase have been identified that have
Downloaded by: Universite de Sherbrooke. Copyrighted material.
Unknown (forms cofibers with type II)
COLLAGEN/UNDBLAD, KORMOS
BICCUXHICXL PROCZSI
9IIAJIXACOLOGIC AOIMTS clueocorticoid staroids Icortlaol, triaacina-
1
2
Translation
3
Prolyl and lysyl
Anino acid analogs (sia-hydroxy-L-prolina, acid) PH inhibitor (athyl-3,4hibitor (Minoxidil ) [H,16-dia«thyl proataSlandin-t,)
5
Eacration
Cslchicina
6
crosa-link xoraation
0-eainopropionitrila
XCTURZ HXXNS TO INHIBIT COLLACIK ]IIOSYVTBESK
3
Tranalation
XAti-aanaa KNA
v«
Frocollagan paptida rtooval
Vrocolla^an paptidaaa inhibitors
Figure 2. Schematic presentation of collagen biosynthetic loci that have been examined for potential pharmacologic manipulation. In addition, two potential sites for intervention are noted.
The most widely studied compound that decreases collagen accumulation is p-aminopropionitrile (BAPN), a drug that inhibits the extracellular enzyme lysyl oxidase. Through its inhibition of this enzyme, collagen molecules are not able to cross-link into small-diameter fibers and form larger waterinsoluble deposits. This chemical has been studied for several decades for its potential utility in clinical practice, with small studies performed in patients with scleroderma,18 esophageal stenosis, 19 as well as tendon adhesions.20 The problem with this compound in these studies, is the high degree of adverse reactions to the systemic ingestion of this compound. Over the past few years, studies in several laboratories have been examining the use of BAPN when applied topically, rather than systemically.21 These studies indicate that systemic toxicity is thus reduced, and preliminary clinical studies are underway. Downloaded by: Universite de Sherbrooke. Copyrighted material.
different collagen type specificities.8-10 In addition to these proteases, numerous other non-specific proteases are able to degrade the collagen molecule, if it is thermally or chemically denatured. The large number of post-translational modification steps for collagen suggest that pharmacologic intervention to control the synthesis of this protein could be achieved at several loci. The difficulty arises in targeting the compound to the site of excess collagen production. Figure 2 indicates where various compounds, both specific and non-specific inhibitors, interact to block collagen secretion and deposition. These compounds include glucocorticoid steroids and interferon, which both produce pleiotropic effects on cellular protein synthesis, including the inhibition of collagen mRNA1112 transcription. A few cell types, such as vascular smooth muscle cells, respond to glucocorticoids by stimulating collagen biosynthesis.13 More specific inhibitors of collagen biosynthesis include proline analogs that are incorporated into the collagen nascent chains during translation, preventing appropriate hydroxylation.1415 This destabilizes the triple helical molecules, which are then rapidly degraded. Along the same lines, several inhibitors of the hydroxylating enzymes prolyl and lysyl hydroxylase have been examined for their potential roles in inhibiting collagen biosynthesis. 1617
Physiologic Role of Collagen It has been recognized for many years that collagen provides the mechanical strength of tissues, particularly type I collagen in non-osseous tissues. However, there are numerous other functions these proteins perform that are currently under active investigation. Type IV collagen molecules contain a flexible nonhelical segment that enables the molecules to form end-to-end dimers which have been described as forming a chicken wire array.22 These two-dimensional arrays then associate in side-to-side aggregates, forming honeycomb structures within the basement membrane. This provides an organized structure to the basement membrane, while allowing channels to course through the tissue, thereby enabling transfer of nutrients and ions across tissues. The extremely long type VII collagen appears to constitute the anchoring fibrils that link the epidermal basement membrane to the underlying connective tissue dermal layer.23 Similarly, type VI collagen has been characterized by immunohistochemical analysis to form a flexible network of fibers that anchor nerves and blood vessels into the surrounding connective tissue.24 This type VI structure is very open and filamentous, tending to surround the vessels and then wrap around interstitial fibers. This contrasts to the unique anchoring fibrils seen by electron microscopy which contain type VII collagen. Of great interest have been studies of the influence of different collagen types and basement membrane components on endothelial cell phenotype. These studies have been undertaken to determine what signals are important in the process of angiogenesis. Several investigators have examined the behavior of capillary and aortic endothelial cells cultured on different extracellular matrices.25-28 The studies
39
have determined that the ability of endothelial cells to form tube structures in vitro and to synthesize cellspecific proteins, is dependent on several aspects of the extracellular matrix. Such factors include the type of collagen present, whether other basement membrane proteins were added (e.g., laminin), and the biophysical characteristics of the matrix. It was found that the responsiveness of endothelial cells to an angiogenic factor (TGF-P) is dramatically altered if the cells . are growing in two-dimensional gels vs. three-dimensional gels. Only in the latter configuration did the cells respond to TGF-p with formation of tube-like structures. These investigations demonstrate the profound effect the extracellular matrix and the particular collagen have on cellular phenotype. Investigations in this laboratory have demonstrated major changes in cellular phenotype in hepatocytes if different extracellular matrices are used as substrata.29 With these cells, the biophysical characteristics of the extracellular matrix appear to be of more influence on cellular phenotype than the specific type of collagen used as a substratum. Current studies are underway to better understand this cell-matrix interaction in hepatocytes.
