No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Inhibition of Collagen Accumulation in Fibrotic Processes: Review of Pharmacologic Agents and New Approaches with Amino Acids and Their Analogues
Abstract
Accumulation of collagen is the major pathologic feature in a variety of fibrotic processes, including dermal fibrosis in progressive systemic sclerosis, morphea, familial cutaneous collagenoma, connective tissue nevi of the collagen type and in keloids. Recent advances in the biochemistry of collagen have allowed us to define specific levels of collagen biosynthesis and degradation at which a pharmacologic intervention can lead to reduced collagen deposition. In this review, we are discussing the mechanisms of action by some of the therapeutic agents currently in use. We further present some new developments involving amino acids and their analogues which could potentially provide us with novel means to reduce the excessive accumulation of collagen in dermal fibrotic processes.
... Based on the knowledge emerging from the studies on regulation of collagen gene expression in normal situations, attempts have also been made to develop pharmacologic approaches to control excessive collagen deposition in fibrotic skin diseases [105,106]. Although many of these compounds have been effective in tissue or cell culture environment, their efficacy in clinical situations has been compromised by toxicity. ...
... Thus, there has been a distinct need for further development of novel approaches to control collagen accumulation in patients with connective tissue abnormalities. The progress in this area of dermatopharmacology was reviewed in the Journal in 1982 [106], but several newer approaches have been developed since then. ...
... The latter observations would explain the compromised wound healing in patients treated with doxorubicin. Inhibition of triple-helix formation has also shown to be the mechanism of action of several proline analogues which inhibit collagen deposition in tissues [106]. The proline analogues, and cis-4-hydroxyl-Lproline and azetidine carboxylic acid in particular, unlike the naturally occurring trans-4-hydroxy-L-proline, are incorporated into newly synthesized preproa-chains in place of prolyl residues [110]. ...
The extracellular connective tissue matrix of the skin is a complex aggregate of distinct collagenous and non-collagenous components. Optimal quantities and delicate interactions of these components are necessary to maintain normal physiologic properties of skin. This overview summarizes the progress made in understanding the normal biology and biochemistry of the extracellular matrix, and will highlight cutaneous diseases with underlying molecular defects in the structure and expression of extracellular matrix components.
... Despite its name's similarity to the widely known antibiotic penicillin, D-penicillamine (DPA) is a byproduct of penicillin without any antibiotic properties [1]. It is a known chelating agent, a chemical compound used to trap or remove heavy metals, such as copper, lead, iron, and mercury, from the body; moreover, DPA also showed positive immunomodulatory and antifibrotic capacities [2][3][4][5]. ...
... 4. Understands that prescribing a compounded product instead of an approved drug should not be done solely for the economic benefit of the veterinarian. 5. Prescribes in an extra-label manner in keeping with the current research and evidence for a specific species. ...
Chelant agents are the mainstay of treatment in copper-associated hepatitis in humans, where D-penicillamine is the chelant agent of first choice. In veterinary medicine, the use of D-penicillamine has increased with the recent recognition of copper-associated hepatopathies that occur in several breeds of dogs. Although the different regulatory authorities in the world (United States Food and Drugs Administration—U.S. FDA, European Medicines Agency—EMEA, etc.) do not approve D-penicillamine for use in dogs, it has been used to treat copper-associated hepatitis in dogs since the 1970s, and is prescribed legally by veterinarians as an extra-label drug to treat this disease and alleviate suffering. The present study aims to: (a) address the pharmacological features; (b) outline the clinical scenario underlying the increased interest in D-penicillamine by overviewing the evolution of its main therapeutic goals in humans and dogs; and finally, (c) provide a discussion on its use and prescription in veterinary medicine from a regulatory perspective.
... However, three cases of isolated plantar collagenoma without associated clinical abnormalities have been reported. [4] In one case of isolated collagenoma, Uitto et al [5] showed that the increased collagen is of the adult type (Type I) and that a local reduction of collagenase might be the cause of the excess collagen. In contrast to cutis verticis gyrata (CVG) with a diffuse involvement of scalp, isolated collagenoma presents as a localized abnormality. ...
... Moreover, it has been demonstrated by ultrastructural and 3 H-proline radioautographic studies on fibroblasts of the rat foot pad that the radioactivity does not appear in vacuoles containing fibrillar collagen, but is transported to the extracellular space (Marchi and Leblond, 1983). Agents that inhibit protein synthesis (cycloheximide; Korner, 1966;Peterkovsky and Tomkins, 1968), the formation of collagen fibrils (α,α-dipyridyl;Dehm and Prockop, 1971;Uitto et al., 1982) or the translocation of procollagen-containing vacuoles (colchicine or vinblastine; Diegelmann and Peterkovsky, 1972;Ehrlich et al., 1974;Scherft and Heersche, 1975;Fernandez-Madrid et al., 1980;Cho and Garant, 1982) had no effect on the volume density of vacuoles containing cross-banded fibrillar collagen (Everts et al., 1985b(Everts et al., , 1989. These studies demonstrated that the latter structures are not related to recently synthesized collagen. ...
Degradation of fibrillar collagen may occur in the extracellular space by enzymes, such as the metalloproteinase collagenase, or in the lysosomal apparatus of fibroblasts following phagocytosis. As the mechanisms involved in the regulation of the latter process are unknown, we investigated possible modulating effects of the cytokines epidermal growth factor (EGF), platelet-derived growth factor (PDGF), interleukin-1 alpha (IL-1 alpha) and transforming growth factor-beta (TGF-beta) on both collagen phagocytosis and the release of collagenase in an in vitro model employing periosteal tissue explants. The data demonstrated that the level of intracellular collagen digestion could be influenced by cytokines: IL-1 alpha inhibited and TGF-beta enhanced phagocytosis of fibrillar collagen by periosteal fibroblasts, whereas the cytokines had an opposite effect on the release of procollagenase. In combination, IL-1 alpha and TGF-beta proved to have an antagonizing effect on either parameter. PDGF and EGF had no effect on phagocytosis or collagenase release. The level of phagocytosed collagen correlated positively with the actual breakdown of collagen as assessed by the release of hydroxyproline but negatively with the level of released procollagenase. Our findings demonstrated that cytokines are able to modulate both the phagocytosis of collagen fibrils by fibroblasts and their subsequent intracellular breakdown, as well as the release of procollagenase, an enzyme considered crucial for extracellular collagenolysis. Moreover, our data show a negative correlation between these two parameters. It is concluded that IL-1 alpha, EGF and TGF-beta may be important in modulating the contribution of the intracellular and extracellular route of collagen breakdown.
... Immunornodulating effects relate to the ability to decrease circulating immunoglobulin concentrations, inhibit chemotaxis of neutrophils and to interfere with Tlymphocyte function. 42 The use of penicillamine as an anti-fibrotic agent in human medicine has been disappointing. Seven prospective, controlled studies of penicillamine use in primary biliary cirrhosis have not demonstrated drug efficacy at doses ranging from 600-1000 m g / d a~.~~ ...
Most chronic liver disorders are accompanied morphologically by the deposition of fibrous tissue within the hepatic parenchyma. This fibrotic tissue compromises hepatic function and contributes significantly to hepatic failure. Fibrosis is a dynamic process associated with the continual deposition and resorption of connective tissue. Therapeutic strategies are emerging whereby this dynamic process can be modulated. Since collagen is the major component of the extracellular matrix deposited in hepatic fibrosis, most anti-fibrotic therapies have been directed toward the control of collagen metabolism. After collagen genes are transcribed and translated into precursor procollagen proteins, a number of post-translational modifications that ensure the deposition of structurally sound collagen within the extracellular matrix occur. A number of drugs can specifically modulate collagen biosynthesis at the transcriptional level or at various post-translational stages. These anti-fibrotic drugs include corticosteroids, azathioprine, penicillamine, colchicine, zinc, prostaglandins, cyclosporine, and interferons. The pharmacologic action of these drugs and the clinical role in veterinary and human fibrotic hepatopathies will be discussed.
... Several other enzymes involved in collagen synthesis have been associated with ¢brotic activity (10). One of them, the lysosomal enzyme beta-galactosidase (b-gal) cleaves hydroxyllysine-galactose from collagen's pro-a chains during collagen synthesis (6,11,12). A second lysosomal enzyme is N-acetylglucosaminidase (N-ACGA) which is activated in collagen synthesis. ...
The lysosomal enzymes N-acetylglucosaminidase (N-ACGA) and beta-galactosidase (beta-gal) are involved in cellular collagen metabolism and may, therefore, be markers of fibrosis in idiopathic interstitial pneumonias, such as idiopathic pulmonary fibrosis (IPF). N-ACGA and beta-gal were analyzed in the bronchoalveolar lavage fluid (BALF) of patients with the histologic pattern of usual interstitial pneumonia (UIP, n=10) and controls (n=9). Cellular distribution in BALF as well as the concentration of TGF-beta a well-known mediator of fibroblast matrix deposition were correlated to the enzyme activities in both groups of patients. We found that both, N-ACGA (UIP: 25.2 nmol/l s +/- 3.4; controls: 73 nmol/l s +/- 1.3) and beta-gal (UIP: 4.7 nmol/l s +/- 0.5; controls: 2.4 nmol/l s +/- 0.3) were elevated significantly in BALF of patients with IPF compared to that of control patients (P<0.003). This increase was paralleled by an increase in neutrophils (IPF: 17.9% +/- 21.8; controls: 5.4% +/- 6.3; P=0.03) and eosinophils (IPF: 2.0% +/- 1.5; controls: 0.2% +/- 0.45; P=0.002) in BALF fluid. In addition, N-ACGA activity correlated closely with lung function (FVC, TLC, and DLCO), transforming growth factor-beta (TGF-beta) in BALF (r=0.77, P=0.008) and activated lymphocytes (r=0.66, P=0.0021). Our findings suggest that measurement of lysosomal enzymes such as N-ACGA may represent a useful indicator of fibrotic activity in IPF.
