The Fibrous Components of Connective Tissue with Special Reference to the Elastic Fiber

The Fibrous Components of Connective Tissue with Special Reference to the Elastic Fiber DAVID A . HALL Nufield Gerontological Research Unit. Department of Medicine. School of Medicine. Leeds. England Page

I. Introduction ..................................................... I1. Morphological Studies on Collagen and Elastic Fibers ............... A . Introduction ................................................. B Histology .................................................... 1. Methods .................................................. 2. Theories of Staining ...................................... C. Morphological Studies on the Pathological Involvement of Elastic Tissues ...................................................... 1. Senile Elastosis .......................................... 2. Ehler's Danlos Syndrome-Rubber Skin ................... 3. Pseudoxanthoma Elasticum ............................... 4. Colloid Millium .......................................... 5 . Obliterative Diseases of Elastic Fibers ..................... D . Structural and Physical Aspects of Connective-Tissue Fibers ... 1. Morphology under the Light Microscope ................... 2. Electron-Microscope Appearance .......................... 3. X-Ray Diffraction Studies on Connective Tissue ............. 4. Physical Properties of Collagen and Elastin ............... 5 . Model Structures for Elastic Fibers ....................... I11. Biochemical Studies on Collagen and Elastic Fibers ................. A . Chemical Composition of Collagen and Elastin ................ 1. Amino Acid Analyses .................................... 2. Polysaccharide as a Component of Connective Tissue ........ 3. Lipid ..................................................... B. Distribution of Collagen and Elastic Fibers ................... 1. Methods of Determination ................................. 2. The Relative Distribution of Collagen and Elastin .......... C. The Enzymatic Susceptibility of Elastin and Collagen ........... 1. The Elastase Complex and Its Physiological Significance ... 2. Specificity of the Enzymes ................................. I V. The Physiology of Connective-Tissue Fibers ....................... A . Fibrogenesis ................................................. 1. Embryonic Tissue ........................................ 2. Wound Healing ......................................... B. The Aging of Connective-Tissue Fibers ....................... 1. Changes in Collagen ..................................... 2. Changes in Elastic Fibers ................................. V . The Production of Elastic Material from Collagen ................. A . Structural Evidence ......................................... 1. Histochemical Evidence .................................. 2. Electron-Microscope Studies ............................... 3. X-Ray Diffraction ........................................

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B. Chemical Evidence ........................................... 1. Alkali Treatment ......................................... C. Evidence for the Heterogeneity of Collagen ................... 1. Sequence Studies ......................................... 2. The Action of Collagenase ................................ 3. The Effect of Phthalate Buffer on Collagen ................. VI. Conclusions ...................................................... VII. References .......................................................

I. INTRODUCTION The year 1955 saw the reintroduction of a concept (Burton et al., 1955; Hall et al., 1955a) that had engaged the interest of histologists since the days of Unna (18%). It had, however, been relegated to the limbo reserved for those hypotheses that have failed to withstand the test of subsequent research. This particular concept concerned the relationship between collagen and elastic fibers, especially with regard to the possibility of direct conversion of the former into elastic fibers in the animal body. Although such a concept could explain much of the histological evidence for the existence of material with tinctorial properties intermediate between those of collagen and elastica, it appeared to receive its death blow with the appearance of the first relatively complete and accurate analyses for the protein constituents of these connective-tissue fibers (Bowes and Kenten, 1948; Stein and Miller, 1938). In view of the reawakened interest in connective tissue, this review should serve a twofold purpose. Not only will it provide one of the original authors with a medium for discussing the present status of the hypothesis, but it will enable him to attempt to fill a gap long apparent in the studies of elastic tissue, namely the examination of the properties of elastic fibers as they fit into the general physiology of connective tissue, especially in comparison with the properties of collagen fibers. In the first three major sections to follow, therefore, evidence is presented to justify the consideration of fibrous components of connective tissue as a group, and not as individuals. In the fourth section is gathered together the not inconsiderable mass of evidence for the consideration of two members of the group-collagen and elastic fibers-as being more intimately related than has hitherto been assumed likely. The literature relating to connective-tissue fibers has been reviewed from the point of view of the elastic fiber, and hence references to collagen are by no means complete. Selection has been made solely to provide an adequate background against which to compare the properties of the elastic fiber.

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11. MORPHOLOGICAL STUDIES ON COLLAGEN A N D ELASTIC FIBERS

A . Introduction Connective tissue contains three major groups of components : cellular structures, fibrous elements, and the amorphous semisolid gel in which they are embedded. A complete coverage of all aspects of connective tissue can be attempted in a review of this nature only at the expense of detail, and in view of the author’s own interests it was decided to restrict the review to a consideration of the fibers alone, and to consider these from the point of view of the elastic fibers and their relationship to the other fibrous element, collagen. Such a narrow division is not completely practicable, and at various stages in the discussion nonfibrous components will be introduced. The origin of the fibers is, for instance, dependent on specific cellular activity, and such activity will have to be considered when dealing with the general question of fibrogenesis. Similarly, quantitative relationships between the fibers and the amorphous component appear to be of considerable importance in considerations of pathological and aging changes (Sobel and Marmorston, 1956). At this point it may be appropriate to introduce the question of nomenclature. Semantics, probably more than any other factor, has obscured the results of connective-tissue research, and it would appear that the time has come for stricter definition, especially in the elastin field. The definitions suggested below are the sole responsibility of the author, but they are essentially similar to those agreed on by a number of workers in the elastin field who met during a symposium on connective tissue in 1956 and whose individual contributions to the subject are recorded in Connective Tissue (Tunbridge, 1957). Elastin: The name given to a derived protein, obtained from elastic tissue by techniques aimed at the removal of as much extraneous material-ther protein, polysaccharide, etc.-as possible, without causing undue degradation of the protein (cf. Partridge and Davies, 1955). The term is also used loosely in general phrases which do not presuppose the precise structural level under consideration. Elastic fibers: A morphological term which can be employed in either physicochemical or histological context to define the intact fibrous element. I t may or may not contain material in addition to the protein elastin (cf. Hall, 1957c, and Partridge et al., 1957). Elastica-staining : An adjectival phrase with purely histological connotations, which presupposes neither a specific fibrous structure nor a given chemical composition. Elastic tissue: A tissue rich in elastic fibers or other elastic-staining material, which exhibits as a, whole the property typical of individual elastic fibers-namely elasticity. Elastomucin: A considerable amount of tautology can be avoided if the mucopolysaccharide associated with the elastic fiber be given the name elustomucin. The

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term was introduced by Hall et al. (1952) to describe a protein-polysaccharide complex, the existence of which could explain a number of facts concerning the structure, enzymatic resistance, and degradation products of elastic fibers. Partridge and Davies (1955) have, however, doubted whether such a complex is a fundamental component of the fiber. Whether such is the case or not, a certain amount of polysaccharide, either by loose bonding or because it is an integral part of the fiber, can be regarded as being more closely associated with the elastic elements of tissue than are the polysaccharides of the ground substance. T o this material the name elastomucin can most usefully be applied. Collagen: The position with regard to collagen is just as confusing, since in this case a single term is used to describe everything from the molecular level to the fiber bundle, whether observed under the electron microscope, the light microscope, or the naked eye. Also there is the added confusion of soluble collagens, both natural and derived. It would appear imperative that the nomenclature of collagen should be rationalized as soon as possible, but in view of the fact that the present review deals mainly with the relationship between elastic fibers and collagen as a fiber species, which it will not in general be necessary to define more rigorously, the author will delegate this task to others. It would appear to be a formidable one, since no general agreement on nomenclature was reached at the symposium mentioned above. Ground substance : The amorphous matrix enveloping the fibrous components will be given this general term. I t is not assumed that it has the same composition throughout the body, and indeed qualitative differences between the polysaccharides present in various sites may have a profound bearing on the physiology of the fibrous components. Our knowledge of the comparative chemistry of connective-tissue ground substances is, despite the activities of Meyer and his co-workers (Meyer and Rapport, 1951), still very fragmentary.

Characterization of connective-tissue components was originally the outcome of extensive histological observation, and it is, therefore, not surprising that in the majority of the earlier reviews and monographs the problem was approached almost entirely from this point of view (e.g., Fleming, 1876 ; Popa, 1936). More recently, with the advent of improved chemical and physical methods, the spectrum of techniques available for the examination of connective-tissue fibers has been broadened, and certain differences in properties between the various fibers have been observed. The characterization of fibers by histological techniques, by its very nature, permitted the identification, in pathological tissue, of intermediate structures with staining properties midway between those of individual fiber species. Chemical analysis,. confined in the first place to nonpathological tissue, tended on the other hand toward more rigorous definitions and consequently resulted in the specialized consideration of the individual fibers rather than the fiber complex as a whole. Hence later reviews have dealt specifically with one particular fiber species (Dempsey and Lansing, 1954 ; Kendrew, 1953), and little comparative work has been reported. Notable exceptions have been the series of conferences organized by the Josiah

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Macy Foundation (Ragan, 1950), the book “Connective Tissue in Health and Disease” ( Asboe-Hansen, 1954), and shorter reviews on the physiology of connective tissue (Baker and Abrams, 1955). Even in these, however, comparison of the properties of the fibers themselves is limited, and in the main collagen has received far more attention than elastin.