JANUARY 1991
The reconstitution of the extracellular matrix following tissue injury is the hallmark of wound healing. This reformation requires significant clearance of damaged collagenous tissue, as well as the subsequent deposition of new collagen and ECM components. The ordered sequence of the events that are required for this process is undergoing more intense investigation, now that several potential methods for optimizing wound healing in compromised individuals are available. A comprehensive review of the fibrogenic component in wound healing has recently been published.30 Figure 3 provides a schematic presentation of the various outcomes following tissue injury. These outcomes depend, in large measure, on the extent of tissue injury and the magnitude of the subsequent inflammatory response to this injury. This presumably results from the highly active growth factors released during the inflammatory phase of tissue repair. These factors, particularly platelet-derived growth factor, transforming growth factor-^, and macrophage-derived angiogenesis factor, are extremely biologically active, and therefore they can initiate a vigorous tissue response. In particular, TGF-0 has been shown to induce fibrosis and angiogenesis when injected subcutaneously, as
TISSUE INJURY
INFLAMMATORY CELLS
INFLAMMATORY MEDIATORS
MESENCHYMAL CELLS
- • RESOLUTION
PARENCHYMAL CELLS
REGENERATION
MIGRATION AND PROLIFERATION
i,m,:g:_
i.nr, isz\3r
g* MATRIX SYNTHESIS-*
TRANSITIONAL
REPAIR
MATURATION AND REMODELING
t FIBROSIS
40
I
FUNCTIONAL REPAIR
TISSUE DESTRUCTION
Figure 3. Flow diagram of biologic processes and matrix formation following tissue injury. (Reprinted with permission from Diegelmann, RF, Lindblad, WJ, Cohen, IK: Fibrogenic processes during tissue repair. In Nimni, ME (ed): Collagen-. Biochemistry and Biomechanics, Vol II. Boca Raton: CRC Press, 1988, pp 113-136)
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 1
COLLAGEN/LINDBLAD, KORMOS
(40 mg/mL) every six weeks, with or without surgical excision. This treatment follows from research which demonstrated that glucocorticoid steroids can selectively inhibit collagen biosynthesis,11 as well as possibly alter degradation in keloids.42 Additional experimental compounds are being examined for potential therapeutic intervention in this lesion. Preliminary studies using topical BAPN have been promising; however, additional work is required to insure a truly positive effect. Other treatments for keloids, including the use of pressure dressings and local irradiation of the lesion, still remain unproven in controlled clinical trials. The Ehlers-Danlos syndrome (EDS) is a group of more than ten connective-tissue disorders that result from genetic anomalies in collagen genes. The classification of these disorders is constantly evolving, as the molecular defects are identified. A current classification scheme is presented in Table 1. The clinical manifestations include skin fragility, joint hypermobility, excessive bruising, abnormal scar formation and, in the severest form which is not perinatally lethal, rupture of viscera and arteries. The incidence of EDS is reported as one in 5,000 births, perhaps an underestimation due to diagnostic difficulties. The managePathologies of Collagen Metabolism ment of these patients is confined to symptomatic A significant clinical problem that results from treatment, with the exception of some patients with altered repair following injury is that of keloid. Keloids EDS type VI. A subgroup of these individuals with are benign proliferative cutaneous lesions consisting reduced lysyl hydroxylase activity can be treated with largely of collagen and extending beyond the edge of high levels of the enzyme cofactor, ascorbic acid, which 43 the lesion. The most common areas of occurrence are compensates for the elevated Km of the enzyme. Pathe anterior chest, ears, jawline, shoulders, and upper tients with EDS type IV must be closely followed to back. Patient complaints are not only cosmetic, but detect development of aortic aneurysms, since surgiitching and burning of the lesion are generally present, cal repair in these patients is difficult because of the with severe pain in some lesions, e.g., sternal keloids. extreme fragility of the vascular tissue. Collagen biosynthetic activity is elevated in these leMarfan's syndrome is an autosomal dominant, sions,38 compared to normal dermis; however, there is inherited connective tissue disorder with characterisno change in collagen types.39 These findings need to tic physical manifestations. These individuals tend to be re-examined, since there has been improvement in be tall, possess arachnodactyly, scoliosis, and pectus techniques in use and expansion in the known forms of deformities, with concurrent cardiovascular and ocular collagen since these studies were