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Effect of putrescine on tissue transglutaminase activity in wounds: Decreased breaking strength and increased matrix fucoprotein solubility
Abstract
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.
... 40 Woodley et al. 41 identified an increase in lysyl oxidase-mediated collagen cross-linking that parallels contraction of fibroblast-populated collagen lattices in vitro. Although there has been extensive research into the role of transglutaminase in wound healing, [42][43][44][45] its importance in contraction has not previously been determined. ...
... However, putrescine showed a marked dose-dependent reduction in P1NP concentration. This is consistent with the published literature, suggesting a reduction in collagen synthesis after topical putrescine treatment of healing wounds 44 and hypertrophic scars. 88 The authors attributed the decrease in collagen synthesis to decreased collagen cross-linking, resulting in an increased pool of soluble collagen and therefore exerting a negative feedback effect on collagen synthesis. ...
... An increase in collagen synthesis initially appears contradictory to published evidence; inhibition of transglutaminase would be expected to increase the pool of soluble collagen and hence decrease collagen synthesis. 44,84 However, transglutaminase knockout cells synthesize significantly increased amounts of collagen III and IV in mesangial cells but deposit reduced amounts of mature insoluble collagen compared with wild-type cells. 89 Nardacci et al. 90 studied carbon tetrachloride-induced liver fibrosis in TG2 knockout mice. ...
Skin graft contraction leading to loss of joint mobility and cosmetic deformity remains a major clinical problem. In this study we used a tissue-engineered model of human skin, based on sterilized human adult dermis seeded with keratinocytes and fibroblasts, which contracts by up to 60% over 28 days in vitro, as a model to investigate the mechanism of skin contraction. Pharmacologic agents modifying collagen synthesis, degradation, and cross-linking were examined for their effect on contraction. Collagen synthesis and degradation were determined using immunoassay techniques. The results show that skin contraction was not dependent on inhibition of collagen synthesis or stimulation of collagen degradation, but was related to collagen remodelling. Thus, reducing dermal pliability with glutaraldehyde inhibited the ability of cells to contract the dermis. So did inhibition of matrix metalloproteinases and inhibition of lysyl oxidase-mediated collagen cross-linking, but not transglutaminase-mediated cross-linking. In summary, this in vitro model of human skin has allowed us to identify specific cross-linking pathways as possible pharmacologic targets for prevention of graft contracture in vivo.
... However, unlike other transglutaminases, TGII binds guanine nucleotides in a 1:1 ratio and hydrolyzes GTP (4,5). This GTPase activity is independent of transglutaminase activity (6) and has been shown to mediate ␣ 1 -adrenergic receptor (␣ 1 AR) stimulation of phospholipase C ␦1, increasing inositol phosphate turnover in TGII-transfected cells (7)(8)(9)(10)(11)(12)(13). Numerous in vitro studies implicate TGII in a wide variety of biological processes, including regulation of cell growth and differentiation (14 -16), apoptosis (17), and tissue repair (18,19), as well as signal transduction via ␣ 1 AR. The role of TGII, however, in regulation of these events in relevant target tissues remains unclear. ...
... The TGII overexpressing mice do have, however, cardiac hypertrophy, diffuse interstitial fibrosis, and depressed ventricular function at rest with normal responsiveness to AR stimulation. The remodeling observed is consistent with the transglutaminase activity of TGII, which in other systems has been implicated in cell growth (15), matrix formation (36), and tissue repair processes (18,19). As has been suggested by studies in heart failure (30,31), alterations in expression or function of TGII may play a role in pathogenesis of the syndrome; based on the current body of work, we conclude that this is probably due to the transglutaminase activity of TGII rather than signaling through PLC. ...
Tissue type transglutaminase (TGII, also known as Gh) has been considered a multifunctional protein, with both transglutaminase and GTPase activity. The role of the latter function,
which is proposed as a coupling mechanism between α1-adrenergic receptors and phospholipase C (PLC), is not well defined. TGII was overexpressed in transgenic mice in a cardiac
specific manner to delineated relevant signaling pathways and their consequences in the heart. Cardiac transglutaminase activity
in the highest expressing line was ∼37-fold greater than in nontransgenic lines. However, in vivo signaling to PLC, as assessed by inositol phosphate turnover in [3H]myoinositol organ bath atrial preparations, was not increased in the TGII mice at base line or in response to α1-adrenergic receptor stimulation; nor was protein kinase Cα (PKCα) or PKCε activity enhanced in the TGII transgenic mice.