B. Histology 1. Methods. The fibers of connective tissue can be divided by histological methods into three main groups : collagen, elastic, and reticular fibers. Although the identity of these fibers in a number of sites has been associated with the appearance of a particular staining reaction, the names by which they are known have been derived from a consideration of their physical, chemical, or morphological properties as they appear en masse in those tissues in which each particular species predominates. One of the main difficulties has been the correlation of chemical and histological observations on those tissues that contain mixtures of fibers, or in which a particular fiber is present only in small amounts (Hall, 1951). Until recently it had been assumed, for instance, that the presence of orceinpositive material in a tissue meant of necessity that elastic fibers were present, or that argyrophilic fibers from all sites consisted of the same type of reticulin. I n spite of these weaknesses, histological methods have been of considerable importance in studies of connective tissues, in providing the basis on which, in many instances, other disciplines have built. Many collagen stains are taken up to a limited extent by elastic fibers. For instance, both fibers are slightly acidophilic and hence stain with eosin. On the other hand, certain collagen stains do differentiate between collagen and elastic fibers. Mallory’s aniline blue stain (1900, 1936), for example, stains collagen blue and elastic tissue red, and similar differentiation occurs under ideal conditions with Masson’s trichrome stain ( 1929). The variability of the staining properties of elastic fibers with these stains, however, rendered it necessary for specific elastic stains to be devised. Two such are Unna’s acid orcein method (1896) and any one of a number of modifications of Weigert’s method (1898). With most of these stqins a small degree of generalized staining of collagen fibers occurs, and the amount of elastica-staining material in sections containing both collagen and elastica from, for example, human aorta, depends on the stain employed, indicating that not all the areas taking up one elastica stain are capable of being stained by another. This implies either that a proportion of the collagen in elastic tissue has different properties from the rest, or that the elastica-staining material is itself heterogeneous. The latter may well be the case, since even with a single stain, e.g., Hart’s (1908) modi-

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fication of Weigert’s stain, not only do elastic fibers in different tissues stain to different shades, but elastic lamellae and elastic fibrils in a single section of aorta are quite different in their stained appearance. Polysaccharide can be demonstrated in elastic tissue by the periodic acid-Schiff ( P A S ) reaction ( McManus, 1956), although in native fibers this effect is slight. This is as would be expected, in view of the fact that, even after exhaustive purification (Partridge and Davies, 1955) under conditions that bring about the partial removal of any elastomucin sheath (Hall, 1957c), not all the polysaccharide is removed (Wood, 1958). Fibers that have been subjected to mild digestion with elastase, however, show the presence of considerable amounts of metachromatic polysaccharide (Balo et ul., 1954; Saxl, 1957a). It would appear that in the intact fiber the polysaccharide in the elastomucin layer (Hall ct aJ., 1952) must be combined so firmly and in such a fashion to the protein that it is incapable either of reacting with periodic acid or of aligning the dye molecules to produce metachromasia. Rinehart and Abul-Haj ( 1951), however, using a modification of Hale’s (1946) method, showed the presence of an outer layer in the intact elastic fiber which was rich in acidic polysaccharide. The P A S reaction on collagen, on the other hand, gives very variable results (positive-Wislocki, 1952 ; negative-Leblond, 1950), but it has been pointed out that slight variations in technique could affect the intensity of staining to such an extent as to account for these two extremes. This makes the histological picture even more confusing, and one must compare these results with those obtained by other disciplines to be able to reach a decision concerning their validity. The observations by Jackson (1954) and Wood (1953) on the physical properties of collagen fibers treated with reagents specific for polysaccharides indicate that, at the intact-fiber level, polysaccharide is associated with the collagen in such an intimate fashion as to effect its physical stabilization. It would appear, therefore, that collagen and elastic fibers are similar in that they both contain polysaccharide. The identity of the polysaccharide is indeterminable by histological techniques, and chemical methods are still incapable of differentiating between polysaccharides derived from two or more components of such a complex tissue. If histological differentiation of intact collagen and elastin is difficult, the separate identification of these two fibers in the presence of partially degraded material derived from either or both is even more unsatisfactory. Unna (1890) classified the structures observable in connective tissue as : collagen, collacin, collastin, elastin, and elacin. H e regarded them as a series of products with tinctorial properties which merged imperceptibly into one another. The same stain was used for all these elements, and it

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was difficult to identify any particular structure unless a full spectrum from collagen to elacin were present for comparison. Fullmer and Lillie (1957) have shown that variations in p H can affect the differential staining properties of collagen elastin. Staining with resorcinol-fuchsin depends on the adsorption of the dye onto the fibers by forces that are antagonized by polar groupings. Collagen and elastin differ chemically, among other things, in the amounts of glutamic and aspartic acid present in each, and at p H values in the range 2 to 3 all the acid groupings on the side chains of elastin are back-titrated, whereas an appreciable number of those of collagen remain ionized. This permits differentiation between elastin and collagen, since the latter repels the dye while the former adsorbs it. At lower p H values, however, all acid side chains are backtitrated on both fibers, and both take up the stain. Variations in pH, however, may occur locally owing to the proximity of acidic polysaccharide, and stains based on phenomena that are susceptible to such changes cannot be universally applicable. Gillman et al. (1954) studied the possibilities of a number of elastica stains as a means of differentiating, not only between collagen and elastin, but also between normal and degenerate elastica-staining material. They discarded many of’ the usual elastica stains-orcein (Unna, 18%), Weigert’s ( 1898), Verhoeff’s ( 1908), Gomori’s aldehyde-fuchsin ( 1950), etc.-but demonstrated that a number of other stains could differentiate between true elastic fibers and other elastica-staining material which they suggested should be called “elastotically degenerate collagen.” Positive reactions with a number of these stains could be correlated with the liberation of polysaccharide from the fibers, during the induction of general elastica-staining properties or the adsorption of polysaccharide onto the surface of degenerate fibers. Gillman’s stains have not been employed to their full as yet, as will be seen from the fragmentary evidence presented in Section 11. C concerning those pathological conditions in which elastoses appear to occur, but their use may facilitate the differentiation of many structures hitherto thought to be similar. 2. Theories of Staining. Braun-Falco (1956) examined the way in which the two stains aldehyde-fuchsin (Gomori, 1950) and resorcinolfuchsin (Weigert, 1898) react with elastic tissue. By blocking reactive groups in both collagen and elastin he demonstrated that the specificity of the staining reaction depends on the availability of polar groups. This is in agreement with the observations of Fullmer and Lillie mentioned above (1957), in which alterations in p H produce a similar effect. BraunFalco concluded that the adsorption of the dye molecule onto the fiber is brought about by the reaction of basic centers in the dye through a dipole with the main chains of the fiber.

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Schwarz and Dettmer (1953) and Dettmer (1952) have shown by electron-microscope examination that the dye is bound to the amorphous matrix of the fiber. They suggest that if this material is destroyed by elastase the fiber no longer stains with Weigert’s stain (see also Lansing, 1951), since the fibrils that lie in the interior of complete fibers do not stain with elastica stains. If this is true, it would be expected that treatment such as that employed by Partridge and Davies (1955), which, according to Hall (1957c), may remove the larger part of the elastomucin coat, would result in the destruction of elastica-staining properties. This is, however, not so (Partridge and Davies, 1955 ; Wood, 1958), but much of the elastica-staining material does appear to be associated with those portions of the fiber that dissolve most rapidly. Sax1 (1957a) showed that elastic fibers still retained their structure after they ceased to take up elastic stains. Sachar et al. (1955) utilized the release of stain from stained elastic tissue as a measure of elastolytic activity, but Findlay (1954) claimed that the staining of elastic tissue with resorcinal-fuchsin produced inhibition of elastolysis. H e drew attention, however, to the facc that this was true only of intact tissue, whereas Sachar et al. used pow. dered elastin. Similarly the observations by Partridge and Wood regarding the retention of staining properties by purified elastin may be due to the presence of the small amount of polysaccharide and p-protein (cf. Section 111. A ) which is still present. These results are not necessarily at variance but may appear to differ on account of the varying degrees of disorganization or partial purification to which the starting material is subjected. Both orcein and resorcinol-fuchsin contain phenolic compounds, and their mode of attachment to elastin would appear to be similar (Michaelis, 1901). Fullmer and Lillie (1956), for instance, have shown that staining with orcein is independent of p H and is not affected by blocking the hydroxyl, amino, carboxyl, or aldehyde groups. No studies similar to those of Braun-Falco for resorcinol-fuchsin regarding the staining of altered collagen have been attempted for orcein, but Tunbridge et al. (1952) pointed out that the partial degradation of collagen by proteolytic enzymes such as pepsin induced in the early stages of attack a high degree of affinity for orcein, and it may well be that exactly similar reactions account for the specificity of both dyes. The main conclusion to be deduced from histological observation, therefore, is that many of the older techniques for differentiation between collagen and elastic fibers, and more especially “elastotically degenerate collagen,” are inadequate. Although recent studies on the mode of action of the stains, when taken in conjunction with chemical studies on the

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connective tissue fibers, may enable more specific stains to be devised, the results so far obtained have been meager. C.

Morphological Studies on the Pathological Involvement of Elastic Tissues

Increases in material having elastica-staining properties have been reported in a number of pathological conditions, especially those in which involvement of the dermis occurs. 1. Senile Elastosis. Kissmeyer and With (1922) found an increase in elastica-staining material in dermis from subjects between the ages of 26 and 40 but pointed out that the changes are most marked in the exposed sites from fair-skinned individuals. This was confirmed by Dick (1947) and by Tattersall and Seville (1950), and, although Ejiri (1936, 1937) did not differentiate between exposed and unexposed skin surfaces, his general findings were in agreement and were supported by the negative observation of Hill and Montgomery (1940) that no changes occur in skin from covered regions of the body. In general there is an increase in elastica-staining material in exposed tissue of elderly subjects, at the expense of collagen-staining fibers. It has been reported (Findlay, 1954) that in the deeper layers of the dermis the finer elastic fibers degenerate with the production of (‘elacin,” a lessening of Weigert-positiveness, and the appearance of islands of PASpositive material. Nearer the surface, masses of PAS-positive material unite to form the elastin-like colloid to which the name (‘collacin” has been given. There is no evidence from histological studies alone, that the elasticastaining material present in these pathological tissues differs from that present in normal tissue except that the material that appears in the upper layers of senile dermis is composed of broad ribbonlike structures as opposed to the fine fibers originally present. An explanation of the different appearance of the fibers and evidence for their origin was first given by Tunbridge et al. ( 1952), who by electron-microscope studies showed that there is no increase in true elastic fibers in senile skin, but that exposed areas contain large quantities of bent and broken collagen fibers coated loosely with amorphous particulate material. They also showed that short periods of treatment of native collagen with pepsin produce material having similar staining properties and a similar appearance under the electron microscope. Lansing ( 1951) and Findlay (1954), however, studying the solubility of these senile fibers under the action of elastase, showed that senile elastica is very susceptible to elastase and hence assumed that normal elastic fibers occur in abundance in senile tissue.