reported. abnormalities. Significant vascular problems are seen Of note, the extracellular processing enzyme, lysyl with these people, including mitral valve prolapse and oxidase, has been reported to be elevated in keloids, regurgitation, aortic valve insufficiency, and aneurysms. with levels three times those of normal dermis.40 This These latter often result in early demise. In addition, could result in increased cross-linking of collagen at Marfan's syndrome is associated with ocular disorders, crucial cell-matrix interaction points, making them particularly bilateral ectopia lentis, accompanied by less susceptible to collagenolytic breakdown,41 and myopia and glaucoma. The molecular defect(s) in this could influence subsequent expression of collagen, as syndrome has not been defined and may result from a problem with collagen and/or elastin. Several studies well as its turnover. The majority of patients seen with keloids are have implicated inadequate lysyl oxidase activity in either black or are descendants of Mediterranean eth- the pathogenesis of this disorder, since extractable collanic groups, although determining if there is a genetic gen is greater from tissues of affected individuals, and 4445 component to this disorder has not proven fruitful. It both desmosine and pyridinoline are decreased. would appear that the genetic component, if there is However, other authors indicate that there is adequate lysyl oxidase activity, with an appropriate amount of one, is but one factor in a multifactorial process. 46 Unfortunately, the recurrence rate following the reactive aldehydes on the collagen molecule. It was surgical removal of a keloid is quite high without phar- concluded in this latter study that there was an inhibimacologic intervention. At present, the recommended tion in the conversion of these aldehydes to cross-links. treatment is intralesional injection of triamcinolone The prevalence of Marfan's syndrome in the popu-
Downloaded by: Universite de Sherbrooke. Copyrighted material.
well as to enhance collagen deposition when applied to healing wounds. 3132 It has been known for a number of years that, in dermal wound healing, overall collagen biosynthesis is elevated within 48 hr of injury, with a continued increase for the next four to six days, before returning to non-injured levels.33 However, this represents the overall biosynthetic capacity of a number of cell types. For example, it was subsequently observed that, as early as 18 hr postinjury, type IV collagen biosynthesis was occurring in the blood clot at the site of damage. 30 This appeared to result from monocytic cells, and it has recently been reported that macrophages could synthesize collagen.34 Since epithelial cells35 and endothelial cells36 also produce collagen, additional studies are required to identify the different sources of collagen in healing wounds, and also to define the factors that regulate the expression of these proteins. In addition, recent studies on collagenolytic activity in healing wounds indicate that this activity may be a primary controlling enzymatic activity in collagen accumulation.37
41
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 1
Major Clinical Manifestations
Connective Tissue Abnormalities
Gravis
AD
Increased collagen fibril diameter
Mitis
AD AD
Soft, hyperextensible skin, easy bruising, poor wound healing, hypermobile joints Similar to type I but less severe Soft skin, no scarring, joint hypermobility Thin, transparent skin, arterial, bowel and uterine rupture Short stature, hernias, moderate joint hypermobility, soft skin Ocular fragility and keratoconus, scoliosis, hypermobile joints, soft skin Joint hypermobility, soft skin, normal scarring
Type
II. Ill
Classification of Connective Tissue Disorders
Inheritance*
V.
Familiar hypermobilityt Arterial (former ecchymotic) X-linked
VI.
Ocular
AR
VII.
Arthrocalasis multiplex
Both AD and AR reported
VIII. IX.
Periodontal Cutis laxa*
AD AD, AR and X-linked recessive were reported
X.
Fibronectin defect
AR
IV
Both AD and AR were reported X-linked recessive
Generalized periodontitis Soft and lax skin, hernias, diverticulas of the gastrointestinal and urinary tract, emphysema, short arms, broad claviculas, hooked nose Similar to type II
Increased collagen fibril diameter Increased collagen fibril diameter Abnormal type III collagen synthesis, secretion or structure Not known Lysyl-hydroxylase deficiency, small collagen fibril diameter Abnormal structure of the aminoterminal cleavage site in pro-al(I) and pro-a2(I) Not known Abnormal copper utilization (low serum copper and ceruloplasmine level) with defect in lysyl-oxidase increased collagen fibril diameter Defect in fibronectin structure
*AD = autosomal dominant; AR = autosomal recessive. tThe most often diagnosed form of EDS. *lt was not classified as part of EDS earlier.