This is in contrast to mice moderately (∼5-fold) overexpressing Gαq, where inositol phosphate turnover and PKC activity were found to be clearly enhanced. TGII overexpression resulted in a
remodeling of the heart with mild hypertrophy, elevated expression of β-myosin heavy chain and α-skeletal actin genes, and
diffuse interstitial fibrosis. Resting ventricular function was depressed, but responsiveness to β-agonist was not impaired.
This set of pathophysiologic findings is distinct from that evoked by overexpression of Gαq. We conclude that TGII acts in the heart primarily as a transglutaminase, and modulation of this function results in unique
pathologic sequelae. Evidence for TGII acting as a G-protein-like transducer of receptor signaling to PLC in the heart is
not supported by these studies.
... However, SPM enhanced the polymerization at 4 mM (Fig. 6). The strongest inhibitory effect of PUT could depend on the short carbon chain length of PUT (C 4 ) compared with SPD (C 7 ) and SPM (C 10 ), because the short length of polyamines could be easy to enter into residues in peptide chain (Dolynchuk et al., 1994;Wu et al., 2000). Considering these different effects of PUT, SPD, and After the TGase-catalyzed reaction, the reaction mixture was mixed with the 50 L of loading buffer containing 2-mercaptoethanol, and heated for 3 min at 100°C. ...
... However, when ODC activity increases during cell growth, the amount of PUT that is a product of ODC increases (Tabor and Tabor, 1984;Pegg, 1986) and can inhibit protein polymerization by TGase via inhibition of formation of -(␥-glutamyl)PUT. However, SPM promotes formation of bis(␥-glutamyl)polyamines by TGase in protein polymerization (Dolynchuk et al., 1994;Wu et al., 2000). ...
It is generally accepted that the nucleosome structure is not static, and that alternative conformations are adopted in response to several stimuli associated with the different functions. Histones are substrates for transglutaminase (TGase), and polymerized histone and polyamine binding histone have been suggested to play important roles in nucleus. We examined whether histone polymerization catalyzed by TGase was influenced by polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM). PUT inhibited histone polymerization, and SPD slightly prevented it. However, SPM slightly enhanced histone polymerization. These results indicate that the nuclear accumulation of the polyamines may play an important role in nuclear remodeling by histone modification. We speculate that histone cross-linking by TGase may be involved in the chromatin structure. Also, we propose that histone cross-linking by TGase may be responsible for the changes in DNA function such as transcription and replication and that TGase may be involved in cell growth and differentiation.
... Recent studies regarding scar formation in skin demonstrated transglutaminase-induced irreversible cross-links of ECM, which cannot be digested by any known enzyme. 18,19 In keeping with this, excessive accumulation of ECM in PVR membranes could result from the formation of irreversible ECM cross-links in fibrocellular membranes. ...
... Previously, it has been reported that topical treatment of incisional skin wounds with putrescine, a competitive inhibitor of transglutaminase cross-linking, caused decreased woundbreaking strength. 19 Furthermore, systemic administration of MDC or spermidine, also competitive inhibitors of transglutaminase activity, decreased healing of gastric and duodenal stress ulcers. 59 Some pharmaceutical compounds seem to inhibit transglutaminase, not only competitively, but irreversibly. ...
Proliferative vitreoretinopathy (PVR) is characterized by the development of epi- and subretinal fibrocellular membranes containing modified retinal pigment epithelial (RPE) cells among others. In the present study, the role of transglutaminases in accumulation of extracellular matrix (ECM) proteins in these membranes was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes.