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The validity of such an assumption is, however, dependent on proof of the absolute specificity of elastase. There was no evidence against such a concept at that time, but subsequent studies have shown that elastase is capable of attacking thermally denatured collagen (Banga, 1953 ; Hall et al., 1953). More recently Hall (1957b) has shown that a soluble fragment of high molecular weight which can act as a substrate for elastase can be obtained from collagen by the action of collagenase. It would appear that certain elastase-resistant polysaccharides and procollagen must be removed from collagen by either thermal or chemical denaturation (Balo et al., 1956) before it can serve as a substrate for elastase. The specific groupings attacked may be similar to those attacked by chymotrypsin, since both enzymes hold certain synthetic peptides in common as substrate (Grant and Robbins, 1957). 2. Ehler’s Danlos Syndrome-Rubber Skin. As the trivial name for this condition implies, it is associated with hyperelasticity of the dermis. In normal skin the dermis consists of bundles of collagen fibrils, which are in themselves virtually inextensible. The mobility and tone of the skin is maintained by the fact that the bundles lie at an angle to one another, so that stresses may be taken up by distortion of the normal “weave” of the collagen bundles, before force is exerted on any individual bundle or fiber. Jensen (1955) has suggested that to account for hyperelasticity one must assume that the network of collagen bundles is abnormal and that the bundles lie roughly parallel to one another. An applied force is thus capable of separating the bundles, with consequent distention of the dermis. This theory, however, fails to take account of two salient factors. The model proposed for the dermis cannot account for the strength of the tissue, on the one hand, and, on the other, the dermis contains unusually large amounts of elastin. If the collagen fibers do not form an interwoven pattern, the strength of the skin can be dependent only on the ground substance, and this could not account for the appreciable, although markedly reduced, stability of the tissues. Histologically, dermis from subjects with Ehler’s Danlos syndrome is characterized by the presence of a high concentration of elastica-staining material, differing from that observed in senile elastosis, however, in that the fibers more nearly resemble those of normal dermis, differing only in number, and Tunbridge et al. (1952) showed by electron-microscope examination that this was in fact a true’elastosis. Sax1 and Graham (in Bourne, 1956), searching for a systemic cause for the condition, showed that elastic tissue from a bulla, excised from the anterior surface of a hypermobile knee, was highly susceptible to the action of the enzyme

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elastase, whereas apparently normal elastic tissue from adjacent regions was almost completely resistant to the enzyme. Such resistance could be imparted to tissue by the adsorption during life of an inhibitory substance from the serum, via the tissue fluids. An increased serum inhibitor level was observed, but the full implications of this have not yet been discussed. 3. Pseudoxanthoma Elasticum. This condition, which is also associated with marked cutis iaxa, has been described as an elastosis on the basis of increased elastica-staining material which shows signs of degeneration. Thomas and Rook (1949) and Hannay (1951) have stated, however, that the “elacin” which is present is too abundant to have originated solely from the elastic fibers originally present, and ascribe the elastica staining to degenerate collagen. Here again, as in senile elastosis, the elastica-staining material after which the condition is named has been shown by electron-microscope studies to be due to degraded collagen (Tunbridge et al., 1952). 4 . Colloid Millium. I n this condition, the elastic fibers originally present in the skin swell, and when stained with Mallory’s phosphotungstic acid hematoxylin stain demonstrate orange masses of swollen material coalescing where the fibers cross (Findlay, 1954). The ultimate stage is an amorphous, nonfibrous mass of PAS-positive material. This dissolves rapidly under the action of elastase, revealing the remains of fibers which have not progressed so far in degradation and which are more slowly dissolved by the enzyme. Findlay suggests that total elastolysis is assisted in the case of colloid millium by the fact that the elastomucin has been separated from the fiber. Since he employed crude elastase preparations which most probably contained both proteolytic and mucolytic components (Hall, 1957a), these observations are in complete accord with present views on elastase action. 5. Obliterative Diseases of Elastic Fibers. In the fibrous tumors of the skin of patients suffering from infection by the helminthic parasite Onchocerca volvulus the elastic fibers of the affected dermis are completely destroyed. I n the burnt-out stage of the disease, although the damaged collagen bundles may re-form perfectly, there is evidence that elastic fibers are not re-formed (Jamison and Kershaw, 1956). In this the condition differs from lathyrism which arises from feeding the seeds of the sweet pea (Lathyrus odoratus), in which case there is evidence that elastic fibers, if already formed, are unaffected, although their initial formation is prevented if the causative agent is administered early enough.

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D. Structural and Physical Aspects of Connective-Tissue Fibers 1. Morphology under the Light Microscope. Examination of unstained preparations of connective tissue under the light microscope demonstrates collagen and elastic fibers as two distinct species. Collagen occurs in bundles of straight or slightly wavy, white fibers which form networks but which show no signs of branching. Elastic fibers, on the other hand, are fine individual elements which follow an irregular course through the tissue lying on and around the bundles of collagen fibrils. They branch and anastomose with one another freely. This description of elastic material refers to the fibers as they occur in dermis and in the interlamellar areas of arterial media, but not to the lamellar structures of the media and the broad fibers of ligament which differ from the fibrils in their staining and physical properties as well as by morphological criteria. The internal organization of these various structures cannot be seen at the level of the light microscope, but certain broad assumptions regarding the arrangement of the various subfibrous components can be made from a consideration of their appearance in polarized light. Collagen fibers from all sources show an appreciable degree of birefringence, but this is not true of elastic components. Thus it may be deduced that the internal structure of the collagen fiber is more highly oriented than that of the elastic fiber. 2. Electron-Microscope Appearance. The deduction referred to above is borne out by an examination of connective tissue under the electron microscope. Collagen fibers characterized by a regularly repeating system of cross-striations at 640-A. intervals (Wolpers, 1944 ; Gross and Schmitt, 1948; Gross, 1949; Wyckoff, 1949) and by finer interband structures (Hoffmann et al., 1952) differ considerably from elastin (Wolpers, 1944 ; Gross, 1949). Hall et al. (1955b) described the gross structure of elastic material from aorta and from ligainentum nuchae and showed that the amorphous structure ascribed to the elastic fiber as a result of observations with polarized light was borne out by electron-microscope studies. In the aorta at least two major components were apparent, one frankly fibrous, the other appearing to consist of a network of fibers covered with an electron-dense, formless coating ; Keech et al. ( 1956) have since reported the existence of many apparently different forms of elastic fiber in dermal preparations. The electron opacity of the outer layer of both the elastic fiber and the lamella has rendered it impossible to obtain more than fragmentary evidence concerning its inner structure from an examination of intact material.

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During fragmentation, however, small areas of the outer sheath are stripped away, revealing a finer fibrillar structure within. This disturbance of the architecture of the elastic structures, which is an essential concomitant of the teasing process, makes it difficult to differentiate between true substructures and those that arise as artifacts in the preparation procedure. The advent of the thin-section technique (Neuman et al., 1949) should enable more detailed localization of smaller elements to be made. Rhodin and Dalhamn (1955), however, examining the lamina propria of the trachea of the rat, showed that little structural detail could be observed within this fiber in the intact state. Hall et al. (1952, 1955b) and Lansing et al. (1952) utilized crude elastase preparations to examine the finer structure of elastic elements. Both groups showed that a finer fibrillar structure was revealed after the removal of the outer electron-opaque coating. The collapse of the structures also appeared to present evidence for the penetration of this amorphous phase in between the individual fibrils. It would, therefore, be more correct to refer to it as an amorphous matrix. Owing to the crude nature of the elastase, it was not possible to stop the reaction after the removal of the matrix, and hence details of the underlying fibrils could not be obtained, since they were degraded simultaneously. Sax1 (unpublished work), using purified enzyme preparations, has been able to demonstrate fine detail in the inner fibrillar structures. 3. X - R a y Diffraction Studies on Connective Tissue. The X-ray diffraction pattern of elastin was first investigated by Kolpak (1935), who found unstretched fibers to give diffuse rings, whereas stretched samples gave a meridional arc corresponding to a spacing of 3 A. and equatorial spots characteristic of spacings of 11.5, 5.9, and 4.6 A. Astbury (1938, 1940) attributed these findings to the presence of small quantities of collagen fibers, which, although themselves fully oriented, were arranged haphazardly in the elastic fiber. At such low concentrations it was impossible to identify a collagen powder diagram superimposed on that of an elastic fiber, but when the collagen fragments were aligned by stretching the elastic fibers the more discrete collagen fiber diagram became visible. Astbury drew attention to the fact that prolonged boiling prevented the observation of a collagen pattern even after stretching. H e felt, however, that elastin should be included in the collagen group of fibers, as opposed to the keratin-myosin-epidermin-fibrinogengroup, and suggested that it might represent a member that was permanently in a contracted state on account of its thermal transition point’s being below that of the animal body. Bear (1944) recorded small-angle X-ray patterns of collagen, as faint arcs on a photograph of beef ligament, but again ascribed these to the

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presence of collagen as an impurity, and in a review of the collagen fiber (1956) suggested that elastin should not be included in the collagen group. When reviewing the position in 1953 Kendrew assessed the evidence as adequate to justify the exclusion of elastin from the collagen group, but electron-microscope studies may necessitate a reconsideration of this point of view. Collagen fibrils have been observed in elastic tissue by a variety of workers, but there is still no direct evidence as to whether structures having the characteristic spacings of collagen at either electron-microscope (640-A.) or X-ray diffraction (2.86-A.) levels constitute an integral part of the elastic fiber structure. 4 . Physical Properties of Collagen and Elastin. The tensile properties of collagen and elastin differ considerably, the Young’s modulus of the elastic fibers being smaller than that of collagen by a factor between 400 and lO,oOO, whereas the extension at break is 20 to 30 times as great (Buck, unpublished results quoted in Burton, 1954 ; Krafka, 1937). Much of the early work on the thermoelasticity of elastic fibers was carried out on whole ox ligament. Meyer and Ferri (1936) and Wohlisch et al. (1943) showed that ligament behaved as a rubberlike solid up to 100% extension and that extensibility was independent of time up to 50% extension. Lloyd and Garrod (1946) later showed that similar elastic properties, which could be represented by a model consisting of a steel spiral spring maintained in a partially compressed state by a rubber band, could also be demonstrated in elastic fibers freed from collagen and ground substance. Wood (1954) examined the effect of various reagents which might remove or destroy polysaccharide present in both collagen and elastic fibers. H e showed that mucopolysaccharides appeared to be of greater importance in stabilizing the collagen component of the tissue than the elastic fibers. H e also reported that the collagen associated with the elastic fibers appeared abnormal in that it could be extended by 70 to 75% and pointed out that Banga (1949) had reported an abnormal collagen in association with elastic fibers on the basis of determination of flow birefringence on the protein extracted from aorta with urea solution. Wood’s recent studies on the tensile properties of reconstituted elastic material (1958) have confirmed that as far as stretching phenomenon are concerned elastic fibers are not dependent on a polysaccharide component for the type of load-extension relationship observed. The dependence of collagen on polysaccharide for its thermal stability and resistance to extension has been studied by Jackson (1954). It would appear that at the fiber level extensibility can be increased, as also can solubility, with a concomitant lowering of the thermal transition tempera-