42
lation is one in 15,000, with only symptomatic treatment available for these individuals. Steroid administration has been used prior to puberty to hasten epiphyseal closure and thereby control excessive height. Routine ophthalmic examinations, blood pressure control, and periodic scanning of the aorta are used to monitor for complications in the Marfan's individual, with replacement of the aorta performed in some cases. Another group of connective tissue disorders resulting from mutations in the collagen genes are the multiple forms of osteogenesis imperfecta (Ol). These collagen disorders may be transmitted as an autosomal dominant or recessive characteristic, depending on the form of OI. The disease is characterized by fragile, osteoporotic bones that break, with minimal trauma. Often, the tendons, ligaments, and fascia are also involved, with characteristically blue sclera present in all forms but type IV. The clinical severity of the disease varies greatly, even within families. OI may manifest as a few fractures in childhood and/or adulthood or may result in more than 50 fractures during the lifetime of the individual. The severest form of the disease is type II OI which is lethal in the perinatal period. With advanced DNA sequence analysis, virtually all cases of OI have now been found to contain a mutation in either the pro-al(I) or pro-a2(I) chains of collagen.47 This was particularly surprising, considering the diverse nature of the disorder, as indicated above. This relationship between gene mutation and disease manifestation has enabled various regions of the collagen molecule to be
mapped, demonstrating the unique requirements for each part of the molecule. Currently, therapy for OI consists of orthopedic interventions, with external bracing and internal splinting of bone deformities. Of particular importance is maintenance of normal muscle tone, with intensive physical therapy and swimming exercises prescribed for these individuals.
CONCLUSIONS This review has attempted to discuss briefly the many advances in our understanding of collagen that have occurred during the 1980's. We are now beginning to appreciate the different physiologic functions performed by these molecules, as well as approaching the problems associated with abnormalities in the collagen genes and their regulated biosyntheses. The next decade should see greater advances in the pharmacologic control of these proteins in the clinical setting.
REFERENCES 1. Murata K, MotoyamaT, SukaM.etal: High production of type VI collagen in multiple fibromatosis with multiple articular dysplasia. Biochem Biophys Res Commun 147:275, 1987 2.
Bentz H, Morris NP, Murray LW, et
JANUARY 1991
BASIC SCIENCE REVIEW
COLLAGEN: A MULTIFUNCTIONAL FAMILY OF PROTEINS
triplets of gly-X-Y, where generally X represents proline and Y hydroxy-L-proline residues, producing a rigid structure as the individual a-chains assemble into a triple helix. What distinguishes the different forms of collagen are the extent and distribution of non-helical regions within the molecule which, in turn, determine the flexibility and biophysical features of the overall protein. Consequently, type I collagen, with no intervening globular regions, is rigid while type IX collagen, with two globular regions, would be expected to be flexible. Type IX collagen is also distinctive, having a glycosaminoglycan side chain, thereby making this collagen a large hybrid complex which, in turn, is covalently linked to type II collagen in cartilage.3 The different collagen types are synthesized from at least 26 genes located on six different chromoCollagen Biochemistry and Pharmacology somes. Many times, the genes which encode for aWhile many people conceptualize the biochemi- chains of one collagen type are present on different cal features of collagen using type I collagen as a chromosomes. For example, the gene for the al(I) prototype, this is only a partial reflection of the entire chain of type I collagen is present on chromosome 17, multi-gene family of proteins. Figure 1 provides a com- while the gene for a2(I) is on chromosome 7.4 These parison of the currently identified 13 types of collagen, different genes are used to produce mature mRNA demonstrating the diverse size and shape of these which is used to translate protein on the rough endomolecules. Each molecule is comprised of three en- plasmic reticulum (RER) of the cell. This initial protein chains, either identical or a heterocomplex of chains. product is termed "preprocollagen," having its leader The molecular weight of the triple helical domain of sequence (denoting a secreted protein) removed as each protein varies greatly. For example, the col- the protein moves through the wall of the RER. Each lagenous component of each type VI collagen ("short- collagen nascent chain contains globular domains at chain" collagen) a-chain is 52 to 62 kD1 or 40 percent of both the C-terminal and N-terminal, which are rethe total a-chain size of 140 kD. In contrast, type VII moved from selected types of collagen. The segments ("long-chain" collagen)2 has 90 percent of its total 170 that are removed are termed "propeptides." kD a-chain as a collagenous domain. It should be Within the cisternae, specific prolyl and lysyl resinoted that all of these proteins contain the hallmarks dues in the nascent protein are hydroxylated by their of collagen, namely a primary structure of repeating respective hydroxylases. These enzymes require fer-
Downloaded by: Universite de Sherbrooke. Copyrighted material.
William J. Lindblad and Arpad I. Kormos Our understanding of the biochemical structure and physiologic role of the collagen protein family has undergone significant advances during the past decade. It is now appreciated that these proteins are responsible for an impressive array of functions, in addition to the maintenance of tissue mechanical strength. Collagen imparts structure to basement membranes, provides information to cells to elicit a specific cellular phenotype, and provides crucial signals during embryogenesis for the appropriate migration and phenotypic expression of cells. This review will attempt to summarize a few aspects of the field of collagen biochemistry, to provide a glimpse at this rapidly expanding area of study.