... However, three cases of isolated plantar collagenoma without associated clinical abnormalities have been reported. [4] In one case of isolated collagenoma, Uitto et al [5] showed that the increased collagen is of the adult type (Type I) and that a local reduction of collagenase might be the cause of the excess collagen. In contrast to cutis verticis gyrata (CVG) with a diffuse involvement of scalp, isolated collagenoma presents as a localized abnormality. ...
The cytokine transforming growth factor beta (TGFβ) has a role in regulating the normal and pathological response to wound healing, yet how it shifts from a pro-repair to a pro-fibrotic function within the wound environment is still unclear. Using a clinically relevant ex vivo post-cataract surgery model that mimics the lens fibrotic disease posterior capsule opacification (PCO), we investigated the influence of two distinct wound environments on shaping the TGFβ-mediated injury response of CD44⁺ vimentin-rich leader cells. At the leading edge of the wound, the substantial fibrotic response of this cell population to a rigid wound environment required endogenous TGFβ. However, TGFβ was dispensable for the role of leader cells in wound healing in the endogenous basement membrane wound environment, where repair occurs in the absence of a major fibrotic outcome. A difference between leader cell function in these distinct environments was their cell surface expression of the latent TGFβ activator, αvβ3 integrin. This receptor localized exclusively to this CD44⁺ cell population when they localize to the leading edge of the rigid wound environment. Providing exogenous TGFβ to bypass any differences in the ability of the leader cells to sustain activation of TGFβ in different environments revealed their inherent ability to induce pro-fibrotic reactions on the basement membrane wound environment. Furthermore, exposure of the leader cells in the rigid wound environment to TGFβ led to an accelerated fibrotic response including the earlier appearance of pro-collagen + cells and alpha smooth muscle actin (αSMA)+ myofibroblasts and increased fibrotic matrix production. Collectively, these findings show the influence of the local wound environment on the extent and severity of TGFβ-induced fibrotic responses, which has important implications for understanding the development of the lens fibrotic disease PCO in response to cataract surgery wounding.
An intestinal epithelial cell line from neonatal rat duodenum (IEC-17) was used as a model for investigating metal toxicity.
In this paper, we have further characterised toxic effects on IEC-17 cells following exposure to two physiological metals, zinc (Zn ²⁺ ) and copper (Cu ²⁺ ) and one non-physiological metal, cadmium (Cd ²⁺ ). Time-effect experiments showed that the duration of the exposure affected the extent of cell damage only when Cu ²⁺ and Cd ²⁺ were used. During the first 48 hours of Zn ²⁺ exposure, the cells were seriously affected, but subsequently were able to recover. On the other hand, a colony forming ability test and morphological observations showed a special sensitivity of this cell line to Cu ²⁺ . A possible explanation is suggested in relation to extracellular matrix formation.
Objectives:To study collagen phagocytosis by human extravillous trophoblast.
Treatment of hepatobiliary disease in dogs and cats often involves the use of multiple drugs for their inflammatory, antifibrotic, cupruretic, hepatoprotectant, antimicrobial, diuretic, procoagulant, or antacid actions. This article reviews the indications for and optimal use of the following agents in the setting of hepatobillary disorders of dogs and cats: glucocorticoids, azathioprine, colchicine, zinc, D-peniclliamine, ursodiol, vitamin E, S-adenosyl-L-methionine, milk thistle (silymarin), carnitine and taurine, antimicrobials, lactulose, spironolactone and other diuretics, vitamin K1, and gastrointestinal protectants.
: Sports-related acquired connective tissue nevus or "athlete's nodule" has been reported under a variety of different names. The lesion develops in response to activity causing chronic or repetitive low-grade pressure or irritation activity, which is often a component of athletic training. A case of "athlete's nodule" in a figure skater has been reported. Our case demonstrates the typical histopathologic findings of deposition of normal-appearing collagen bundles arranged haphazardly within the dermis as described in previous reports. Additionally, results of CD34, colloidal iron, and elastic tissue staining have been described. The macular clinical presentation with the absence of significant epidermal change is a unique presentation of athletic nodule. It is the goal of the authors to create increased awareness among pathologists and clinicians in identifying these lesions and to expand the clinical presentation to include nonprotuberant lesions.
Proline analogues inhibit procollagen triple helix formation and are antifibrotic in vivo. Efficacy of the proline analoguecis-4-hydroxy-L-proline (cHyp) on vascular collagen accumulation is improved by in vivo delivery in liposomes. This effect may be due to local release of drug from liposomes taken up by vascular endothelium. To test this postulate, we used a co-culture system to assess the antifibrotic effect of cHyp in liposomes taken up by endothelium (upper well) by measuring inhibition of growth of smooth muscle cells and fibroblasts (lower well). We also studied whether release of cHyp was prolonged in poly(ethyleneglycol) (PEG)-conjugated liposomes compared to liposomes not conjugated with PEG (control liposomes). In fibroblasts, free (unencapsulated) cHyp (1 mg/ml) added to the upper well inhibited growth for 3 days; an equivalent dose of cHyp in control liposomes inhibited growth for 4 days. cHyp in PEG-liposomes produced greater growth inhibition than cHyp in control liposomes. cHyp in liposomes did not inhibit growth of smooth muscle cells more than free cHyp. Washing free cHyp from endothelium after 1 day incubation restored growth of smooth muscle cells whereas washing liposomes containing cHyp failed to restore cell growth. These results suggest that liposomes enhance drug efficacy of cHyp by prolonging the release of drug from endothelium.
N-Oxaloglycine (3) is an alpha-ketoglutarate (1) analogue that is a competitive inhibitor of prolyl 4-hydroxylase (EC 1.14.11.2). A study of the structure-activity relationships of some other oxalo derivatives shows that substitution on the glycine moiety modulates activity stereoselectively and that if the omega-carboxylate is homologated or replaced by either acylsulfonamides or anilide, then activity is sharply reduced. This sensitivity to these changes is contrasted with the relative insensitivity of another putative alpha-ketoglutarate analogue, pyridine-2,5-dicarboxylic acid (2), and the implication is discussed that compounds of both series are unlikely to bind to prolyl hydroxylase in the same way even though both inhibit the enzyme competitively.
Neurofibromas, the hallmark of neurofibromatosis, are cutaneous or subcutaneous tumors consisting of proliferating neural structures and the adjacent extracellular matrix. The predominant extracellular matrix component is collagen which comprises approximately 70% of the dry weight of tissue. Significant quantities, approximately 3% of the dry weight, of proteoglycans are also present, while few, if any, elastic fibers can be demonstrated either by structural or biochemical means. The expression of procollagen genes in neurofibromas can be detected by assay of types I, III, and IV procollagen mRNAs. The levels of mRNA specific for type I procollagen are about 10 times more abundant than those of types III and IV procollagen mRNAs. It appears then that the growth and development of neurofibromas are dependent on continuous expression of collagen genes. Consequently, a pharmacologic approach that would limit collagen gene expression in cutaneous neurofibromas could be potentially beneficial for patients with neurofibromatosis.
Inhibition of the 4-Hydroxylation of Proline ResiduesInhibition of Collagen Triple Helix Formation by the Administration of Proline AnaloguesInhibition of the Cleavage of PropeptidesInhibition of Cross-Link FormationConclusions
ReferencesDiscussionProlyl and Lysyl HydroxylasesN-and C-Proteinase InhibitorsTargeting of Antifibrotic AgentsInhibition of Cross-LinkingStimulation of Collagenase ActivityRegulation of Early Events in Collagen SynthesisAssessment of New Antifibrotic DrugsReferences
An increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high-molecular-weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (αA and βB) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. (a) The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. (b) The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. (c) Immuno-electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day-8 hepatocytes in primary culture.
Hypertrophic scarring and keloid formation are clinical problems with effectively limited solutions. Although numerous methods have been devised to combat them, this article focuses on promising pharmacologic strategies that target collagen metabolism. Laboratory investigations of amino-acid analogs, procollagen peptides, D-penicillamine, and pentoxifylline have demonstrated them to be effective inhibitors of collagen synthesis in cultured cells and/or in animal models. Clinical trials of intralesional administration of interferons have shown impressive reductions in the size and collagen production of keloids. Furthermore, interference of extracellular matrix-enhancing cytokines, such as TGF-beta, may be an effective solution to keloids and hypertrophic scars. Additional research of soluble cytokine receptors, autoantibodies to cytokines, cytokine receptor antagonists, and cytokine-binding molecules may lead to the development of better therapeutic agents.