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ture, by procedures that remove polysaccharide. Hall and Reed (1957) ascribed the variations in thermal stability which could be associated with differences in collagenase susceptibility to changes in the polysaccharide content of the fibers rather than to fundamental changes in amino acid analysis. 5. Model Structures for Elastic Fibers. The nature of the internal fibrillar structure of elastin has been in doubt for some time. Hall et al. (1952, 1955b) could not determine the fine detail of fibrils released by elastase because of the simultaneous degradation of fibrils and amorphous matrix. Gross (1949) claimed to have revealed helical structures under the action of trypsin, although these were later shown to be contaminants derived from trypsinogen (Franchi and De Robertis, 1951 ; Gross, 1951). Lansing et al. (1952) also identified helical fibrils in the elastase degradation products of the elastic fibers of ligament. Another possibility was introduced by the observations of Schwarz and Dettmer (1953), who claimed to have revealed striated fibrils akin to collagen by treatment of aortic tissue with elastase. These observations led Banga (1953) to suggest that collagen fibrils constituted the core of all elastin fibers. She drew attention to the fact that workers other than Schwarz and Dettmer had invariably employed elastic fiber preparations obtained by heat treatment of elastic tissue. She showed that thermally denatured collagen is susceptible to elastase action, and hence any collagen at the center of heated elastic fibers would be altered to an elastase-susceptible form. This suggestion was questioned by Hall et al. (1953), who pointed out that Schwarz and Dettmer did not present adequate evidence that the collagen fibers surrounding the elastica had been removed prior to attack by elastase, although they agreed with the premise that thermally denatured collagen could act as substrate for the enzyme. Keech and Reed (1957a) showed that elastic fiber preparations obtained from aorta or ligament by treatment with boiling 2% acetic acid (Gross, 1949; Hall et al., 1952, 1955b) were complex structures from which collagen fibers could be liberated by short periods of treatment with collagenase. Based on the foregoing morphological evidence, and supported by biochemical studies, a model structure for elastic fibers was proposed by Hall ( 1 9 5 7 ~ ) . H e suggested that elastic fibers are essentially biphasic-an outer layer which contains polysaccharide and protein surrounding an inner layer which consists solely of protein. Romhanyi (1955), on the other hand, suggested that elastin might consist of at least three concentric cylindrical structures. H e based his argument on the apparent changes in diameter of elastic fibers after treatment with either aniline or phenol or after staining with resorcinol-fuchsin.

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Romhanyi’s experiments indicate that it is difficult by purely optical means to define the exact boundary of the fiber, since different reagents reveal “cylinders” of varying diameters. Hall ( 1 9 5 7 ~ )drew attention to this phenomenon when comparing the results of enzymatic studies on elastic fibers with those of Partridge and co-workers (1955) using purely chemical methods. The main difference between the two sets of results could be ascribed to the fact that the amount of tissue included within the hypothetical outer surface of the fiber could be varied in such a way as to include or exclude the majority of the polysaccharide-rich material. Since fibers from which the majority of the polysaccharide had been removed still retained their structure, Partridge suggested that polysaccharide was not a necessary component. A similar conclusion has been advanced by Wood (1958) on the basis of physical determinations on reconstituted elastic material. Many of the properties of reconstituted elastin differ quantitatively, however, from those of the native fibers, and, although this may be an indication of the degree of main chain hydrolysis which occurs during solution with oxalic acid, it might also represent the effect of the removal of an outer structure rich in polysaccharide. Morphological and enzymatic studies, therefore, indicate that the line of demarcation between the native elastin fiber and the surrounding ground substance should be drawn in such a position that an appreciable amount of mucopolysaccharide is included, and that the presence of this material may have a considerable part to play in the stabilization of the fiber. Chemical and physical examination of the fibers, on the other hand, dealing with properties which appear to be those of the protein alone, indicate that the fiber is delineated by a cylindrical surface enclosing only fibrous protein. These two sets of views are not necessarily in conflict, since the same properties are not selected for comparison.

111. BIOCHEMICAL STUDIES ON COLLAGEN AND ELASTIC FIBERS A . Chemical Composition of Collagen and Elastin Prior to the last ten or twenty years, analyses of protein- and polysaccharide-containing tissue preparations have not been dependable. This has been especially true of insoluble structures such as collagen and elastin. Amino acid analyses of the entities which purport to be the pure proteins from these tissues have become available only recently, as methods of analysis have improved, but even now results must be considered in relationship to the methods of preparation employed. The position with respect to polysaccharide is even more complex. Carbohydrate may be present in connective tissue either free or combined in the amorphous

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phase, or combined either loosely or by firmer linkages with the fibrous structures themselves. Methods of preparation may well bring about only partial fractionation of these different species of polysaccharide, especially if two or more are combined in any one site. 1. Amino Acid Analyses. Studies of the amino acid analysis of collagen preceded those on elastin because of the greater ease with which analyses could be performed on the derived protein, gelatin. Recent analyses for collagen (Bowes et al., 1955) have justified the assumption that the conversion from collagen to gelatin is accomplished without the removal of amino acids or small peptides. Hence it must be assumed that the recent discovery of between 20 and 30% of a protein with an entirely foreign analysis in gelatin (Russell, 1957) implies the presence of a similar degree of heterogeneity in collagen itself. The analysis of elastin has until recently been accomplished only by the removal of all other extraneous material by methods depending on the relative inertness of elastin to chemical attack. Thus Stein and Miller (1938) stated that elastic tissue could be boiled for prolonged periods in water, dilute acids, or alkalies or strong urea solutions without any variation in the amino acid analysis of the residue. Hall (1951, 1955) showed, however, that this was only partially true. Analyses such as those obtained by Lansing et al. (1951) for old aortic elastin are dependent on the reagent employed for purification. The material by which the amino acid composition of such preparations differed from the classical elastin analysis is resistant to boiling 2% acetic acid but dissolves after prolonged treatment with boiling 40% urea solution. Even the resistance of young elastin to boiling urea solutions is dependent on the tissue-solvent ratio. It would appear, therefore, that with the exception of the classic amino acid analysis for ox ligamentum elastin, young aortic elastin, etc., which appears constant, all variant analyses for elastin are a function of the method of purification. The analysis of native elastin can be compared and shown to agree substantially with that of soluble elastin preparations obtained by the action of oxalic acid on purified elastic fibers (Adair et al., 1951). Partridge et al. (1955 ; 1957) showed that elastin dissolves rapidly in boiling 0.25 M oxalic acid with the release of acidic acids, and the production of two species of soluble protein (a- and p-elastins). Chemical studies indicated that the two derived proteins, although they differ considerably in molecular weight ( 6 7 , O and 5500) , do not show marked differences in the number of N-terminal residues. Partridge et al. suggested that the p-protein consists, on the average, of two chains containing 27 residues, and the a-protein of seventeen chains with 35 residues each. These chains appear to be linked laterally to one another by acid-resistant cross linkages,

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the nature of which is still obscure. The rates of production of these two fragments preclude the formation of the smaller 0-fraction from the larger a-component, although Bowen (1953) provided evidence that such a relationship might occur in the case of fractions obtained from urea-soluble elastin. In general these findings may be taken as indicating that the gross structure of elastic fiber is heterogeneous. If this is so, however, the analytical evidence would indicate that both species of protein, although differing in size and chain organization, are chemically similar. Hall ( 1 9 5 7 ~ )suggested that the 0-protein is present in the amorphous coat and matrix of the fiber, and the more highly polymerized a-material constitutes the internal fibrillar element. No evidence has as yet been advanced which suggests that elastin is chemically heterogeneous such as would appear to be the case with collagen and gelatin (Russell, 1957), but there is increasing evidence for structural heterogeneity (cf. also Section 11. D ) . Even doubts as to the unitary nature of both collagen and elastin, however, do not invalidate comparisons of the amino acid composition of the two proteins if adequate specifications of the methods of preparation are provided. Figures are now available which account for 96% of the amino acids of collagen and 106% of those of elastin (Bowes et al., 1955; Partridge and Davies, 1955), and it appears unlikely that significant changes in these values will be introduced by subsequent work. Collagen and elastin differ from most other proteins in that the sum of their glycine and proline residues together amount to 33 and 41%, respectively, of the total number of residues. Another qualitative similarity is the presence of hydroxyproline in both proteins. The values are, however, quantitatively different. Collagen with 11.07 g. of residues per 100 g. has seven times as much hydroxyproline as elastin. This again may be due to heterogeneity in the elastic fiber, since the small amount of hydroxyproline present in elastin could be accounted for by the retention of collagen in the elastin preparation (cf. Section 11. D ) . No method of preparation which has so far been employed, however, has resulted in the complete removal of hydroxyproline. Harkness et al. (1957) showed that in dog artery the amount of hydroxyproline in elastin may vary from 2 to 1% from animal to animal, but lower values were not obtained. Neither protein contains tryptophan, tyrosine, or cystine in significant amounts, but there the similarity ceases. Elastin is predominantly nonpolar, only 0.1% of the amino nitrogen being provided by polar amino acids, as opposed to the 28.9% of collagen. Their place is taken by monoaminomonocarboxylic acids, especially those of larger residue weight. The net result of these differences on the titration curves of collagen and elastin has been discussed by Bowes and Kenten (1948) and Bendall (1955).