Department of Pharmaceutical Sciences, Wayne State University, Detroit and The Wound Healing Center, Division of Plastic and Reconstructive Surgery, Medical College of Virginia, Richmond Reprint requests-. Dr. Lindblad, Dept. of Pharmaceutical Sciences, Wayne State University, 721 Shapero Hall, Detroit, MI 48202 Accepted for publication August 14, 1990 Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
37
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 1
JANUARY 1991 FUNCTION
hyali membr
/ •O 'O
Hyali
Eimi la r t o type I diafibe rs
e.g.
fibr
V
All t
XI
Hyali ne cartilage
eshwoi k
la r t o type III Unkn ow n (may fc rm co
nt membrane
fibrils
Oi
Anchoring fibrils
Unknown (fc
Hypertrophic car
Unkn
Descemet's membr
Unkn
Figure 1. Comparison of different collagen proteins, with each type drawn approximately to scale. Bars indicate triple helical segments and circles, globular regions. Closed segments remain in the mature collagen protein, while open portions indicate regions that are removed during post-translational modifications. The bar indicates lOOnm. (Reprinted in modified form with permission from Burgeson, RE: New collagens, new concepts. Ann Rev Cell Biol 4:551, 1988)
rous iron, ascorbate, molecular oxygen, and a-ketoglutarate as cofactors in the hydroxylation process, with the extent of hydroxylation dependent on the amount of time the non-helical peptides reside in the cistemae. 5 Following the hydroxylation of lysyl residues, sugar residues are added to the hydroxylysyl groups. This glycosylation process is catalyzed by two specific glucosyl and galactosyl transferases.5 Three appropriate nascent chains then spontaneously assemble into a triple helix, effectively stopping modifications of the protein by these enzymes. The stability of the collagen helix is determined by inter-chain hydrogen bonding resulting from the proper hydroxylation of prolyl residues. Consequently, any condition that interferes with this process can dramatically alter the thermal stability of the collagen molecule which, in turn, decreases its denaturation temperature and increases proteolytic breakdown. Scorbutic individuals manifest connective tissue defects partly due to this impairment in collagen hydroxylation. As a control mechanism, a portion of newly synthesized collagen is constantly degraded within the cell, prior to secretion.6 This presumably reflects the degradation of protein with structural defects, although the level can be altered as a means of control38 ling total secreted collagen.
The triple helical procollagen molecule is then secreted into the extracellular space surrounding the cell, where additional processing occurs. For the fibrillar forms of collagen, the propeptides at the C- and N-terminal of the collagen molecule are removed by specific endoproteinases. These endoproteinases, some forms as large as 500 kD,7 rapidly remove the C-terminal propeptides and the majority of the N-terminal propeptides before the individual collagen molecules begin to self-assemble into small-diameter fibers. Once the residual N-terminal propeptides are removed, these fibers can grow by merging with other small-diameter fibrils. At some point after the time of extrusion of the procollagen molecules from the cell, and before large aggregates are formed, lysyl and hydroxylysine residues in the collagen molecules are oxidatively deaminated by the enzyme lysyl oxidase. This deamination enables crosslinking between collagen molecules to occur spontaneously, leading to the formation of water-insoluble fibrils of collagen. Once collagen has assumed its native triple helical configuration, only one class of enzyme at physiologic pH and temperature is able to specifically cleave the molecules, namely, collagenase. Several different species of collagenase have been identified that have
Downloaded by: Universite de Sherbrooke. Copyrighted material.
Unknown (forms cofibers with type II)
COLLAGEN/UNDBLAD, KORMOS
BICCUXHICXL PROCZSI
9IIAJIXACOLOGIC AOIMTS clueocorticoid staroids Icortlaol, triaacina-
1
2
Translation
3
Prolyl and lysyl
Anino acid analogs (sia-hydroxy-L-prolina, acid) PH inhibitor (athyl-3,4hibitor (Minoxidil ) [H,16-dia«thyl proataSlandin-t,)
5
Eacration
Cslchicina
6
crosa-link xoraation
0-eainopropionitrila
XCTURZ HXXNS TO INHIBIT COLLACIK ]IIOSYVTBESK
3
Tranalation
XAti-aanaa KNA
v«
Frocollagan paptida rtooval
Vrocolla^an paptidaaa inhibitors
Figure 2. Schematic presentation of collagen biosynthetic loci that have been examined for potential pharmacologic manipulation. In addition, two potential sites for intervention are noted.