In a controlled study in 109 female rats we evaluated the effect of soluble D-Penicillamine in doses of 10 mg/ml or 100 mg/ml on capsular formation around semipermeable 2 cm3 mini-prostheses. This was compared with methylprednisolone 1 mg/ml or 10 mg/ml, and a group given saline served as controls. The drugs were injected into the lumen. Capsular wet weight and capsular tensile strength were measured after a period of 40 days. Rats given D-Penicillamine showed a significant, dose-dependent, reduction in wet weight and tensile strength compared with the saline group. There was no significant difference between the groups given D-Penicillamine and those given steroids. Topical treatment with diffused D-Penicillamine can significantly reduce the amount of capsular formation around silicone implants. This drug, which is highly specific for the systemic treatment of fibrotic diseases, should be evaluated further to use in reducing capsular formation.
Athlete's nodules are connective tissue nevi of the collagen type (collagenomas) which appear as thick dermal masses at sites of chronic trauma. They have been described as occurring on the dorsum of the feet, knees, and knuckles of surfers, boxers and marble players. Recurrent minor blunt trauma and pressure are aetiological factors in this condition. We report a 54-year-old man with dermal nodules on the dorsal surface of his feet. These lesions initially appeared during his participation as a player on a high school football team. They were attributed to the chronic pressure of his high-laced athletic sneakers and frequent minor injuries to the involved areas. We describe the successful treatment of our patient's lesions by surgical excision. We also discuss the differential diagnosis and other therapeutic options for athlete's nodules.
Epidermal growth factor and cis-hydroxyproline specifically inhibited synthesis of type 1 collagen, a major gene product of the differentiated dental mesenchymal cells (odontoblasts). In tandem, synthesis of enamel proteins, specific gene products of differentiated dental epithelial cells (ameloblasts), was also inhibited. Under these culture conditions, total protein synthesis in tooth organs was not inhibited but rather increased. Inhibition curves of the gene products specific for epithelial and mesenchymal phenotypes were quite similar, indicating coordinate and intimately associated regulation of gene expression under conditions that perturb cytodifferentiation.
The effect of topically applied N-5', an inhibitor of chemical mediator release from mast cells, on the carrageenin-air-pouch inflammation was studied. The formation of granulation tissue, the accumulation of exudate and the number of infiltrating cells were significantly reduced by the treatment with N-5' (100 mg/kg). The collagen content in granulation tissue was dose-dependently reduced without affecting the noncollagen protein and DNA content by treatment with N-5'. At a dose of 100 mg/kg of N-5', prolyl hydroxylase activity in the tissue was significantly decreased. The selective inhibition of collagen accumulation in granulation tissue resulted from reduction of collagen biosynthesis in vivo. N-5' did not directly inhibit collagen synthesis by diploid fibroblasts, but inhibited fibroblast proliferation in culture. Such results indicate that one of the inhibitory mechanisms of collagen accumulation by N-5' in inflamed sites may involve the inhibition of fibroblast proliferation.
Collagens are a structurally and functionally heterogenous group of proteins encoded by a family of genes that share evolutionary history. Collagen gene expression is regulated both in developmental, tissue-specific manners as well as in response to a variety of biologic and pharmacologic inducers. In the present review we have attempted to synthesize a conceptual overview of the available information from studies aimed at deciphering the molecular mechanisms of collagen gene expression. We have chosen to focus our discussion mainly, although not exclusively, to observations relating to type I collagen gene for a number of practical reasons. The underlying theme that emerges from this survey of the literature is that the regulation of collagen gene expression is complex, utilizing transcriptional, posttranscriptional and translational mechanisms. Although the transcriptional control mechanisms that involve activation and modulation of collagen gene transcription by RNA polymerase II appear to predominate, preferential stabilization of collagen mRNAs and modulation of translational discrimination appear to play significant roles in the regulation of collagen biosynthesis under some physiological situations. Molecular organization of the regulatory regions of collagen genes reveal a mosaic of subdomains with overlapping sequence motifs, involved in positive and negative transcriptional regulation. The precise identity of the cis-acting subdomains of the promoter/enhancer-proximal DNA of collagen gene and how they interact with the trans-acting nuclear protein(s) have yet to be elucidated and will remain the focus of future studies.
The effects of proline analogues, L-3,4-dehydroproline and L-azetidine-2-carboxylic acid, on collagen synthesis by cultured 3T6 fibroblasts have been studied. Prolyl hydroxylase activity was partially inhibited in cells cultured with dehydroproline for 24 h, resulting in the synthesis of collagen in which the proline was underhydroxylated. Azetidine had no effect on prolyl hydroxylase and less effect on the degree of hydroxylation of proline. Fibroblasts grown in the presence of either analogue and fixed in-situ contained greatly distended cisternae of the rough endoplasmic reticulum. Proline analogues otherwise caused few ultrastructural changes in the cells. Treated cells which had been handled more roughly during preparation for electron microscopy contained many large cytoplasmic vacuoles in addition to dilated cisternae. Our results indicate that the major effect of the proline analogues was the inhibition of prolyl hydroxylation. However, electron microscopy of the treated cells revealed hitherto unreported cytoplasmic damage.
Lysyl oxidase, the only enzyme involved in collagen crosslinking, is shown to be present in embryos of the sea urchin Strongylocentrotus purpuratus. The enzyme specific activity increases over six-fold during development, showing the greatest rise during gastrulation and prism larva formation. The enzyme is inhibited by the specific inhibitor, beta-aminoproprionitrile (BAPN). Continuous BAPN treatment of S. purpuratus and Lytechinus pictus embryos from late cleavage stages onward increases the amount of noncrosslinked collagen present in prism larvae. When BAPN is added at the 128- or 256-cell stage it causes developmental arrest at the mesenchyme blastula stage. Embryos can be maintained in the arrested state for at least 96 h and will resume normal development and morphogenesis following BAPN removal. If BAPN is added after the mesenchyme blastula stage, it has little adverse effect on development; consequently nonspecific toxic effects of the drug are unlikely. The results suggest that lysyl oxidase and collagen crosslinking play a vital role in primary mesenchyme migration, gastrulation, and morphogenesis during sea urchin development and indicate that BAPN may be very useful in studying the extracellular matrix-cell interactions at the cellular and molecular level.
Corticosteroids are one of the most widely used group of drugs in the world today. The beneficial effects on the illness being treated must be weighed against the many adverse reactions (Table 1), which may be affected by the route of administration—topical, oral, intravenous, intramuscular, or intralesional. The major adverse reactions through systemic administration will be considered in this chapter.Corticosteroids are hormones produced and released by the adrenal cortex and controlled by the pituitary through release of adrenocorticotropic hormone (ACTH). Hormonal steroids may be classified as those having important effects on intermediary metabolism (glucocorticoids), those having primarily salt-retaining activity (mineralocorticoids), and those having androgenic and estrogenic activity. The major glucocorticoid in man is cortisol, and the major mineralocorticoid is aldosterone (Table 1).Glucocorticoid hormones exert their effect by binding to a specific receptor site located on the cell surface and entered by simple diffusion into the cytoplasm of the cell. This complex is translocated to the nucleus, binds to chromatin, and causes formation of specific mRNAs which then mediate the response to the glucocorticoid.1
Trabeculectomy fails to control the intra-ocular pressure adequately in a proportion of patients. Approaches to solving this problem have involved modifications of surgery, histological studies of tissue from failed and functioning blebs, animal studies, and in vitro investigations of some of the basic processes of wound healing. This paper reviews the current state of investigations in these disciplines with particular reference to wound healing in this specialised site.
Immunohistochemical analysis of collagen types within the external anal sphincter muscle was carried out. The analysis revealed increases in types I, III, and V collagen in RVC affected animals. In addition, staining for type II collagen was positive in affected and carrier individuals but was negative in normal animals. This finding may represent a novel method for the detection of animals heterozygous for the RVC condition.
Zusammenfassung
Rectovaginale Constriction (RVC) bei Jersey‐Rindern IV. Immunohistochemische Untersuchungen des Kollagens
Es wurde eine immunohistochemische Analyse der Kollagen‐Typen im externen analen Sphincter durchgeführt. Dabei stellte sich eine Zunahme der Kollagen‐Typen I, III und V bei RVC‐Tieren heraus. Ferner war die Färbung für Typ‐II‐Kollagen bei Tieren mit RVC sowie bei RVC‐Trägern im Gegensatz zu normalen Tieren positiv. Aufgrund dieses Befundes können zukünftig bezüglich der RVC heterozygote Tiere möglicherweise identifiziert werden.
In this chapter we will briefly review the structure of the normal airway and the extracellular matrix components from which
it is composed. The role of the fibroblast/myofibroblast in maintaining this structure will be discussed, together with the
evidence for their role in remodelling the airway wall in asthma and COPD. Finally we discuss the potential for pharmacological
modulation of fibroblast function as a potential means of reversing the changes in airway structure in these diseases.