PVR membranes were incubated with dansyl-cadaverine to demonstrate active transglutaminase. Localization of tissue transglutaminase (tTgase), its reaction product epsilon-(gamma-glutamyl)-lysine, and fibronectin was investigated immunohistochemically. Colocalization was studied with a confocal laser scanning microscope. PVR membranes were also analyzed by RT-PCR for the presence of tTgase mRNA. In vitro, RPE cells were treated with transforming growth factor-beta2 (TGF-beta2), basic fibroblast growth factor, interleukin-6, and interleukin-1beta. Their effect was studied using immunohistochemistry and Northern and Western blot analyses.
Transglutaminase activity and expression of tTgase were present in all PVR membranes. Staining was most prominent at the rim of the membranes. The enzyme was colocalized with epsilon-(gamma-glutamyl)-lysine and fibronectin. No staining differences were found between epi- and subretinal membranes. Although native RPE cells contained only a basal level of tTgase mRNA, the expression and activity of tTgase was increased under culture conditions and further stimulated by TGF-beta2 treatment.
The findings demonstrate that in PVR membranes tTgase is present and functionally active. The amount and activity of this enzyme appears to be related to the differentiation state of the RPE cells and their stimulation by TGF-beta2, a growth factor known to be increased in the vitreous of PVR. Intervention at this pathway may open a new approach for PVR prevention and therapy.
... The scratch wound closure assays used in this study suggest a TG2 inhibitor may carry a risk to normal wound healing, as suggested by prior studies using various fibroblasts from TG2 KO mice and TG2 siRNA studies [19]. Additionally, evidence from a study using topical application of putrescine (a non-specific TG inhibitor) on rat dermal wounds suggests that TG2 loss affects wound tensile strength [53]. To understand this risk with zampilimab, we carried out dermal punch and linear wound investigations in treated cynomolgus monkeys and microscopically examined the wounds over time. ...
Fibrotic remodeling is the primary driver of functional loss in chronic kidney disease, with no specific anti-fibrotic agent available for clinical use. Transglutaminase 2 (TG2), a wound response enzyme that irreversibly crosslinks extracellular matrix proteins causing dysregulation of extracellular matrix turnover, is a well-characterized anti-fibrotic target in the kidney. We describe the humanization and characterization of two anti-TG2 monoclonal antibodies (zampilimab [hDC1/UCB7858] and BB7) that inhibit crosslinking by TG2 in human in vitro and rabbit/cynomolgus monkey in vivo models of chronic kidney disease. Determination of zampilimab half-maximal inhibitory concentration (IC50) against recombinant human TG2 was undertaken using the KxD assay and determination of dissociation constant (Kd) by surface plasmon resonance. Efficacy in vitro was established using a primary human renal epithelial cell model of tubulointerstitial fibrosis, to assess mature deposited extracellular matrix proteins. Proof of concept in vivo used a cynomolgus monkey unilateral ureteral obstruction model of chronic kidney disease. Zampilimab inhibited TG2 crosslinking transamidation activity with an IC50 of 0.25 nM and Kd of <50 pM. In cell culture, zampilimab inhibited extracellular TG2 activity (IC50 119 nM) and dramatically reduced transforming growth factor-β1-driven accumulation of multiple extracellular matrix proteins including collagens I, III, IV, V, and fibronectin. Intravenous administration of BB7 in rabbits resulted in a 68% reduction in fibrotic index at Day 25 post-unilateral ureteral obstruction. Weekly intravenous administration of zampilimab in cynomolgus monkeys with unilateral ureteral obstruction reduced fibrosis at 4 weeks by >50%, with no safety signals. Our data support the clinical investigation of zampilimab for the treatment of kidney fibrosis.
... Bowness et al. (16,17) reported TG activity was present at sites of wound healing and that inhibition of TG by putrescine caused decreased breaking strength and increased solubility of the repairing wound tissue (18). However, the cellular expression, distribution, and metabolic fate of the TG at sites of wound healing in relationship to the expression of cytokines and migration of inflammatory cells were not investigated. ...