The most widely studied compound that decreases collagen accumulation is p-aminopropionitrile (BAPN), a drug that inhibits the extracellular enzyme lysyl oxidase. Through its inhibition of this enzyme, collagen molecules are not able to cross-link into small-diameter fibers and form larger waterinsoluble deposits. This chemical has been studied for several decades for its potential utility in clinical practice, with small studies performed in patients with scleroderma,18 esophageal stenosis, 19 as well as tendon adhesions.20 The problem with this compound in these studies, is the high degree of adverse reactions to the systemic ingestion of this compound. Over the past few years, studies in several laboratories have been examining the use of BAPN when applied topically, rather than systemically.21 These studies indicate that systemic toxicity is thus reduced, and preliminary clinical studies are underway. Downloaded by: Universite de Sherbrooke. Copyrighted material.
different collagen type specificities.8-10 In addition to these proteases, numerous other non-specific proteases are able to degrade the collagen molecule, if it is thermally or chemically denatured. The large number of post-translational modification steps for collagen suggest that pharmacologic intervention to control the synthesis of this protein could be achieved at several loci. The difficulty arises in targeting the compound to the site of excess collagen production. Figure 2 indicates where various compounds, both specific and non-specific inhibitors, interact to block collagen secretion and deposition. These compounds include glucocorticoid steroids and interferon, which both produce pleiotropic effects on cellular protein synthesis, including the inhibition of collagen mRNA1112 transcription. A few cell types, such as vascular smooth muscle cells, respond to glucocorticoids by stimulating collagen biosynthesis.13 More specific inhibitors of collagen biosynthesis include proline analogs that are incorporated into the collagen nascent chains during translation, preventing appropriate hydroxylation.1415 This destabilizes the triple helical molecules, which are then rapidly degraded. Along the same lines, several inhibitors of the hydroxylating enzymes prolyl and lysyl hydroxylase have been examined for their potential roles in inhibiting collagen biosynthesis. 1617
Physiologic Role of Collagen It has been recognized for many years that collagen provides the mechanical strength of tissues, particularly type I collagen in non-osseous tissues. However, there are numerous other functions these proteins perform that are currently under active investigation. Type IV collagen molecules contain a flexible nonhelical segment that enables the molecules to form end-to-end dimers which have been described as forming a chicken wire array.22 These two-dimensional arrays then associate in side-to-side aggregates, forming honeycomb structures within the basement membrane. This provides an organized structure to the basement membrane, while allowing channels to course through the tissue, thereby enabling transfer of nutrients and ions across tissues. The extremely long type VII collagen appears to constitute the anchoring fibrils that link the epidermal basement membrane to the underlying connective tissue dermal layer.23 Similarly, type VI collagen has been characterized by immunohistochemical analysis to form a flexible network of fibers that anchor nerves and blood vessels into the surrounding connective tissue.24 This type VI structure is very open and filamentous, tending to surround the vessels and then wrap around interstitial fibers. This contrasts to the unique anchoring fibrils seen by electron microscopy which contain type VII collagen. Of great interest have been studies of the influence of different collagen types and basement membrane components on endothelial cell phenotype. These studies have been undertaken to determine what signals are important in the process of angiogenesis. Several investigators have examined the behavior of capillary and aortic endothelial cells cultured on different extracellular matrices.25-28 The studies
39
have determined that the ability of endothelial cells to form tube structures in vitro and to synthesize cellspecific proteins, is dependent on several aspects of the extracellular matrix. Such factors include the type of collagen present, whether other basement membrane proteins were added (e.g., laminin), and the biophysical characteristics of the matrix. It was found that the responsiveness of endothelial cells to an angiogenic factor (TGF-P) is dramatically altered if the cells . are growing in two-dimensional gels vs. three-dimensional gels. Only in the latter configuration did the cells respond to TGF-p with formation of tube-like structures. These investigations demonstrate the profound effect the extracellular matrix and the particular collagen have on cellular phenotype. Investigations in this laboratory have demonstrated major changes in cellular phenotype in hepatocytes if different extracellular matrices are used as substrata.29 With these cells, the biophysical characteristics of the extracellular matrix appear to be of more influence on cellular phenotype than the specific type of collagen used as a substratum. Current studies are underway to better understand this cell-matrix interaction in hepatocytes.