This article reviews the scientific basis for the certain factors that delay wound repair in the clinical setting. A brief history of wound healing is given, followed by a discussion of endogenous local factors (bacterial infection, hypoxia, foreign body, and desiccation) and endogenous systemic factors (nutritional deficiencies, aging, coagulation disorders, and the Ehlers-Danlos syndromes) associated with poor wound repair. Also reviewed are the mechanisms by which exogenously administered agents (glucocorticoids, antineoplastic agents, and anticoagulants) may delay healing. Commonly used topical antimicrobials, their spectrum of activity, and evidence of effects on wound healing are examined. Finally, properties of commercially available wound coverings and wound care in the future are discussed.
Conversion from procollagen to collagen is a specific process that is a requirement for proper alignment of collagen molecules to form functional fibers. This process is catalyzed by at least three structurally and functionally distinct enzymes cleaving collagen types I–III. The cleavage processes possibly taking place in the more recently discovered collagen types are not known to any extent at this time.
Two amino-terminal proteinases, one cleaving type I and type II procollagens and the other cleaving type III procollagen, have been purified close to homogeneity, and the more unspecific activity of carboxy-terminal proteinase has been isolated from several tissues. In our experimental model, however, cleavage of the carboxy-terminal propeptides of types I and III procollagen is differently affected by lysine. This suggests the presence of at least two distinct enzymes for the removal of carboxyl-terminal propeptides.
The regulation of the reaction process from procollagen to collagen is not well known at present. The importance of the phenomenon in terms of fibril formation, however, is demonstrated by several elegant studies in vitro; and certain genetic disorders in which this process is defective demonstrate the significance in vivo. Moreover, the factors shown to effect the cleavage process may be potentially beneficial in the treatment of the pathological processes with abnormal collagen accumulation such as fibrosis.
In this paper we briefly review the current knowledge of the converting enzymes, including some very recent findings of our laboratory as well as the evidence presented for the biological significance of the conversion process.
A case of plantar fibromatosis that responded to five monthly intralesional steroid injections is reported. Improvement was noted after 3 to 4 months of therapy. Intralesional steroid injections may represent an alternative to surgery in patients with plantar fibromatosis or Dupuytren's contractures.
Several attempts have been made to develop antifibrotic drugs for human use, but their success has been limited. The present data suggest that peroral zinc treatment has a direct and selective inhibitory effect on carbon tetrachloride-induced collagen accumulation in rat liver. Zinc did not normalize the carbon tetrachloride-induced increases in either liver relative weight, liver total protein content, fat accumulation, or the standard liver function tests, but it did efficiently inhibit liver collagen accumulation. It also reduced skin and liver collagen content and urinary hydroxyproline excretion in normal growing animals, indicating that the inhibition is not limited to the fibroproliferative inflammation associated with carbon tetrachloride injury. Neither inhibition of polysomal protein synthesis nor increased degradation of mature collagen fibers was found to play any major role in the effect of zinc. Instead, a plausible mechanism is inhibition of proline hydroxylation.
Several structural analogues of proline have been shown to be incorporated into proteins in place of proline. As a consequence, the proliferation of cells in culture and the extracellular deposition of collagen in animal systems are reduced. In this study, the effects of two proline analogues, cis-4-hydroxy-L-proline and L-azetidine-2-carboxylic acid, on the growth parameters and procollagen production by cultured normal human skin fibroblasts were examined. The results indicated that incubation of the cells with the analogues reduced the rate of fibroblast proliferation and lowered the plating efficiency. Further experiments demonstrated that fibroblasts in the presence of L-azetidine-2-carboxylic acid synthesized procollagen polypeptides which were not in a triple-helical conformation, as judged by limited pepsin proteolysis. Also, a significantly increased fraction of the newly synthesized collagenous peptides was in a dialyzable form, suggesting increased degradation of the nonhelical chains. The rate of translation of collagenous polypeptides and the preprocollagen messenger RNA activity in the cells were not affected by the analogues. The proline analogues thus appear to inhibit the production of procollagen on the posttranslational level by preventing the polypeptides from folding into a stable triple-helical conformation. The nonhelical polypeptides are then readily susceptible to proteolysis leading to reduced deposition of extracellular collagen fibers. Similar experiments were also performed with fibroblasts cultured from patients with active progressive systemic sclerosis. Quantitatively and qualitatively comparable inhibition of procollagen production by L-azetidine-2-carboxylic acid was noted with scleroderma cells as with control fibroblast cultures. The results suggest, therefore, that proline analogues may, in the future, prove useful in limiting excessive collagen deposition in scleroderma and other forms of dermal fibrosis.
Treatment with methylprednisolone (MP) of patients with acute myocardial infarcts remains a controversial topic; although some studies have shown that MP reduces infarct size, others have shown that it alters the healing process. To determine whether short-term MP limits infarct size and whether it alters the healing process of function, we investigated effects of different doses of MP or infarct size, scar formation, and cardiac function. Experimental infarction was produced in 23 anesthetized open-chest dogs by ligation of the proximal left anterior descending coronary artery. Group 1 dogs (n = 8) received MP 50 mg/kg iv 15 min and 3, 24, and 48 hr after occlusion (high MP); group 2 (n = 7) received MP 30 mg/kg iv 15 min after occlusion (low MP). Group 3 (n = 8) received saline (control). After 6 weeks of coronary occlusion, two-dimensional (2D) echocardiograms were performed, dogs were killed, and their hearts were examined. Regional function, expressed as percent of change in the area of the left ventricular cavity calculated from short-axis 2D echocardiograms, was markedly reduced in the high MP group with the percent of change in the area of 23.3 ± 4.9% compared with 38.4 ± 3.9% in the control group (p < .05) and with 40.5 ± 4.4% in the low MP group (p < .05). A ratio obtained by dividing infarct thickness by noninfarcted wall thickness was lower in the high MP group (0.42 ± 0.04 mean ± SEM) vs both the low MP group (0.95 ± 0.05) and the control group (0.88 ± 0.07; p < .01). Infarct extent was smaller in the low MP group, with the percent of infarcted endocardial circumference of 19.0 ± 1.9% vs the high MP group (30.0 ± 3.5%; p < .05). The percent of infarcted endocardial circumference in the control group was 25.0 ± 1.8% (p = NS). Scars in the treated and control groups were similar histologically and by hydroxyproline content. Therefore, high MP, but not low MP, caused marked scar thinning associated with reduction in regional function, without a change in collagen content.
Topical application of putrescine, a transglutaminase inhibitor, for 3 days directly to rat skin wounds produced a significant average decrease of 48 percent in wound breaking strength in test animals from 8 pairs studied between day 5 and day 10 after wounding. No external or systemic toxic effects of putrescine were seen with localized topical application of 50 mM putrescine for 3 days in any of the test rats (n = 12), and no systemic toxicity was seen in rabbits (n = 4) after topical exposure to 50 mM putrescine for 3 weeks. Quantitation of tritiated fucose incorporation in rat wound explants from 10 pairs of rats revealed that a significant overall decrease in radiolabeled glycoprotein production of 23 percent occurred when putrescine was present; in addition, the fraction of tritiated glycoprotein which was soluble in buffer was significantly increased, while that in the buffer-insoluble fraction decreased. This study suggests that putrescine inhibits tissue transglutaminase-mediated cross-linking of fucoprotein in the extracellular wound matrix and supports a role for this process in the generation of incisional wound strength.
Based on experimental and clinical evidence indicating that the anti-oxidant agent liposomal Cu/Zn superoxide dismutase (Lipsod) is an effective anti-inflammatory drug and possibly might be effective in reducing late radiation-induced tissue injury, a clinical trial using Lipsod to treat long-standing radiation-induced fibrosis (RIF) was begun at the Necker Hospital, Paris in May 1984. Thirty-four patients presenting 42 distinct palpable zones of RIF involving the skin and underlying tissues were treated from May 1984 to January 1986 and followed for an average of 5 years (range, 14-89 months). Lipsod was administered over 3 weeks in twice weekly i.m. injections of 5 mg for a total of 30 mg. Patients underwent two physical examinations by independent physicians at each check-up. Parameters noted included determination of the density of the palpated fibrotic block and the dimensions of the projected cutaneous surface. The extent of change in the fibrotic zone was expressed as the ratio of the sum of the dimensions (L + W) and the ratio of the uncorrected areas (L x W) of the projected cutaneous surface before and after treatment. Changes in density were noted and scored. All patients showed some clinical regression of fibrosis. In most patients, clinically assessable regression begun during the third week of treatment and was maximum by 2 months. The mean decreases in the linear dimensions (L + W) and in the area (L x W) of the projected cutaneous surface were 41 +/- 30% and 57 +/- 26%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
The properties of collagen are affected by the replacement of Pro by imino acid analogues. The structural effect of the low-level local substitution of L-azetidine-2-carboxylic acid (Aze) has been analyzed by computing the energy of CH3CO-(Gly-Pro-Pro)4-NHCH3 triple helices in which a single residue of one strand has been replaced by Aze. When Aze is in position Y of a (Gly-X-Y) unit, low-energy local deformations are introduced in the triple helix, i.e., it becomes more flexible. On the other hand, the flexibility of the triple helix is not increased with Aze in position X. The energy of the triple helix to coil transition is not changed significantly by this amount of substitution. In an earlier study, we have demonstrated that the regular substitution of Aze in every tripeptide distorts or destabilizes the triple helix to a large extent [A. Zagari, G. Némethy, & H. A. Scheraga (1990) Biopolymers, Vol. 30, pp. 967-974]. Thus, it appears that a high level of substitution is required to cause the observed chemical and biological effects of Aze on collagen.