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The latter, using rigorously purified material, showed that the number of polar groups which could be calculated from the titration curve for elastin were in close agreement with the analytical figure for these amino acids, thus indicating that in the preparations employed (Partridge and Davies, 1955) no other polar groupings were present. 2. Polysaccharide as a Component of Connective Tissue. The evidence for the presence of polysaccharide as an integral part of collagen or elastic fibers is as yet mainly inferential and not analytical, the main reason being the difficulties experienced in determining whether the bonds holding the polysaccharide in close association with the fibers are sufficiently strong to justify the classification of the polysaccharide as a structural component. Part of the polysaccharide can, however, be so classified. Both collagen and elastin can withstand a considerable degree of chemical treatment without losing their fundamental fibrous structure. After such treatment a small amount of polysaccharide can still be identified in the protein preparations (collagen: 0.42%, Grassmann et ul., 195713; elastin: 0.3%, Partridge and Davies, 1955; 0.1, Wood, 1958). The identity of the sugars obtained by hydrolysis of this polysaceharide has been determined in the case of collagen, glucose and galactose (Grassmann and Schleich, 1935 ; Gross et al., 1952), fucose (Glegg et al., 1953), and glucosamine (Schneider, 1940, 1949). In elastin Lloyd (in Bourne, 1956) has shown that a small amount of polysaccharide remains inseparable from the protein even after prolonged extraction with hot sodium chloride and cold calcium chloride solutions. A further fraction remains attached to protein; but the complex can be extracted by neutral calcium chloride solution. This material is insoluble in buffer solutions after removal of the calcium chloride by dialysis but can be made to pass into solution under the action of elastase without the liberation of aldehyde groups. Among polysaccharides more easily, extractable from connective tissue, Meyer has identified hyaluronic acid, chondroitin sulfates A, B, and C, heparatin sulfate, and kerotosulfate. Since both collagen and elastin are present in the ligament, and the interfibrous spaces are filled with polysaccharide-rich ground substance, it is difficult to assess the exact site of attachment of any one polysaccharide. This can, however, be attempted by histochemical and electron-microscope methods. Rinehart and AbulH a j (1952) have shown the existence of acid polysaccharide on the surface of the elastic fibers, and Hall et al. (1952, 1955b) have shown that elastase can remove acid polysaccharide from the surface and interfibrillar regions of elastic lamellae in aorta before destroying the underlying protein structure.

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3. Lipid. The involvement of lipid in the structure of connectivetissue fibers has been suggested for some time. Reticulin contains, in addition to the collagen protein, a mixture of lipid and polysaccharide (Kramer and Little, 1953), and Little and Windrum ( 1954) have reported that a relatively high proportion of the native collagen fiber consists of myristic acid. Lansing et al. (1952) suggested that elastase was, in fact, a lipase on the grounds that under the action of the enzyme Sudanophilic droplets of low density were liberated from elastic tissue. Sax1 (1957a) recognized the presence of both neutral and acid lipid in whole elastic tissue, but pnly the neutral lipid was liberated by elastase, the acidic lipid being destroyed. The addition of serum protein resulted in the hydrolytic fission of the neutral fat. B. Distribution of Collagen and Elastic Fibers 1. Methods of Determination. Early studies on the distribution of the various connective-tissue fibers were performed by histologists employing differential staining methods. The results obtained were sufficiently accurate to justify classification of tissues as predominantly collagenous, elastic, or of mixed composition containing appreciable concentrations of both fibers. Quantitative values for the relative proportions of elastic fibers were difficult to obtain, however. The advent of chemical analysis permitted numerical values to be ascribed to the relative concentrations of the separate components, and, based on these figures, many theories relating tissue architecture to function were propounded. The indiscriminate use of these values may be no more justifiable than the use of visual assessment of stained areas of a section. Employed with circumspection, however, chemical analyses of tissue can present evidence of considerable importance in studies of the changes occurring in tissue during differentiation, growth, and senescence or with the onset of pathological conditions. The apparent inertia of elastic fibers to chemical attack proved an important property in devising methods of analysis. Most procedures have consisted in treatment with boiling water, acetic acid, or alkali, or autoclaving with these reagents (Lowry et al., 1941; Neuman and Logan, 1950). I n choosing appropriate conditions, the period of treatment was determined to ensure the removal of all extractable material. Gross (1949) and Hall et al. (1952), for instance, stated that treatment with acetic acid brought about the removal of all morphologically discernible collagen from elastic tissue. This method has been criticized by Partridge and Davies (1955) as being too drastic. I t is, however, possible that their apparently milder reagents bring about preferential removal of polysaccharide-rich fragments from the elastic fiber itself. Retention of collagen or ground

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substance, on the one hand, or removal of part of the elastic fiber, on the other, tends to produce extremes of analytical values, which differ considerably. Moreover, the existence in aorta, in addition to collagen and elastin, of a third component which could not be dissolved by acetic acid but which was more soluble in boiling 40% urea solution than true elastin indicated the fallacy of applying any one analytical procedure to a tissue (Hall, 1951, 1955). Since the material is retained with elastin after extraction with acid or water, all methods based on this type of procedure give erroneous values for elastin content in aorta. The amount of elastin in the tissue can be calculated either from the dry weight of the residue or from the hydroxyproline content of a hydrolyzate of the residue (Harkness et al., 1957), the collagen content being calculated from hydroxyproline determinations on the extracted protein. Harkness et al. showed that hydroxyproline content of the elastin varied by over 100% in twelve dogs, thus making it difficult to determine by this method the amounts of elastin present. Similarly, figures for collagen extracted by p H 5 phthalate buffer (Hall, 1957b) calculated on the hydroxyproline content, when compared with the actual amount of protein extracted, present the paradoxical situation that the collagen content of the extracted protein is 175%. Hence all numerical values for collagen/ elastin ratios in tissue must be considered in relationship to the method of analysis. 2. The Relative Distribution of Collagen and Elastin. Despite these drawbacks to the assessment of numerical values for collagen and elastin content, interesting observations have been made, notably the contribution of Harkness et al. (1957). These workers, studying the collagen and elastin content of the aorta of dogs, employed autoclaving with water followed by boiling with decinormal alkali to remove first the collagen, and second a fraction--"dry material other than collagen and elastin"-which they did not analyze. They reported that in adult dogs, the ratio elastin/ elastin plus collagen remains roughly constant at 50 to 60% from the aortic valves to within some 5 cm. of the diaphragm. The proportion of elastin then decreases rapidly and remains at a value between 25 and 30% throughout the abdominal aorta and the iliac, femoral, and saphenous arteries. A similar drop occurs at the point of departure of the arteries at the upper confines of the thorax. In young animals the drop is from 70% to 60% (newborn), 50% (3 weeks), and 35% (6 weeks).

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C. The Enzymatic Stcsceptibility of Elastin and Collagen

1. The Elastase Complex and Its Physiological Significance. The earliest positive observations on the action of mammalian enzymes on elastic tissue were reported by Ewald (1890), but he employed crude enzyme preparations, and it was not until 1949 that Balo and Banga isolated an enzyme in a partially purified form from pancreas (1949a). This appeared to be specific for elastic fibers. Their studies arose from a search for the causative agent for the degradation of elastic fibers and lamellae such as occurs in aortic media during arteriosclerosis. The activity of the enzyme can be controlled by a component of serum which has positive inhibitory activity, and Balo and Banga (1949b) suggested that the decrease in the inhibitor which could be correlated with the onset of arteriosclerosis could account for an apparently unchecked activity of the enzyme resulting in medial degeneration. Lansing (1955) pointed out that the decrease in inhibitor content could also be correlated with increased age of the patient and might have no direct connection with the onset of degeneration, a point of view which was also accepted by Balo and Banga themselves (1953) and which constrained them to suggest that elastase might be concerned with the synthesis of elastic fibers as well as their disruption, and that it might be a failure of synthesis that accounted for the changed appearance of the elastic fibers in arteriosclerosis. The whole question of the physiological significance of the enzyme is bound up with the ultimate results of as yet unsuccessful attempts to prove that it is present in the circulating blood. If we assume that elastic fibers are synthesized within the body, some elastoclastic mechanism must also be operative if generalized elastosis is not to occur. In the adult animal (Slack, 1954) the turnover of glycine in the elastic tissues is very small, and it may well be that the amounts of enzyme necessary for this low rate of catabolism may lie below the threshold of analysis. There may, however, be positive removal of elastase from the circulation by the formation of a triple complex between enzyme, substrate, and inhibitor (Saxl, 1957a), and this may be of significance in those pathological conditions in which resistance to elastase attack is apparent. A considerable amount of contradictory circumstantial evidence regarding the systemic or digestive role of elastase has been reported recently. Lansing et al. (1953), studying the teleost fish Lophius piscatorius, in which the pancreas is located in two anatomically separate sites, showed that elastase is secreted only by islet cells. Further evidence for this was obtained by Carter (1956), who reduced the elastase content of dog pancreas by the administration of cobalt. Hall et al. (1952) also reported

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their inability to identify elastase in the pancreas of the human subjects who were diabetic. Balo and Banga (1950), however, stated that in the pancreas elastase occurred in conjunction with an inhibitor which could be removed by dialysis or by acid treatment. Grant and Robbins (1955) also identified an inactive elastase in pancreas and suggested that the removal of the “inhibitor” represented the conversion of a zymogen to the free enzyme. Kokas et al. (1951) demonstrated the presence of the zymogen in pancreatic juice and hence assumed that elastase was a product of acinar tissue. From these conflicting reports it is evident that the exact location of the cells that synthesize and secrete elastase remains in doubt. 2. Specificity of the Enzymes. Balo and Banga (1949b) reported that the elastin-elastase system was specific in so far as native elastin fibers cannot be degraded by any other of a considerable variety of enzymes. The selective removal of elastin and failure to attack collagen suggest that elastase may be specific; this is, however, not the case. Elastase activity is not restricted to the solubilization of a single substrate. This more general activity was first recorded by Banga (1953) and Hall et al. (1953) , who simultaneously reported that thermally denatured collagen could also act as substrate for the enzyme. The latter group of workers also claimed that elastase could degrade the insoluble proteins of the lens. These observations were confined to relatively crude enzyme preparations which might also contain other proteolytic enzymes. It has been pointed out that similar differences exist between the susceptibility to trypsin of collagen fibers before and after thermal denaturation, and the effects observed may not be typical of elastase. Other evidence is, however, available for the interaction between elastase and partially degraded collagen. Both Findlay (1954) and Dempsey and Lansing (1954) quoted the elastase susceptibility of the elastica of senile elastosis as indicating that true elastic fibers were synthesized during the onset of the senile changes. Tunbridge et al. ( 1952), however, showed by electronmicroscope studies that this material was in fact denatured and partially degraded collagen. Balo et d. (1956) showed that the metacollagen (cf. Section V. A) was also susceptible to elastase, and Hall has demonstrated ( 195713) that certain high-molecular-weight material obtained from collagen after its dissolution by collagenase (CZ. wekhii) is broken down into smaller molecules by e1astase.l Treatment of whole elastic tissue with elastase at the lower optimum pH of 7.8 (Hall, 1957a) results in the liberation of metachromatic 1 The general question of the importance of the polysaccharide in determining the susceptibility of elastic fibers in aorta to elastase attack has recently been discussed by Yu and Blumenthal (1958).