JANUARY 1991
The reconstitution of the extracellular matrix following tissue injury is the hallmark of wound healing. This reformation requires significant clearance of damaged collagenous tissue, as well as the subsequent deposition of new collagen and ECM components. The ordered sequence of the events that are required for this process is undergoing more intense investigation, now that several potential methods for optimizing wound healing in compromised individuals are available. A comprehensive review of the fibrogenic component in wound healing has recently been published.30 Figure 3 provides a schematic presentation of the various outcomes following tissue injury. These outcomes depend, in large measure, on the extent of tissue injury and the magnitude of the subsequent inflammatory response to this injury. This presumably results from the highly active growth factors released during the inflammatory phase of tissue repair. These factors, particularly platelet-derived growth factor, transforming growth factor-^, and macrophage-derived angiogenesis factor, are extremely biologically active, and therefore they can initiate a vigorous tissue response. In particular, TGF-0 has been shown to induce fibrosis and angiogenesis when injected subcutaneously, as
TISSUE INJURY
INFLAMMATORY CELLS
INFLAMMATORY MEDIATORS
MESENCHYMAL CELLS
- • RESOLUTION
PARENCHYMAL CELLS
REGENERATION
MIGRATION AND PROLIFERATION
i,m,:g:_
i.nr, isz\3r
g* MATRIX SYNTHESIS-*
TRANSITIONAL
REPAIR
MATURATION AND REMODELING
t FIBROSIS
40
I
FUNCTIONAL REPAIR
TISSUE DESTRUCTION
Figure 3. Flow diagram of biologic processes and matrix formation following tissue injury. (Reprinted with permission from Diegelmann, RF, Lindblad, WJ, Cohen, IK: Fibrogenic processes during tissue repair. In Nimni, ME (ed): Collagen-. Biochemistry and Biomechanics, Vol II. Boca Raton: CRC Press, 1988, pp 113-136)
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 1
COLLAGEN/LINDBLAD, KORMOS
(40 mg/mL) every six weeks, with or without surgical excision. This treatment follows from research which demonstrated that glucocorticoid steroids can selectively inhibit collagen biosynthesis,11 as well as possibly alter degradation in keloids.42 Additional experimental compounds are being examined for potential therapeutic intervention in this lesion. Preliminary studies using topical BAPN have been promising; however, additional work is required to insure a truly positive effect. Other treatments for keloids, including the use of pressure dressings and local irradiation of the lesion, still remain unproven in controlled clinical trials. The Ehlers-Danlos syndrome (EDS) is a group of more than ten connective-tissue disorders that result from genetic anomalies in collagen genes. The classification of these disorders is constantly evolving, as the molecular defects are identified. A current classification scheme is presented in Table 1. The clinical manifestations include skin fragility, joint hypermobility, excessive bruising, abnormal scar formation and, in the severest form which is not perinatally lethal, rupture of viscera and arteries. The incidence of EDS is reported as one in 5,000 births, perhaps an underestimation due to diagnostic difficulties. The managePathologies of Collagen Metabolism ment of these patients is confined to symptomatic A significant clinical problem that results from treatment, with the exception of some patients with altered repair following injury is that of keloid. Keloids EDS type VI. A subgroup of these individuals with are benign proliferative cutaneous lesions consisting reduced lysyl hydroxylase activity can be treated with largely of collagen and extending beyond the edge of high levels of the enzyme cofactor, ascorbic acid, which 43 the lesion. The most common areas of occurrence are compensates for the elevated Km of the enzyme. Pathe anterior chest, ears, jawline, shoulders, and upper tients with EDS type IV must be closely followed to back. Patient complaints are not only cosmetic, but detect development of aortic aneurysms, since surgiitching and burning of the lesion are generally present, cal repair in these patients is difficult because of the with severe pain in some lesions, e.g., sternal keloids. extreme fragility of the vascular tissue. Collagen biosynthetic activity is elevated in these leMarfan's syndrome is an autosomal dominant, sions,38 compared to normal dermis; however, there is inherited connective tissue disorder with characterisno change in collagen types.39 These findings need to tic physical manifestations. These individuals tend to be re-examined, since there has been improvement in be tall, possess arachnodactyly, scoliosis, and pectus techniques in use and expansion in the known forms of deformities, with concurrent cardiovascular and ocular collagen since these studies were reported. abnormalities. Significant vascular problems are seen Of note, the extracellular processing enzyme, lysyl with these people, including mitral valve prolapse and oxidase, has been reported to be elevated in keloids, regurgitation, aortic valve insufficiency, and aneurysms. with levels three times those of normal dermis.40 This These latter often result in early demise. In addition, could result in increased cross-linking of collagen at Marfan's syndrome is associated with ocular disorders, crucial cell-matrix interaction points, making them particularly bilateral ectopia lentis, accompanied by less susceptible to collagenolytic breakdown,41 and myopia and glaucoma. The molecular defect(s) in this could influence subsequent expression of collagen, as syndrome has not been defined and may result from a problem with collagen and/or elastin. Several studies well as its turnover. The majority of patients seen with keloids are have implicated inadequate lysyl oxidase activity in either black or are descendants of Mediterranean eth- the pathogenesis of this disorder, since extractable collanic groups, although determining if there is a genetic gen is greater from tissues of affected individuals, and 4445 component to this disorder has not proven fruitful. It both desmosine and pyridinoline are decreased. would appear that the genetic component, if there is However, other authors indicate that there is adequate lysyl oxidase activity, with an appropriate amount of one, is but one factor in a multifactorial process. 46 Unfortunately, the recurrence rate following the reactive aldehydes on the collagen molecule. It was surgical removal of a keloid is quite high without phar- concluded in this latter study that there was an inhibimacologic intervention. At present, the recommended tion in the conversion of these aldehydes to cross-links. treatment is intralesional injection of triamcinolone The prevalence of Marfan's syndrome in the popu-