Biologic consequences of silicone implantation may include changes in host connective tissue metabolism. Lysosomal beta-galactosidase (beta-GAL) activity, which is a sensitive marker of fibrotic diseases and may be a useful marker of collagen turnover, was examined in the serum of rats with implanted silicones. No significant difference in spectrofluorometrically determined enzyme activity was demonstrated in rats subjected to dorsal submuscular pocket dissection without implantation and corresponding nonoperative controls. Rats with implanted solid silicone elastomer or free polydimethylsiloxane gel (both components obtained from mammary implant) revealed enhanced activity of serum beta-GAL. Higher enzyme activity was observed in animals with implanted silicone gel with a peak level of 2.73 +/- 0.08 pmol/30 min/ml 16 weeks after implantation. Increased collagen deposition and capsular thickness was demonstrated around implanted gel material as compared with that around elastomer shell. Animals with implanted absorbable and nonabsorbable materials, polyglactin and Teflon (polytetrafluoroethylene), respectively, after initial increase of beta-GAL activity demonstrated enzyme activity within the normal range. Findings indicate that there is enhanced lysosomal beta-GAL activity after silicone implantation in rats. Clinical relevance and its possible significance as a predictor or indicator of local or systemic fibrosis after silicone implantation seems worthy of further investigation.
To study collagen phagocytosis by human extravillous trophoblast.
First-trimester extravillous trophoblastic cell lines and primary trophoblast cell preparations were cultured in vitro with collagen-coated fluorescent latex beads and fluorescent-labeled collagen. Confocal microscopy was used to demonstrate internalization of collagen and beads. The effect of cytochalasin B, temperature, metabolic inhibitors, and cytokines was studied by culturing trophoblast cells with tritiated collagen. Acridine orange was used to stain for lysosomal compartments, and histochemical methods were used to demonstrate acid phosphatase in trophoblast cells.
Both cell lines and primary culture cells internalize collagen and beads. Confocal microscopy unequivocally localized the phagocytosed material to the intracellular compartment. Inhibition by cytochalasin B and culture at 4C of uptake of [3H] collagen suggested that the process was phagocytosis. Cytokines and growth factors did not affect phagocytosis. Lysosomal compartments and acid phosphatase appear to colocalize.
The continuous remodeling and turnover of collagen which occur in a wide variety of tissues under both physiologic and pathologic conditions are thought to be mediated by two pathways: one external (involving release of proteolytic enzymes), and the other internal (involving phagocytosis). Similar remodeling events are likely to occur during trophoblast invasion. Although current views emphasize the importance of the extracellular pathway, we postulate, on the basis of our findings, that both pathways are used, with the internal pathway probably being dominant. We hypothesize that the proteolytic enzymes (extracellular pathway) disrupt collagen matrices, thereby facilitating phagocytosis. It is teleologically sound to conceive of a dominant intracellular pathway, as it allows for more precise control of the process of invasion and is economical, as the products of collagen degradation can be used as energy sources or building blocks.
Various means of skin injury in athletes are examined, supplementing those discussed in Cutaneous Manifestations of Disease Part 1 (November/ December). Skin injury due to mechanical means such as corns, calluses, talon noir (calcaneal petechiae), tennis toe, joggers nipples, and piezogenic pedal papules will be discussed followed by a thorough discussion of environmental means of skin injury. There are a variety of ways that the environment may affect athletes both during competition and years after the competition is finished. The discussion will conclude with a look at performance-enhancing drugs and their effect on an athletes skin, and how the practitioner can better appreciate and perhaps prevent the long-term sequelae of drug abuse.
Hsp47 is a collagen-specific molecular chaperone whose activity has been implicated in the pathogenesis of fibrotic diseases. Here, we describe the development of an assay for screening libraries of chemical compounds for inhibitors of Hsp47. A preliminary screen of 2080 compounds identified four that demonstrated inhibitory activity against Hsp47 in vitro, with IC(50) values ranging from 3 to 27 muM. Compounds identified through this method may provide the basis for development of novel antifibrotic therapeutics.
Immunohistochemical staining with monoclonal antibody to tissue transglutaminase was used to study cryostat sections of human skin wounds. The enzyme was found in acute wounds and chronic hypertrophic scars but not in normal mature scars. Because tissue transglutaminase is responsible for the formation of isopeptide cross-links, a two-stage high-performance liquid chromatographic analysis was used to quantitate the epsilon (gamma-glutamyl) lysine cross-link produced by various types of wound tissues. Eighteen patients with hypertrophic scars between 6 months' and 10 years' duration after injury underwent a double-blind trial with putrescine 50 mmol/L in a eutectic vehicle for 2 months under nonocclusive dressings (Biofill). For the control portion of the same or different scar, sham vehicle and non-occlusive dressing were simultaneously applied. Both scars were harvested at biopsy or elective revision surgery 2 months later. After homogenization and exhaustive proteolysis, digests were studied with the use of high-performance liquid chromatography analysis. The results of treatment were a significant decrease in the levels of isopeptide cross-link formation from 0.018 +/- .006 nmol/micromol amino acids in untreated scars to 0.008 +/- .001 nmol/micromol amino acid in the treated group (p < 0.05). The isopeptide cross-link content in treated scars was nearly as low as that in normal mature scars (0.003 +/- 0.001 nmol/micromol amino acid). These results show that cross-link formation by tissue transglutaminase activity is inhibited during treatment of hypertrophic scar by putrescine. These results support the possible therapeutic use of topical putrescine in the treatment of hypertrophic scar formation.
Genetic defects at the level of transscription and translation may explain why very little type II procollagen is synthesized by patients with type IV of Ehlers-Danlos syndrome. Similary defect synthesis of type I collagen may explain the decrease in the ratio of type I collagen to type III collagen in osteogenesis imperfecta. The synthesized type I collagen in this disorder is unusually sensitive to pepsin, possibly due to an amino acid substitution in the pro-alpha chain of type I procollagen. Defects in intracellular processing include a decrease in lysyl hydroxylase in the type VI variant of Ehlers-Danlos syndrome. Defects in extracellular processing include a decrease in procollagen aminoprotease seen in dermatosparexis and in Ehlers-Danlos syndrome type VII. A deficiency in the enzyme lysyl oxidase is seen in type V Ehlers-Danlos syndrome. A defective cross-linking of collagen may also explain the tissue changes in Marfan's syndrome and in homocysteinuria. Mechanisms for gene selection of collagen may be influenced by environmental factors such as lysosomal enzymes which can change the collagen synthesized by chondrocytes from type II to type I. Scurvy is a classical example of how collagen biosynthesis can be altered at the posttranslational level. Several agents have been used in an attempt to inhibit fibrosis. Glucocorticosteroids can inhibit the synthesis of collagen and other proteins. Penicillamin and beta-aminopropionitrile can inhibit the cross-linking of collagen, the latter via and inhibition of lysyl oxidase. Prolin analogues as cis-hydroxyprolin have been used to inhibit the intracellular post-translational enzymes. Colchicine disrupts microtubules and slows the rate of secretion of procollagen and it may also increase the secretion of collagenase.
NH2-terminal extension peptides of type I and type III procollagens were isolated from dermatosparactic and normal fetal calfskin, respectively. Cell culture experiments showed that the globular domains of the tested procollagen peptides were biologically active but that peptides from the helical region of collagen had no effect. The peptides were added to the incubation medium of calf fibroblasts along with radioactive precursor amino acids, and the amount of newly synthesized collagen was determined. The experiments indicated that procollagen peptides exerted a feedback-like inhibitory effect specific for the synthesis of collagen. Neither degradation of collagen, hydroxylation of collagen alpha chains, nor synthesis of noncollagenous proteins were affected. Synthesis of type II collagen by calf chondrocytes was not reduced. In addition, it was shown that procollagen peptides from calf were equally effective when added to human fibroblast cultures, an observation that could be of considerable medical interest.
Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.
Fibroblasts isolated by enzymic digestion of chick embryo tendons were incubated for several hours in suspension under conditions in which they were in steady state in terms of the synthesis and secretion of procollagen. Under these conditions, the cells synthesized and secreted about 630 microgram of procollagen/10(9) cells/h. The cells were labeled with [14C]proline for 15 to 120 min and then the kinetics of secretion were followed by chasing the label and assaying the 14C-peptides digestible by collagenase in the cells and in the medium. The results demonstrated that secretion of collagenase-digestible peptides did not follow the kinetics of a single first order process but suggested at least two pseudo-first order process with half-times of 14 and 115 min. The [14C]procollagen secreted during 0 to 30 min and 90 to 120 min of chase was the same in terms of the ratio of pro-alpha1 to pro-alpha2 chains, the size of the pro-alpha chains, the extent of interchain disulfide bonding, the extent of prolyl hydroxylation, and the degree of helicity as tested by resistance to pepsin digestion. Addition of ascorbic acid to the incubation medium increased slightly the extent of prolyl hydroxylation but did not alter the kinetics of secretion. The results suggested that the kinetics of secretion are influenced by a two-compartment system in which at least one metabolic pool contributing to the secretory process is present as a "side pocket."