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material from the fibers, and also from the surrounding ground substance. A similar effect has been observed by Rinaldini (1958), who has employed elastase preparations rich in mucolytic activity to separate aggregated cells for tissue culture. It would appear that the linkage between polysaccharide and protein is attacked and that either the same polysaccharide is present in ground substance as in the fiber, or the mucolytic enzyme is no more specific than the proteolytic component. Grant and Robbins (1957) advanced the hypothesis that the role of elastase in metabolism might be that of an endopeptidase on the basis of a comparison between the activity of various elastase preparations, trypsin, and chymotrypsin on a variety of substrates. Elastase in a relatively high degree of purity showed appreciable activity with acetyl-L-tyrosine ethyl ester ( A T E E ) as substrate, thus demonstrating its similarity to chymotrypsin. Under similar conditions, however, elastase also digested casein and hemoglobin, and thus its A T E E activity might be due to the presence of chymotrypsin in the elastase preparations. On the other hand, the most active elastase preparation was one precipitated from solution by dialysis, a phenomenon not observed with chymotrypsin. This possible relationship has not yet been fully elucidated.

IV. THE PHYSIOLOGY OF CONNECTIVE-TISSUE FIBERS A . Fibrogenesis The question of elastic-fiber formation in vivo is not difficult to review, since little if any concrete evidence exists from which the mode of fibrogenesis can be determined. Much of the evidence discussed below is negative and can be assessed only by comparison with the positive findings available for collagen production. Two main lines of approach have been examined : (1) the production of fibers in actively growing and differentiating tissue, and (2) the replacement of lost tissue in wound healing. These aspects have been studied by reference to fibrogenesis in whole organisms and in tissue culture. 1. Embryonic Tissue. The fibroblast has been identified as the instigator of fibrogenesis, although the actual site of fiber formation has long been in doubt. It was originally suggested that fibrin fibers originating as extrusions of fibroblast cytoplasm were converted into collagen fibers extracellularly. This hypothesis was based on early histological studies and has 'now been completely discounted, although Buck (1953) has suggested that fibrin may, by its contraction, play a part in stretching and orienting the collagen fibers during their production. In an actively differentiating tissue such as embryonic mesenchyme, the production of fibers is far in excess of the cellular content, and it would appear unlikely

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that all the fibers could originate within the cells themselves in a fibrous state. Although Doljanski and Romlet (1933) claimed that in tissue culture formation of collagen fibers could be initiated in areas far removed from the cells, the work of Porter (1951) and Fitton-Jackson (1956) has shown that the fibroblasts do have a primary role to play in fibrogenesis, and fiber formation starts in the immediate vicinity of the cells. Fitton- Jackson ( 1954) showed that certain cytoplasmic granules within the fibroblast, which contain both protein and mucopolysaccharide, are associated with the formation of intercellular material. It is suggested (Fitton-Jackson, 1956) that the fibroblasts evolve globular proteins similar in composition to collagen, and polysaccharides from which the ground substance is derived. In the region outside the cell, the initial stages in fiber formation take place with the production of a primitive collagen fiber with a band periodicity of about 210 A. At this stage each fibril is surrounded by a region of relatively low electron density, but as the fibers increase in diameter the size of this region decreases. The increasing diameter of the fibers during growth appears to be due to an uptake of material from the ground substance, and this latter thus assumes a fundamental role in fibrogenesis. It would appear proved that the formation of the initial fibrils requires the interaction of collagen precursors and some component of the environment. The exact nature of the precursors evolved by the fibroblast is in doubt, but Harkness et al. (1954) have shown that material extracted from tissues by neutral salts and having many properties of collagen, including that of being able to act as starting material for the regeneration of collagen fibrils, demonstrates a far higher rate of turnover for glycine than do other collagenous fractions of the tissue. One is left with the hypothesis that callagen fibers are formed from a soluble precursor evolved by the fibroblast, by combination with some component of the extracellular mass, but the way in which these phenomena are controlled is as yet unknown. If the mode of formation of collagen fibers is little understood, that of elastic fibers is shrouded in even deeper mystery. First, although there have been numerous claims, no one has conclusively proved the existence of an elastoblast. Robb-Smith (1954) states that elastic fiber production always follows the appearance of collagen in the embryo and in healing wounds. It would appear likely that a similar extracellular process to that which brings about the formation of collagen fibers initiates the fibrogenesis of elastic fibers. Either the fibroblast is induced to produce a variant of its normal precursor, or, as was suggested by Hall et d. (1955a),

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elastic fibers may be evolved by the interaction of a selection of the precursor fragments secreted by the elastoblast, with certain components of the ground substance. This presupposes that the precursor material secreted by the fibroblast is not a single protein species but a mixture of polypeptides of differing amino acid composition, such that the selection of a certain number affords the necessary building bricks for the production of elastic fibers instead of collagen. It is interesting in this context to recall that Schultz (1922) suggested that new elastica-staining material was derived from a similar source to collagen but was saturated with the polysaccharides of ground substance. Hass (1939) thought that elastic fibers might be formed from fibroblastic products as a fibrillary membrane at lipid interfaces in the surrounding tissue. Lipid is always closely associated with elastic elements, and such a mode of fibrogenesis might explain the deposition of elastic lamellae in vascular walls. Elastin fibers cannot be obtained in tissue culture from undifferentiated mesenchyme, but they can be caused to proliferate in differentiated tissue in which they already exist (Maximow, 1929). Even adult tissue, however, may not always be capable of supporting the regeneration of elastic fibers. For instance, keloid or even normally regenerating scar tissue contains little or no elastica. 2. Wound Healing. In early studies of tissue regeneration in the healing wound, as in the case of embryonic differentiation, suggestions were made that plasma clots or fibrin fibrils (Baitsell, 1946; Nageotte and Guyon, 1930) were converted into collagen fibrils. Here again, however, the only role the fibrin is likely to play would appear to be that of a matrix or template in association with which fibrogenesis may occur, and which by its physical properties may exert orienting forces on the fibers during synthesis. The most important observations on wound healing have been studies on the retention of granuloma tissue in wounds in animals with vitamin C deficiency. Wolbach and Howe (1926) first showed that wounds in ascorbic acid-deficient animals failed to heal. Danielli et al. (1945) showed that, although at low levels of ascorbic acid intake large amounts of reticulin were formed, the appearance of the wound was abnormal. The metachromatic material which is present in the granuloma decreases as the wound heals, owing to fiber formation, but in scorbutic subjects the metachromatic granulomatous tissue remains. Elster ( 1950), Robertson (1952), and Perrone and Slack (1951) showed that ascorbic acid was not necessary for the maintenance of already formed collagen fibers but appeared to be required for fibrogenesis. The properties and constitution

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of newly formed tissue have been studied by Robertson and Schwartz (1952) and by Jackson ( 1957), making use of the considerable amount of granuloma tissue which can be induced by injection of carrageenin solutions. Jackson showed by tracer studies that during the development of the tissue neutral salt-soluble collagen was produced first, and this was then consolidated into insoluble collagen, probably by combination with a tyrosine-rich mucopolysaccharide (Bowes et al., 1956). The amount of soluble collagen extracted from the fibers by acid increases with the progress of the granuloma, showing that the initial stages of the consolidation of the neutral salt-soluble material into the fibers are not complete. Jackson also reported the presence of a water-soluble hydroxyproline-containing fraction, whose function in fibrogenesis he was unable to determine. Since Robertson and Schwarz (1952) have shown that in scorbutic granuloma (which is a permanent phenomenon as opposed to the transient nature of the condition induced by carrageenin) the main protein constituent is devoid of hydroxyproline, it may be that the fragment rich in hydroxyproline combines with the products of fibroblastic activity at a late; stage in the production of the neutral salt-soluble collagen. Elastic fibers often do not occur in scar tissue at all, and never occw early, a@ an explanation of this might be that the presence of the hydroxyproline-cantaining fraction in granuloma tissue produces a situation in which the whole of the products of fibroblastic activity go to the production first of neutral salt-soluble collagen, and then insoluble collagen fibrils, whereas: in normal tissue in the absence of excessive amounts of the hydroxyqroline-containing material part of the fibroblastic products will be avaitable for the synthesis of elastic fibers.