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well as to enhance collagen deposition when applied to healing wounds. 3132 It has been known for a number of years that, in dermal wound healing, overall collagen biosynthesis is elevated within 48 hr of injury, with a continued increase for the next four to six days, before returning to non-injured levels.33 However, this represents the overall biosynthetic capacity of a number of cell types. For example, it was subsequently observed that, as early as 18 hr postinjury, type IV collagen biosynthesis was occurring in the blood clot at the site of damage. 30 This appeared to result from monocytic cells, and it has recently been reported that macrophages could synthesize collagen.34 Since epithelial cells35 and endothelial cells36 also produce collagen, additional studies are required to identify the different sources of collagen in healing wounds, and also to define the factors that regulate the expression of these proteins. In addition, recent studies on collagenolytic activity in healing wounds indicate that this activity may be a primary controlling enzymatic activity in collagen accumulation.37
41
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 1
Major Clinical Manifestations
Connective Tissue Abnormalities
Gravis
AD
Increased collagen fibril diameter
Mitis
AD AD
Soft, hyperextensible skin, easy bruising, poor wound healing, hypermobile joints Similar to type I but less severe Soft skin, no scarring, joint hypermobility Thin, transparent skin, arterial, bowel and uterine rupture Short stature, hernias, moderate joint hypermobility, soft skin Ocular fragility and keratoconus, scoliosis, hypermobile joints, soft skin Joint hypermobility, soft skin, normal scarring
Type
II. Ill
Classification of Connective Tissue Disorders
Inheritance*
V.
Familiar hypermobilityt Arterial (former ecchymotic) X-linked
VI.
Ocular
AR
VII.
Arthrocalasis multiplex
Both AD and AR reported
VIII. IX.
Periodontal Cutis laxa*
AD AD, AR and X-linked recessive were reported
X.
Fibronectin defect
AR
IV
Both AD and AR were reported X-linked recessive
Generalized periodontitis Soft and lax skin, hernias, diverticulas of the gastrointestinal and urinary tract, emphysema, short arms, broad claviculas, hooked nose Similar to type II
Increased collagen fibril diameter Increased collagen fibril diameter Abnormal type III collagen synthesis, secretion or structure Not known Lysyl-hydroxylase deficiency, small collagen fibril diameter Abnormal structure of the aminoterminal cleavage site in pro-al(I) and pro-a2(I) Not known Abnormal copper utilization (low serum copper and ceruloplasmine level) with defect in lysyl-oxidase increased collagen fibril diameter Defect in fibronectin structure
*AD = autosomal dominant; AR = autosomal recessive. tThe most often diagnosed form of EDS. *lt was not classified as part of EDS earlier.
42
lation is one in 15,000, with only symptomatic treatment available for these individuals. Steroid administration has been used prior to puberty to hasten epiphyseal closure and thereby control excessive height. Routine ophthalmic examinations, blood pressure control, and periodic scanning of the aorta are used to monitor for complications in the Marfan's individual, with replacement of the aorta performed in some cases. Another group of connective tissue disorders resulting from mutations in the collagen genes are the multiple forms of osteogenesis imperfecta (Ol). These collagen disorders may be transmitted as an autosomal dominant or recessive characteristic, depending on the form of OI. The disease is characterized by fragile, osteoporotic bones that break, with minimal trauma. Often, the tendons, ligaments, and fascia are also involved, with characteristically blue sclera present in all forms but type IV. The clinical severity of the disease varies greatly, even within families. OI may manifest as a few fractures in childhood and/or adulthood or may result in more than 50 fractures during the lifetime of the individual. The severest form of the disease is type II OI which is lethal in the perinatal period. With advanced DNA sequence analysis, virtually all cases of OI have now been found to contain a mutation in either the pro-al(I) or pro-a2(I) chains of collagen.47 This was particularly surprising, considering the diverse nature of the disorder, as indicated above. This relationship between gene mutation and disease manifestation has enabled various regions of the collagen molecule to be
mapped, demonstrating the unique requirements for each part of the molecule. Currently, therapy for OI consists of orthopedic interventions, with external bracing and internal splinting of bone deformities. Of particular importance is maintenance of normal muscle tone, with intensive physical therapy and swimming exercises prescribed for these individuals.
CONCLUSIONS This review has attempted to discuss briefly the many advances in our understanding of collagen that have occurred during the 1980's. We are now beginning to appreciate the different physiologic functions performed by these molecules, as well as approaching the problems associated with abnormalities in the collagen genes and their regulated biosyntheses. The next decade should see greater advances in the pharmacologic control of these proteins in the clinical setting.
REFERENCES 1. Murata K, MotoyamaT, SukaM.etal: High production of type VI collagen in multiple fibromatosis with multiple articular dysplasia. Biochem Biophys Res Commun 147:275, 1987 2.
Bentz H, Morris NP, Murray LW, et