Procollagen synthesized by freshly excised chick enbryo leg tendons is efficiently processed by proteolytic removal of first the amino propeptides and then the carboxyl propeptides. The same processes proceed in confluent short term cell cultures derived from such tendon explants; in sparse cultures cleavage of the amino propeptides predominates. Separate amino and carboxyl procollagen peptidase activities were demonstrated by specific assays in enzymes obtained from cell culture media by ammonium sulfate precipitation, ion exchange chromatography, and velocity sedimentation. Both enzymes are inhibited by EDTA and 1:10 phenanthroline but not by inhibitors of serine proteases. Evidence is provided that the proteolytic scissions are specific and similar to the physiologically occurring processes. The collagen telopeptides left after cutting by the enzymes can participate in lysyl oxidase-induced cross-linking. The enzymes can remove propeptides from cross-linked procollagens without destroying these links which occur through telopeptides. The enzymes act on the separated amino and carboxyl portions of procollagen fragmented by vertebrate collagenase and can act on procollagens which have been associated as well as on molecules in solution.
Fibroblasts isolated by enzymic digestion of chick embryo tendons have previously been used to examine the kinetics for the secretion of procollagen (Kao, W. W.-Y., Berg, R. A., and Prockop, D. J. (1977) J. Biol. Chem. 252, 8391-8397). The results indicated that the kinetics approximated the sum of two first order processes with half-times of 14 and 115 min. Here, the same fibroblasts were incubated in the presence of 1.53 mM cis-4-hydroxyproline, an analogue of proline, or in the presence of 0.3 mM alpha,alpha'-dipyridyl, an inhibitor of prolyl hydroxylase, so that the cells synthesized procollagen which could not assume a triple helical conformation characteristic of procollagen. Measurements of the secretion of nonhelical procollagen indicated that the kinetics for secretion differed from the kinetics for the secretion of procollagen and approximated a single first order process with a half-time of approximately 130 min. The nonhelical procollagen synthesized and secreted in the presence of either cis-4-hydroxyproline or alpha,alpha'-dipyridyl consisted of disulfide-bonded pro gamma chains of type I procollagen. The results suggested that the intracellular nonhelical procollagen was present in a single metabolic pool and secretion from this pool occurred with a different rate-limiting step than for helical procollagen. Further results indicated that nonhelical procollagen had a high affinity for prolyl hydroxylase and the affinity for the enzyme was greatly reduced if the procollagen was allowed to assume the triple helical conformation characteristic of normal procollagen. The results are consistent with the hypothesis that the secretion of procollagen is influenced by its conformation-dependent interaction with prolyl hydroxylase or other post-translational enzymes.
The neutral salt-soluble collagen which accumulates in the tissues of animals treated with penicillamine does not differ from
normal in amino acid composition, specific optical rotation, and melting temperature. It has higher intrinsic viscosity, possibly
due to a contamination with higher molecular weight aggregates originating from the depolymerization of insoluble collagen.
In contrast to the aldehyde-deficient lathyritic collagen, the penicillamine collagen has an aldehyde content greater than
normal and rapidly forms stable cross-links in vitro.
Binding studies, involving film and equilibrium dialysis, revealed a significant interaction between collagen and compounds
with a free α-aminothiol structure. The binding capacity of various collagens tested is proportional to their aldehyde content,
and reduction with NaBH4 eliminates this interaction. Reduction of these aldehydes, amidination of the ε-amino groups of lysine and hydroxylysine,
or addition of sodium bisulfite (10-4 m) causes neutral salt-soluble collagen to behave like lathyritic collagen.
It is postulated that the inhibition of cross-linking caused by penicillamine in vivo and in vitro involves a reversible interaction with the aldehydes present in tropocollagen to form a thiazolidine type complex, since
compounds with adjacent free sulfhydryl and amino groups are necessary for activity.
The solubilizing effect exhibited by α-amino-β-thiols on an incompletely cross-linked form of insoluble collagen as well as
the generation of soluble collagen with a high aldehyde content can be attributed to the splitting of a Schiff's base intermediate.
The structure which is responsible for the initial stabilization of the collagen fiber can be reduced with NaBH4, rendering the collagen insoluble in penicillamine.
Rats with subcutaneously implanted polyvinylalcohol sponges and with inflicted skin incision wounds received a single injection of β-aminopropionitrile at four dosages ranging from 40 to 1 mg per 100 g body wt. At 6, 24, and 48 hr after the injection, the activity of lysyl oxidase, extractability of collagen into neutral salt, and bursting strength of the wound were tested. We found that even the lowest dose of BAPN tested significantly inhibited lysyl oxidase activity for 6 hr; with larger dosages the inhibition lasted longer, at 40 mg BAPN, at least 48 hr. The magnitude and duration of lysyl oxidase inhibition by BAPN was reflected in the extractability of collagen and bursting strength of the wound. The data suggest that minimal dose of BAPN would be clinically effective if either the metabolism of the drug is reduced (by monoaminoxidase inhibitors) or by sustained release of BAPN from delivery systems.
A model system consisting of highly purified lysyl oxidase and reconstituted lathyritic chick bone collagen fibrils was used to study the effect of collagen cross-linking on collagen degradation by mammalian collagenase. The results indicate that synthesis of approx. 0.1 Schiff-base cross-link per collagen molecule results in a 2--3-fold resistance to human synovial collagenase when compared with un-cross-linked controls or samples incubated in the presence of beta-aminopropionitrile to inhibit cross-linking. These results confirm previous studies utilizing artificially cross-linked collagens, or collagens isolated as insoluble material after cross-linking in vivo, and suggest that increased resistance to collagenase may be one of the earliest effects of cross-linking in vivo. The extent of intermolecular cross-linking among collagen fibrils may provide a mechanism for regulating the rate of collagen catabolism relative to synthesis in normal and pathological conditions.
To assess potential abnormalities in collagen metabolism in systemic scleroderma, skin fibroblast lines from patients with this disease were established and compared to control cell lines derived from healthy subjects. For studies on the biosynthesis of procollagen, the cells were incubated with [(14)C]proline in a medium supplemented with ascorbic acid and beta-aminopropionitrile, and the synthesis of nondialyzable [(14)C]hydroxyproline, in relation to DNA or cell protein, was taken as an index of procollagen formation. Five of eight scleroderma fibroblast cell lines demonstrated procollagen biosynthesis rates significantly higher than the controls, and the mean rate of procollagen synthesis by scleroderma fibroblasts was about twice that of the control cells. Control experiments demonstrated that the specific activity of the intracellular free proline was not different in scleroderma and control fibroblasts, and the mean population doubling times of the scleroderma and the control fibroblast cell lines were the same. The relative synthesis of the genetically distinct procollagens was examined by isolating type I and type III procollagens from the cell culture medium using DEAE-cellulose chromatography. The ratios of type I/III procollagens in scleroderma cell lines did not differ from the controls. The helical stability of the collagenous portion of type I and type III procollagens, estimated by the resistance of (14)C-collagen to limited proteolytic digestion with pepsin under nondenaturing conditions, was the same in both scleroderma and control cultures. The capacity of the cells to synthesize enzymatically active and immunologically reacting collagenase was also studied; no marked differences in these parameters could be observed. The results suggest that cultured skin fibroblasts from patients with scleroderma demonstrate a metabolic abnormality expressed as increased synthesis of type I and type III procollagens in a normal ratio. This abnormality may play a role in the excessive accumulation of collagen in the skin and other organs affected in scleroderma.
Long-term treatment of patients with generalized progressive scleroderma by means of inhibitors of connective-tissue biosynthesis brings about total or subtotal regression of dermal sclerosis in 40.8%, partial regression in 33.1%, arrest of progression without regression in 14.8%, while in 11.3% it had no effect whatsoever. The drugs used were D-penicillamine, benzyl-penicillin-diethyl-aminoethylesterhydro-iodide, glutamine, hydralazine, chlorpromazine, L-dopa, diphenylhydantoin, and corticosteroids. Disease activity before, during and after treatment was indicated by the urinary fractions of high-molecular hydroxyproline and hydroxylysine containing peptides and of uronic acid, break-down products of collagen and acid glycosaminoglycans of connective-tissue ground substance. The prospects were better for young patients than for old, for those with a short history than for the longstanding disease cases, and for those having a large total dose than for those who had less. If left untreated, scleroderma progresses inexorably.
The biosynthesis of collagen involves a number of unique post-translational modifications which are catalyzed by many specific enzymes. The main steps in collagen biosynthesis are transcription and translation, hydroxylations of prolyl and lysyl residues, glycosylations of hydroxylysyl residues, chain association and disulphide bonding, triple helix formation, secretion of procollagen into the extracellular matrix, conversion of procollagen into collagen, specific aggregation of collagen molecules and crosslink formation. Information about these modifications has rapidly increased during recent years, and initial information is available about the regulation and possibilities of specific pharmacological control of collagen biosynthesis at the level of these stages. Elucidation of the biochemical defect in an inborn error of collagen biosynthesis in man was reported for the first time in 1972 and subsequently several additional defects have been characterized. Alterations in collagen biosynthesis are also found during growth and ageing, as well as in many acquired pathological states: data about the nature of such changes in now rapidly accumulating.