B. The Aging of Connective-Tissue Fibers 1. Changes in Collagen. Electron-microscope studies have shown that the perifidic striations of collagen fibers at 640 A. are universally apparent in tissues at all ages from 1 hour to 89 years (Gross and Schmitt, 1948). I n embryonic tissue, however, Porter ( 1951) and Fitton-Jackson (1954) have shown that fibrils with cross striations of 210-A. periodicity are visible. The exact nature of the structural patterns which bring about these changes in electron opacity at regular intervals along the fiber axis is as ye$ unknown, but it would appear that there is no change in this structure after the fiber has become fully mature. The change observed in the transition from embryonic to infant tissue may represent the process of stabilization, which Bowes et al. (1956) have suggested may be due to the association of the collagen precursor, or its immediate solid successor with a mucopolysaccharide. The presence of mucopolysaccharide sur-

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rounding infant fibers is well substantiated, and there is evidence that at least in certain tissues this material decreases with age (Happey et al., 1953 ; Sobel and Marmorston, 1956). Chemically, little evidence has been advanced to indicate changes in amino acid analysis with age, although Hall and Reed (1957), examining a small population of normals, showed evidence for a trend in the hydroxyproline content toward lower values with increasing age. By studying the argyrophilia which appears to be associated with the polysaccharide content of collagen fibers, Schwarz (1957) has been able to obtain evidence for the lateral aggregation of collagen molecules and fibrils to form larger fibers resulting in an apparent “crystallization” in the adult fibers. This “consolidation” or “crystallization” is accompanied by a change in the reactivity of the fibers toward disruptive reagents. Orekhovitch et al. (1948) showed that a far smaller amount of acid-soluble collagen could be obtained from adult dermis than from young tissue, and Banfield (1952) has rendered these observations more precise by studying the effect of dilute acetic acid on pure collagen preparations. H e was thus able to show that, not only is there less extractable material in old tissue, but the fibers themselves are more resistant to extraction. The earliest stage in this series of structures of increasing stability is represented by the granuloma tissue induced by carrageenin (Jackson, 1957) in which an increasing amount of material can be extracted with acid throughout the whole period of fibrogenesis and resorption. 2. Changes in Elastic Fibers. The structure of the elastic fiber is so variable that it is not easy to determine whether degradation is associated with age purely on morphological grounds. Much of the apparently degenerate elastin, Unna’s so-called elacin, which can be seen in a variety of elastoses of the dermis has been shown (Tunbridge et al., 1952) to consist of degraded collagen. Where the conditions are not so advanced as to warrant the term “elastosis,” little change is observed (Hill and Montgomery, 1940). It is mainly in the arteries that age changes have been observed in elastic tissue. In the media of arteriosclerotic vessels, marked changes are observed in the morphology and tinctorial properties of the elastic fibers (Carlson, 1949) and in the amounts of polysaccharide associated with them. Sax1 (1957b) has shown that the area of aortic media most susceptible to elastase attack varies with age. In middle age groups, the region most markedly attacked by the mucolytic fraction of elastase is the upper third of the media in which lipid changes occur in atheroma. Lansing et al. (1951) have shown that changes begin to be apparent in aortic elastin between the ages of 15 and 25 years and hence may be taken as being dependent on the maturity of the subject and as

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antecedent to the more gross changes associated with atherosclerosis. The fact, however, that the changes appear in the main to be restricted to those arteries in which atherosclerotic changes ultimately occur may indicate that they are in effect the early stages of a pathological process, and not solely aging phenomena. Biochemically, one of the characteristic changes in elastic fibers is their increased affinity for calcium, which may rise from 0.6% in the first decade to an average value of 6.8% by the seventh to eighth decade, and in extreme cases even to values in excess of 13%. Running parallel with these changes are apparent variations in the amino acid composition of the elastin. Lansing et al. report that there is an increase in the concentration of aspartic and glutamic acids, which they suggest provides the site for the binding of increasing concentrations of calcium. Their amino acid analyses indicate a rise in certain amino acids, and a decrease in others, and, in addition to those shown to increase by Lansing, the hydroxyproline content is also raised (Hall, 1951, 1955). Hall also showed that treatment with boiling urea solution was capable of separating from a preparation of aortic elastin a fraction rich in hydroxyproline, aspartic acid, glutamic acid, arginine, lysine, and histidine, leaving behind a substance which had the classic amino acid analysis of young elastin or ox ligamentum nuchae elastin. These observations led to the suggestion that the preparation of “old elastin” described and analyzed by Lansing et al. is, in fact, a mixture of two proteins, one of which has a far higher dicarboxylic amino acid content than either elastin or Lansing’s “old elastin.” This component is, however, closely attached to the true elastin, and hence the whole complex separates together. Small numbers of anisotropic fibers can be observed in dermal or vascular tissues from elderly subjects (Hall et al., 1958). These fibers appear to consist of a protein core enveloped in cellulose, with the complex fibresis similar to the cellulose fibers occurring in the tunic of the ascidians (Meyer et al., 1951). O n the basis of this it is suggested that with aging the organism may revert to a more primitive metabolic level, and degraded collagen fibrils become coated with cellulose.

V. THE PRODUCTION OF ELASTIC MATERIALFROM COLLAGEN A. Structural Evidence 1. Histochemical Evidence. The original observations of Burton et d. (1955) which led to the promulgation of the conversion hypothesis were made on dermal preparations purified according to the method of Neuman ( 1949).

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After treatment with buffer solutions of p H range 7 to 10.4 for prolonged periods of time, pancreatic enzyme concentrates, and solutions of phthalate buffer ( p H 5) with or without the addition of sodium metaperiodate, significant changes occurred in the tissue powder. Masses of amorphous material and high concentrations of anisotropic lamellae were observed in close association. When the reagents were perfused under pressure through thin discs of dermis (Hall, 1956), the spatial relationship of the amorphous material and the anisotropic structures was more easily seen. A certain proportion was free, some loosely adhering to the lamellae, and other lamellae were completely coated with it. This amorphous material stained deeply with Hart’s modification of Weigert’s stain. I n addition to these masses of amorphous Weigert-positive material, large numbers of structures histologically indistinguishable from elastin, in the form of wavy black-staining fibrils, were observed. 2. Electron-Microscope Studies. At the electron-microscope level, the transformation of the collagen fibrils was equally obvious. Collagen fibrils appeared in all stages of degeneration, from the condition in which only the edges of the fiber appeared to have lost their sharp outline, to one of complete gelatinization. Some, however, never reached this stage of total degeneration but became coated with amorphous material. It has been suggested (Smith, 1957) that the structures observed, to which the term “elastin-like” was applied on account of their marked similarity to this material, are in fact collagen fibers coated with gelatin. A comparison of the appearance of these fibers with the frankly gelatinized structures illustrated in Keech and Reed (1957a) indicates the significant difference between the two types of collagen degradation. Gelatinization is accompanied by the appearance of diffuse ill-defined masses hardly distinguishable from the background. The production of elastica is also characterized by the appearance of masses of amorphous material, but these are sharply defined, are electron-opaque, and stand out clearly from the background. Fibers exist (Burton et al., 1955, Fig. 2) in which part is converted to elastica, the remainder retaining its collagenous characteristics. Keech and Reed (1957a) compared the effect of boiling water on the appearance of collagen that has not been subjected to alkaline treatment with that of elastica produced in this way. Untreated collagen gelatinizes, whereas the synthetic elastica merely consolidates and assumes an appearance even more nearly like that of true elastic fibers. Attention was drawn to the similarity of these structures to the products of heat treatment on the irregular electron-opaque structures obtained from young collagen after treatment with collagenase. Disruption of the opaque outer layer reveals an essentially elastic struc-

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ture. Keech et al. (1957a) suggest that these “moth-eaten fibers” represent an intermediate stage in the conversion of collagen to elastin. Keech and Reed ( 1957b) also show that collagenase, elastase, hyaluronidase, and ultrasonic radiation all have the same effect in producing elastin-like material from the “moth-eaten fibers.” Balo et al. (1956) suggest that the residue from collagen fibers, after the removal of polysaccharide and procollagen, either by thermal or chemical denaturation, has a similar appearance to the synthetic elastica produced by alkaline treatment of collagenous tissue. They do not, however, feel justified in calling the material elastin, but give it the name metacollagen. Since the starting material employed by these workers consisted of rat-tail tendons, essentially free of ground substance, it is possible that the material to which they give the name metacollagen is, in fact, not identical to synthetic elastica. The production of elastica-staining areas and material identifiable as elastic fibers under the electron microscope is a more efficient process in whole tissue than in purified collagen. Hence, the ground substance may play an important role in the production of synthetic elastica-staining material, and it may be that metacollagen differs from elastica in the absence of this component. 3. X - R a y Diffraction. As mentioned in Section 11. D, it appears that collagen and elastin differ in that collagen has a highly oriented structure producing a complex array of arcs on an X-ray diffraction photograph, whereas elastin in the unstretched state gives a picture which is representative of an amorphous structure. Ramachandran and Santhanan ( 1957), however, have reinvestigated the X-ray diffraction pictures obtained from collagen, chemically altered collagen, and elastin and have suggested not only that elastin should be classed with collagen but that its molecular structure may well be built up of a triple-chain structure similar to that of collagen (Ramachandran and Kartha, 1954; 1955a, b ; Ramachandran, 1956). They showed that two diffuse rings of 4.4-A. and 2.2-A. spacings and a sharper ring at 11 to 12 A. are typical of elastin. The 2.2-A. spacing is reported for the first time and appears to be similar to rings that occur in pictures obtained from native and thermally shrunk collagen and gelatin. They claim even greater similarity between elastin and collagen fibers denatured by treatment with nickel nitrate and calcium chloride. Since certain of these reactions are reversible, it would not appear likely that the identity of the two sets of X-ray pictures is indicative of conversion from collagen into elastin such as is implied by Burton et al. (1955) and Hall et al. (1955a). If conversion does occur, the material studied by Ramachandran and Santhanan may represent a state in which the correct structural align-

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ment is attained, without the removal of those portions of the molecule which are redundant to the elastin structure.