The effects of lead upon collagen synthesis and proline hydroxylation were examined in the Swiss mouse 3T6 fibroblast. The results indicate that lead reduces proline hydroxylation in stationary phase cultures of 3T6 cells, resulting in increased cellular retention of unhydroxylated procollagen. Inhibition of proline hydroxylation by lead was prevented by increasing the extracellular molar ratio. Interference by lead in the hydroxylation of proline in logarithmic phase cultures of 3T6 cells resulted in increases in the 0.5 n HClO4 soluble/insoluble hydroxyproline ratio. This was attributed to an increase in the rate of breakdown of lead-induced unhydroxylated procollagen. Kinetic analysis of the lead-iron interaction with proline hydroxylase suggests that the mechanism is competitive.
Human skin procollagenase has been isolated, in pure form, from the medium of fibroblasts cultured in the presence or absence of added serum. Purification was achieved using a combination of cation-exchange (phosphocellulose or carboxymethylcellulose) and gel-filtration chromatography. Two forms (60 000 and 55 000 daltons) of the procollagenase were detected by electrophoresis in sodium dodecyl sulfatepolyacrylamide gels and could be separated by chromatography on Ultrogel AcA-44. Each form was converted to active enzyme by trypsin, producing species of 50 000 and 45 000 daltons, respectively. An autoactivation process also occurred, which yielded active enzyme without a detectable change in molecular weight. Procollagenase also was found in organ cultures of human skin but only when serum was added to the medium. This suggests that a serum-inhibitable proteolytic system is present in these cultures which, like trypsin, converts procollagenase to the active enzyme forms that can be isolated from serum-free organ culture medium. The collagenase species obtained from either fibroblast or organ culture medium were chromatographically and electrophoretically identical.
When chick frontal bone cells in culture were exposed to d,l-3,4 dehydroproline, the specific activity of prolyl hydroxylase was markedly reduced, but the concentration of the protein antigenically related to prolyl hydroxylase was not decreased. The specific activity of purified prolyl hydroxylase from cells grown in d,l-3,4 dehydroproline was significantly lower than that of control cells. Preincubation of a homogeneous preparation of chick embryo prolyl hydroxylase with collagenous peptides containing [14C]d,l-3,4 dehydroproline resulted in a time-dependent decrease in the enzymatic activity. These observations suggest that the in vivo reduction in prolyl hydroxylase activity by dehydroproline could be either due to an interaction of the enzyme with collagenous peptides containing dehydroproline and/or the synthesis of an aberrant form of prolyl hydroxylase with decreased enzymatic activity.
The purpose of this review is to present the mechanisms for normal collagen biosynthesis and degradation in the skin. Within this framework, cutaneous diseases resulting from aberrations in these mechanisms will be discussed.
Soft tissue x-ray techniques were used to measure skin thickness as influenced by the chronic usage of topical corticosteroids. In a double-blind study commercial preparations of 1% hydrocortisone (HC), 0.1% triamcinolone acetonide (TA), and a placebo cream were compared for their ability to produce atrophy in normal human forearm skin. After 8 weeks of topical application of the creams, only TA produced clinically apparent atrophy. The average percent decreases in skin thickness measured after 8 weeks of treatment with placebo, HC, or TA were 6.0%, 6.0%, and 17.1%, respectively. During the first week after cessation of treatment the clinical appearance of the skin began to improve and by 1 month all treated skin areas had essentially returned to pretreatment thickness.
Since collagen biosynthesis was last reviewed in the Journal in 1972, much new information has been gained about the relatively complex pathway by which it is synthesized. The authors consider this new information and some of its consequences in terms of the following questions: (1) What is the structural information in the collagen molecule, and how does it determine the structure of the collagen fibril? (2) What are the structure and function of the precursor form of collagen known as procollagen? (3) How is procollagen synthesized and used to assemble collagen fibrils? (4) How do defects in biosynthesis explain genetic diseases of collagen? (5) How is collagen synthesis regulated under normal conditions and in acquired diseases?
Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts. However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.
Familial cutaneous collagenoma is an inherited condition characterized by the presence of multiple dermal nodules symmetrically distributed on the trunk and upper arms. In this study, six patients, the proband, his four siblings and a niece, representing a kindred of fifty-two subjects, were examined for aymptomatic cutaneous nodules mainly on the back and chest. The individual lesions varying from a few millimetres to several centimetres in size, were indurated, and showed minimal epidermal changes. Histologically, the nodules were characterized by an excessive accumulation of dense, coarse collagen fibres in the dermis. The elastic fibres appeared diminished in number, and in some areas they were abnormally thin and fragmented. The lesions, therefore, were connective tissue naevi of the collagen type. On the basis of the family history and histological observations the patients were diagnosed as having familial cutaneous collagenoma. Examination of the family pedigree indicated that the dermal nodules in familial cutaneous collagenoma were inherited in an autosomal dominant pattern. It was also observed that the lesions had an onset at the age of 15 to 19 years, and their number increased significantly during pregnancy. It is conceivable that familial cutaneous collagenoma is an inherited condition whose expression may be under a hormonal control.
Peptides prepared from the amino termini of pro alpha 1(I) and pro alpha 1(III) collagen chains inhibit the production of pro alpha 1(I) and pro alpha 2 by rat calvaria rna in a reticulocyte cell-free system. The synthesis of other proteins was not altered, suggesting a specific effect on collagen production. Various peptides from the helical region of the alpha 1(I) chain did not alter translation. These studies, taken together with earlier studies showing inhibition of collagen synthesis by cells in culture receiving the amino-terminal peptides, are consistent with a regulatory function in collagen synthesis for the amino-terminal peptides from procollagen.
Previously, several proline analogs have shown to be incorporated into protein and, in particular, into procollagen polypeptides. Here a new technique was used to determine the extent to which two proline analogs, cis-4-hydroxy-l-proline and cis-4-fluoro-l-proline, replaced proline and hydroxyproline in newly synthesized pro-α and pro-γ chains of procollagen. Matrix-free chick embryo tendon cells, when incubated with 1.53 mm, cis-4-hydroxy-l-proline, synthesized collagenous polypeptides in which from 13 to 19% of the total imino acid residues were replaced with the analog. Incubation of cells with 1.50 mm, cis-4-fluoro-l-proline resulted in the synthesis of polypeptides in which 27% of the imino acid residues were replaced by the analog. With lower concentrations, proportionally less of the analog was incorporated into protein. The observations here extend previous indications that proline analogs in relatively low concentrations may have a specific effect on the synthesis of collagen.
The development of late radiation fibrosis in rat lung after a single dose 750, 1000, 1250, or 1500 rad of ..gamma.. radiation was studied. The concentration of hydroxyproline was measured to evaluate collagen deposition in irradiated and control lungs. At 120 days after irradiation a linear dose--response curve can be established. Colchicine, known to modify collagen secretion and deposition, was used to modify lung fibrosis after irradiation. It was found that collagen deposition can be reduced only if colchicine is injected when the lung collagen is being synthesized, namely around 90 days after irradiation with these doses. Lung compliance at intervals up to 150 days after irradiation is reduced mainly as a result of increase in lung weight, not collagen deposition.
PRIMARY cultures of cells from various tissues have become important
tools for many experimental studies, but attempts to prepare primary
cultures of specialised cell types are frequently frustrated by the
tendency of fibroblasts to grow more rapidly than other cells. During
studies on procollagen biosynthesis by fibroblasts, it has been found
that if the cells are incubated with any one of four proline analogues,
the analogues are incorporated into protein and the incorporation of the
analogues into procollagen polypeptides prevents the chains from folding
into a triple helicial conformation1-7. We report here that
if fibroblasts are grown in the presence of the proline analogue
cis-hydroxyproline, their rate of growth is markedly limited. This may
provide a simple technique for removing fibroblasts from cultures of
mixed cell populations.
Contraction and epithelization are two phenomena of "wound healing" retarded by corticosteroids. It is unclear how these agents affect collagen synthesis and wound remodeling. The methods used by several authors to conclude that steroids inhibit collagen synthesis are questioned. Therefore, collagen synthesis was measured in cultured steroid-treated chick embryo calvaria, 5 day open wounds in treated rats, and intralesionally injected human keloids. Collagen synthesis was suppressed only by long-term administration of massive systemic doses of a sustained release form of methylprednisolone (Depo-Medrol). Large, intermittent doses of corticosteroids (triamcinolone and Solu-Medrol) did not alter collagen synthesis. Because human keloids become softer and smaller following intralesional administration of triamcinolone without lowering the normally high rate of collagen synthesis, we hypothesize that corticosteroids enhance collagen degradation.
During the early stage of generalized morphea there is an inflammatory reaction involving primarily the fat around the eccrine sweat glands and the subcutaneous tissue. The cellular infiltrate consist of lymphocytes, plasma cells, and histocytic type cells. Lymphoid follicles with germinal centers were also noted. The inflammatory stage is followed by the replacement of the subcutaneous tissue by hyalinized connective tissue. This process is probably responsible for the skin induration in this disease.
The administration of cortisol, oxyphenylbutazone and indomethacin to rats results in a marked and abrupt loss of cutaneous collagen from these animals. This collagen loss was associated with the appearance of both collagenolytic and proteolytic activities in the extracellular, extrafibrillar compartment of the skin. Cycloheximide and 5,5-diphenylhydantoin pretreatment inhibited both the cutaneous collagen losses and the appearance of these enzyme activities in the skin.Kinetic studies have shown that within 4 hr after anti-inflammatory drug administration, peak concentrations of both collagenolytic and proteolytic activities were reached in the skin. These enzymatic activities were profoundly depressed by simultaneous administration of puromycin, cycloheximide and actinomycin-D.Monolayers of cultured strain-L fibroblasts neither contained nor released either proteolytic or collagenolytic activities. Within 4 hr after administration of cortisol, idomethacin or oxyphenylbutazone, both types of enzymatic activities appeared within these cell cultures.
The effect of hydrocortisone acetate, fluocinolone acetonide, fluclorolone acetonide, betamethasone-17-valerate and fluprednyliden-21-acetate on collagen biosynthesis was studied both in vivo and in vitro. In experiments in vivo, test substances and [14C]proline were injected on the chorioallantoic membrane of 11-day-old chick embryos. In experiments in vivo, chick embryo tibiae were incubated in a medium containing the corticosteroids to be tested and [14C]proline. In both types of experiments, the formation of [14C]hydroxyproline was taken as a measure of the rate of collagen biosynthesis. In addition, the effect of these corticosteroids on the activity of protocollagen proline hydroxylase was tested.[14C]hydroxyproline formation in vivo was inhibited by all the corticosteroids tested, and no clear difference in the degree of inhibition could be observed between the various corticosteroids. in vitro, betamethasone-17-valerate inhibited hydroxyproline formation more than the other substances tested. The concentration of corticosteroids required to inhibit collagen formation was considerably higher in vivo than in vitro. The corticosteroids tested did not affect the activity of protocollagen proline hydroxylase in the bones. In all these experiments, total protein synthesis, measured as incorporation of total 14C-radioactivity into the bones, was inhibited by corticosteroids to the same extent as hydroxyproline formation. The results seem to suggest that corticosteroids inhibit collagen biosynthesis by inhibiting the formation of polypeptide precursors of collagen.
Cells liberated from the tendons of chick embryos by controlled enzymic digestion were used to examine the synthesis of the polypeptide chains of collagen separately from the secretion of collagen in the precursor form known as procollagen. When the cells were incubated under anaerobic conditions, the synthesis of collagen hydroxyproline was completely inhibited but the rate of protein synthesis remained about the same as the control rate for about 30 min. Under these conditions the cells synthesized the non-hydroxylated precursor known as protocollagen and the protocollagen was retained in the cells. By incubating the cells with [14C]proline under anaerobic conditions and then exposing the system to air, it was possible to follow the intracellular hydroxylation of [14C]protocollagen to [14C]procollagen. The conformation of the intracellular [14C]protocollagen and [14C]procollagen was examined by proteolytic digestion under conditions in which the triple-helical portion of collagen is resistant to digestion. When digestion with either pepsin or α-chymotrypsin was carried out at either 30 ° C or 37 °C, all of the [14C]protocollagen was digested. After the same protein was hydroxylated to [14C]procollagen by exposing the cells to O2 a large fraction of the protein became resistant to proteolysis and was secreted. The results suggested that the intracellular hydroxylation of collagen polypeptides converts them from a non-helical to a helical form and that the helical conformation probably is required for the protein to be secreted at an optimal rate.
Hydrocortisone and dexamethasone (9α-fluoro, 16α-methyl prednisolone) prevent the appearance of collagenase in cultures of normal human skin, human rheumatoid synovium and rat uterus. Hydrocortisone is maximally inhibiting at 10−7M and dexamethasone at 10−8M in culture medium. Neither steroid is an inhibitor of enzyme activity. The loss of collagenase activity in cultured tissue is not accompanied by detectable inhibition of protein synthesis. Reduction of enzyme activity in culture medium is concomitant with a parallel cessation of tissue collagen degradation, indicating that the tissue fails to produce active collagenase in the presence of physiologic levels of glucocorticoids.
We have used intradermal injections of corticosteroids into normal and ultraviolet light (UVL)-induced inflamed human skin to evaluate the inherent atrophy producing potential as well as the vasoconstrictor potency of selected compounds. Two corticosteroids, desonide and triamcinolone acetonide, which differ only by the presence of a fluorine atom, exhibited similar vasoconstrictor potency but only the fluorine containing steroid produced severe persistent atrophy. Hydrocortisone 17-valerate and desonide produced a mild, transient atrophy while hydrocortisone produced none. Triamcinolone acetonide and desonide were more effective than hydrocortisone and hydrocortisone 17-valerate in producing vasocontriction in this model system of inflammation. The production of atrophy always greater in the UVL-induced inflamed skin than in normal control skin.
Cultures of dividing skin fibroblasts from normal and sclerodermatous human skin have permitted estimations of soluble collagen concentration, net collagen accumulation, cell-doubling times, and the comparison of morphologic and ultrastructural characteristics. In vitro, the scleroderma fibroblast produces more soluble collagen, synthesizes collagen more rapidly, and fourfold more of its protein synthetic activity is directed to collagen production than in the normal skin fibroblast. Cell-doubling times and morphologic and ultrastructural observations of cells in culture have not provided clues to the nature of the biologic defect in the regulation or activation of collagen synthesis by the scleroderma fibroblast.
Fibroblasts were incubated with analogs of proline or lysine and the thermal stability of procollagen molecules containing the analogs was investigated using pepsin digestion at different temperatures as an enzymatic probe of conformation. The procollagens containing either 4-cis-hydroxy-l-proline, 3,4-dehydroproline, or 4,5-trans-dehydrolysine were less stable than normal procollagen and these abnormal collagens were largely in a non-triplehelical conformation within the cells at 37 °C. These results support the idea that procollagen molecules which are not in a triple-helical conformation are not secreted at a normal rate. Procollagens containing both 4,5-trans-dehydrolysine and a proline analog were much less stable than molecules containing a single type of analog. This result suggests that simultaneous administration of both types of analogs may have a greater effect on collagen accumulation in whole-animal experiments than administration of a single analog.
The effects of hydrocortisone acetate, fluocinolone acetonide, fluclorolone acetonide, betamethasone-17-valerate, fluprednyliden-21-acetate and flumethasone pivalate on the biosynthesis of human skin collagen were studied in vitro. Skin specimens were incubated in a medium containing a test substance and radioactive proline, and the formation of radioactive hydroxyproline in nondialysable proteins was taken as an index of the rate of collagen biosynthesis.Hydroxyproline formation was inhibited by all the corticosteroids tested in concentrations of 30 μg/ml or higher. The effect on hydroxyproline formation was smallest with hydrocortisone acetate and most pronounced with betamethasone-17-valerate in all concentrations used. In general, the corticosteroid-induced inhibition of collagen biosynthesis was found to be dose-dependent. The differences in the degree to which collagen formation is inhibited by the different corticosteroids may have relevance to the extent of the local side-effects, such as atrophy of skin, reported to be produced by fluorinated corticosteroids.
L-Azetidine-2-carboxylic acid (AZC), an analogue of proline, has been shown to partially ameliorate hepatic cirrhosis induced in rats by CCl(4). AZC caused a diminution in formation of collagen in the liver accompanied by a relative decrease in the pool of free proline. The synthesis of noncollagenous proteins in the livers of treated rats did not appear to be affected.
Chick embryo cartilage incubated with dl-3,4-dehydroproline (3,4-dehydroproline) synthesized collagen containing the analogue in place of proline. It was not possible
to determine the degree to which 3,4-dehydroproline replaced proline, but under comparable conditions 14C-dl-3,4-dehydroproline was incorporated at about one-fifth the rate of 14C-l-proline. The hydroxyproline content of the collagen was decreased because the analogue replaced prolyl residues which were
normally hydroxylated, and because the proline still incorporated was not hydroxylated to the same extent as in control tissues.
With tissues incubated with 14C-proline and 40 to 100 µg per ml of dl-3,4-dehydroproline the hydroxylation of 14C-proline in collagen was decreased to one-third to one-half of the control value. The hydroxylation of 14C-lysine in collagen was depressed to about the same degree. The analogue did not inhibit protocollagen proline hydroxylase,
and gel filtration indicated the proteins synthesized in the presence of the analogue were of the same size as proteins from
control tissues. The 14C-hydroxyproline content of collagen containing 3,4-dehydroproline could not be increased by incubating it with excess protocollagen
proline hydroxylase.
Collagenous polypeptides prepared by incubating cartilage with puromycin and 3,4-dehydroproline formed stable enzyme-substrate
complexes similar to complexes formed with polypeptides which did not contain 3,4-dehydroproline. However, the polypeptides
containing 3,4-dehydroproline differed from polypeptides which did not contain the analogue in that they remained attached
to the enzyme after incubation with excess enzyme and cofactors. This observation may explain the decreased hydroxylation
of collagen containing the analogue in that tight binding of the enzyme to a 3,4-dehydroproline residue at one position in
a substrate polypeptide may sterically hinder the hydroxylation of adjacent prolyl or lysyl residues in the same molecule.
Autoradiographic experiments indicated that collagen containing 3,4-dehydroproline was not extruded from cartilage cells as
rapidly as normal collagen.