B. Chemical Evidence

1. Alkali Treatment. Alkaline buffers, especially borate, p H 8.7, have been employed (Burton et al., 1955; Hall, 1956) to bring about an apparent conversion of collagen into elastin-like structures. The early studies were concerned mainly with an assessment of the hydroxyproline relationships of the tissues and the extracts. Hydroxyproline-rich protein fractions were obtained by the exhaustive extraction of tissue preparations for periods of up to 3 days. Division of the extracts according to time of extraction produced protein fractions of varying hydroxyproline content. The period during which the protein with the highest hydroxyproline content was extracted varied with the age of the subject, being early in the young tissue and appearing only after protracted extraction in elderly subjects. Continuous fractionation of the extract by perfusing tissue with alkaline buffer under pressure permitted the collection of five protein fractions. Of these, three had appreciable hydroxyproline content. One had a hydroxyproline content of between 30 and 40%, and the other two were similar to collagen. Hall (1956) showed that a similar amount of material was extracted from whole calf skin, and purified tissue with borate buffer and similar amounts of elastin were obtained in the residue, but the physical properties of the residue from the former was more nearly like the native elastic fiber than that obtained from purified tissue. C. Evidence for the Heterogeneity of Collagen One of the major criticisms against the acceptance of the hypothesis that collagen may be converted in vivo into elastic fibers arises from a consideration of their respective amino acid compositions. Collagen is characterized (Bowes et al., 1956) by the presence of high concentrations, of glycine, proline, and hydroxyproline which together account for 437’0 of the residues in the molecule. Elastin, on the other hand, although containing roughly similar amounts of glycine and proline, is relatively free of hydroxyproline (1.270). It has also a far lower concentration of polar amino acids. Their place is .taken by the monoaminomonocarboxylic acids all of which are increased in amount, especially valine, which is present in seven times as high a concentration in elastin as in collagen (Partridge and Davies, 1955). Thus any conversion of collagen into elastin would have to be associated with the removal of a large proportion of the hydroxyproline and polar amino acids, and all the valine would have to remain in the residue. In view of the relative valine concentrations, this

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cannot represent more than one-seventh of the original collagen, and Harkness et al. (1957) have pointed out that the efficiency of the reaction in terms of turnover of one protein species to the other can only be of a similar order. Tristram (1957) and Smith (1957) have also doubted whether such conversion could proceed without complete hydrolysis to the amino acid level followed by resynthesis. Such a sequence of reactions could hardly be accepted as explaining the phenomenon reported by Burton et al. (1955) in which, after perfusion with borate buffer at p H values between 8 and 9, collagen fibers were directly converted to elastinlike material. Not only would complete hydrolysis followed by resynthesis be unlikely to effect the production of the elastin-like material at the same site as the original collagen fibers, since the released amino acids would be leached from the tissue by the perfusing fluid, but also it is inconceivable that sufficient energy could be available in such an in vitro system for the resynthesis of the necessary peptide bonds. Similarly, if such a series of reactions were accepted as an explanation of a possible in vivo conversion (Hall et al., 1955a), the elastin could not be said to be derived directly from the collagen fibers, since the released amino acids would join the amino acid pool, and those employed in the resynthesis could have come from the catabolism of any tissue, or from some exogenous source. Such profound degradation was indeed never envisaged by the authors of the hypothesis, and it is of interest to consider how such a concept arose. 1. Sequence Studies. Bergmann and Niemann (1936), basing their hypothesis on the fragmentary analyses then available, suggested that collagen consisted of a repeating tripeptide containing glycine, proline, or hydroxyproline, with another amino acid as the third component. An assessment of this suggestion in association with wide-angle X-ray diffraction data enabled Astbury (1940) to devise the first structural model for collagen. Since then, small-angle X-ray diffraction studies and polarized infrared observations, both on collagen and on synthetic peptides rich in either glycine or proline, have permitted the evolution of more-complex structures for collagen (Pauling and Corey, 1953; Ramachandran and Kartha, 1955a, b ; Rich and Crick, 1955 ; Cowan et al., 1955) which ascribe a triple helical structure to collagen. As a relic of the Bergmann-Niemann hypothesis, however, the initial assumption was made that a repeating triad of amino acids occurred in the molecular chain. Schroeder et al. (1953, 1954) and Kroner et al. (1953, 1955) suggested that the original gly-pro or (hypro)-R structure was untenable, since they were able to isolate, from among the peptides obtained by partial hydrolysis of collagen, a tetrapeptide gly-pro-hypro-gly. They also suggested on the basis of their

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evidence that there might be regions that were devoid of pyrrolidine residues. Even the possibility of regions of varying analysis did not prevent these authors from adhering to the spirit of the Bergmann-Niemann hypothesis, if not to its letter. They replaced the triad by a tetrad, however, on very flimsy evidence. The hydroxyproline content of the tetrapeptide units was only 1.17% of the total hydroxyproline content of the collagen, but Kroner et al. (1955) report the existence, in their partial hydrolyzates, of three tripeptides in which hydroxyproline is flanked by other nonpyrrolidine amino acids. The hydroxyproline content of these tripeptides-ala-hypro-gly, gly-hypro-gly, and ser-hypro-gly-together represents 2.28% of the whole hydroxyproline content of the molecule. Their decision to ascribe one particular structure to the whole of the collagen molecule on the basis of an analysis of just over 1% of the total and in the face of evidence in support of another structure representing over 2% of the molecule would appear to be unjustifiable. Evidence from larger molecules derived by partial enzymatic degradation of collagen has been presented by Grassmann et al. (1957a). They obtained five or six peptides of considerable chain length (ranging from 20 to 79 amino acid residues). These showed marked variations in amino acid analysis, and, as a general rule, it was possible to show that areas rich in proline and hydroxyproline were devoid of polar amino acids, and vice versa. Even greater variations could be observed between the individual peptides. For instance, one with a chain length of 79 residues contained 11 proline and 12 alanine residues, whereas another polypeptide (43 residues long) contained only 3 alanine residues to 10 prolines. Even if the larger peptide contained the one with fewer residues, the alanine content of the 35 extra residues would have to be 25% of the total, as opposed to the 7% in the smaller peptide, or the 8% of the whole molecule. Similarly, a decapeptide containing 4 arginine and 5 glycine residues was identified. There would appear to be evidence in favor of marked heterogeneity in the collagen molecule, yet here again the fractions considered by Grassmann et al. only amount to 4.2% of the whole protein, and hence strict extrapolation to an analysis for the whole molecule is not justifiable. Grassmann points out, however, that areas of some 30 amino acid residues, completely devoid of polar groups, such as appear to exist, could represent structurally repeating elements, whose length would be roughly similar to one turn of the triple helix of Ramachandran and Kartha and of the same order of magnitude as a single light interperiod band in an electron micrograph. On the whole, it would appear that until more extensive portions of the collagen molecule have been analyzed, there is little reason for assuming

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more than a marked degree of heterogeneity in the molecule. One fact does appear to emerge. Rigidly repeating units of triad or tetrad nature cannot account for the whole of the chain structure of collagen. 2. The Action of Collagenase. The hydrolysis procedures utilized for the preparation of the peptide fractions reported above have of necessity broken down the proteins to a considerable degree. Much of the material obtained is useless for sequence studies, being in the form of free amino acids. Other workers, employing collagenase, have performed digestions which have resulted in the production of polypeptides of much longer chain length. Although it is impossible at this stage to obtain sequence data for these compounds, their gross analyses have been sufficient to demonstrate even further the existence of a considerable degree of heterogeneity in collagen. Mandl (private communication) has isolated a polypeptide with a molecular weight of some 7000 which contains neither hydroxyproline nor proline. This peptide was derived from collagen by treatment with a collagenase preparation from CZ. histolyticum. I n similar studies employing the enzyme from Cl. weZchii, Hall (1957b) reported the preparation from collagen of a polypeptide of high molecular weight (retained by a dialysis membrane) which was relatively devoid of hydroxyproline. Apparently the enzyme acted preferentially on those regions of the molecule that were rich in hydroxyproline. 3. The Effect of Phthulate Buffer on Collagen. When it was first suggested that elastin-like material could be produced from collagen (Burton et al., 1955 ; Hall et al., 1955a), it was reported that alkaline extraction of collagen resulted in the degradation of the fibers with the release of protein fragments rich in hydroxyproline. These results were amplified by Hall (1957b), who showed that perfusion of tissue with borate buffer brought about the release of a number of polypeptide units one at least of which contained a content high in hydroxyproline. Veis and Cohen ( 1956), however, suggested that collagen should be considered as a collection of “segments of varying length and cross section due to differences in cross-link distribution and the lateral ordering of side chains. The segments are chain networks held together by sets of acid stable bonds, while the segments contain and are held in the gross structure by acid labile bonds and physical forces. All bonds are, however, alkali stable.” It would appear, therefore, that alkali treatment of collagen would be the most likely to produce partial degradation products of sufficiently high a degree of complexity for the evolution of another structure to be accomplished without recourse to peptide bond formation. This indeed is what was observed. Electron micrographs of collagen treated with buffers of

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varying p H showed the presence of elastic fiber masses only within a circumscribed p H range. Since this was in the region of low alkalinity, a necessary factor in the conversion might be the availability of polysaccharide, rendered soluble by the mildly alkaline conditions. At lower p H little polysaccharide would be extracted; at higher p H it would be destroyed. It was of significance, therefore, that phthalate buffer was found to have a specific effect. Hall (1957b) reported that extraction of collagen with phthalate results in the removal of material rich in hydroxyproline. The extracted material differs from that isolated from collagenase-digested collagen, however, in that the majority of the hydroxyproline remains with the high-molecular-weight material.

VI. CONCLUSIONS In the earlier sections of this review evidence was presented indicating the desirability of considering the elastic fiber and the elastic lamellae as members of a group of fibrous components of connective tissue. Collagen appears to be relatively constant in composition throughout the tissues, although even here the differences discovered between collagenous tissue at various ages may be mirrored in variations from tissue to tissue. As yet the major studies on chemical composition and physical properties of collagen have been carried out on dermis and tendon, in view of the ease with which collagen from these sites can be obtained in a pure state. For elastin, evidence for variability already exists. True elastic fibers from dermis or aorta may be similar in composition to the classical elastin of ligament, but the aorta also contains components that appear to be similar to elastin in many of their properties but have markedly dissimilar amino acid analysis. I n the last part, evidence is reviewed for the concept that, as well as being members of the same group of fibrous proteins, collagen and elastic fibers may be even more closely related. The points of similarity are as follows : ( 1) They have a common source in the same fibroblasts ; (2) they contain certain amino acid sequences in common which act as specific foci of attack for elastase ; (3) collagen on degradation picks up material from the ground substance, which may be polysaccharide alone, or may include polypeptides to give a protein with many of the physical properties of elastin. The evidence against this concept is based mainly on amino acid sequence studies for collagen. These are still, however, so incomplete as to preclude their use as a true basis for criticism. The only fact that has emerged from them is that areas of heterogeneity do exist in collagen, and these may be sufficiently extensive to permit conversion of the type sug-

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gested to occur without recourse to “cataclysmic degradation” (Tristram,

1957).

The evidence from conditions such as lathyrism or onchocerciasis are more difficult to discount. If collagen can be converted into old elastin, then even the prevention of elastic fiber formation in lathyrism or the complete destruction of the elastic network in onchorcerciasis should not prevent the ultimate replacement of the lost elastic fibers by the body, with collagen fibers as source. Two possibilities present themselves : either the collagen in these conditions becomes highly resistant to attack, and hence cannot take part in elastin production ; or true elastic fibers are required as a matrix before the old elastin can be laid down. Full elucidation of the problem will no doubt have to await intensive sequence studies on collagen fibers, collagen degradation products, elastic fibers, and “old elastin.”

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