... Mice lacking the gene for TG2 (TG2KO) do not show an evident abnormal skin phenotype, possibly because other members of TGs family could compensate the loss of this enzyme (De Laurenzi and Melino 2001;Nanda et al. 2001). Several experimental evidences indicate that TG2 could have a role in tissue repair (Dolynchuk et al. 1994;Telci and Griffin 2006;Verderio et al. 2004). Wound healing studies performed on rat punch biopsy model, demonstrate an increased TG2 expression and activity at the site of wound throughout the healing (Haroon et al. 1999). ...
Transglutaminases (TGs) are a family of enzymes that catalyse the formation of isopeptide bonds between the γ-carboxamide groups of glutamine residues and the ε-amino groups of lysine residues leading to cross-linking reactions among proteins. Four members, TG1, TG2, TG3, and TG5, of the nine mammalian enzymes are expressed in the skin. TG1, TG3 and TG5 crosslinking properties are fundamental for cornified envelope assembly. In contrast, the role of TG2 in keratinization has never been studied at biochemical level in vivo. In this study, taking advantage of the TG2 knock-out (KO) and TG1 heterozygous mice, we generated and characterized the epidermis of TG1-TG2 double knock-out (DKO) mice. We performed morphological analysis of the epidermis and evaluation of the expression of differentiation markers. In addition, we performed analysis of the amino acid composition from isolated corneocytes. We found a significant change in amino acid composition in TG1KO cornified cell envelopes (CEs) while TG2KO amino acid composition was similar to wild-type CEs. Our results confirm a key role of TG1 in skin differentiation and CE assembly and demonstrate that TG2 is not essential for CE assembly and skin formation.
... Failure of the termination of the wound response, will eventually lead to the dysfunction and failure of organs. The crosslinking activity of TG2 was a focus in the formation of fibrosis in a number of different organs, including lung (Griffin et al., 1978a), liver (Kuncio et al., 1998) , heart (Small et al., 1999), vasculature (Dolynchuk et al., 1994), and kidney (Johnson et al., 2003;Johnson et al., 1997b;Skill et al., 2001). ...
This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without proper acknowledgement.
... Arginase catalyses the conversion of arginine to ornithine and urea. Ornithine is an essential precursor for collagen and polyamines synthesis [18], both required for wound healing processes [19][20][21][22][23][24][25]. Moreover, arginase seems to influence the immune response, another important contributor involved in tissue repair. ...
Arginine supplementation has been identified as advantageous in experimental wound healing. However, the mechanisms underlying this beneficial effect in tissue repair remain unresolved. Animal studies suggest that the beneficial role of arginine supplementation is mediated, at least in part through NO. The latter component mediates processes involved in tissue repair, including angiogenesis, epithelialization and collagen formation. This prospective study is performed to investigate arginine metabolism in acute surgical wounds in man. Expression of enzymes, known to be involved in arginine metabolism, was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation. Plasma and wound fluid levels of arginine metabolites (ornithine, citrulline, nitrate and nitrite = NOx) were measured using High Performance Liquid Chromatography. Expression of iNOS, eNOS, arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue. Arginase-1 concentration was measured in plasma and wound fluid using ELISA. Arginase-2 was determined using Western blot analysis. We observed increased levels of citrulline, ornithine, NOx and arginase-1 in wound fluid when compared with plasma. Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma. iNOS was expressed by neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells upon wounding, whereas eNOS reactivity was observed in endothelial cells and fibroblasts. Arginase-1 was expressed in neutrophils post-wounding, while arginase-2 staining was observed in endothelial cells, keratinocytes, fibroblasts, macrophages and neutrophils. For the first time, human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin.
... In fact, many studies support this function of transglutaminases in wound healing situations. It has been reported that the topical treatment of incisional skin wounds with putrescine, resulting in competitive inhibition of transglutaminase cross-linking, causes decreased wound breaking strength (43). Furthermore, the systemic administration of MDC or spermidine, also competitive inhibitors of transglutaminase cross-linking, decreases healing of gastric and duodenal stress ulcers (44). ...
Since transglutaminases create covalent gamma-glutamyl-epsilon-lysine cross-links between extracellular matrix proteins they are prime candidates for stabilizing tissue during wound healing. Therefore, we studied the temporo-spatial expression of transglutaminase activity in skin regenerating from cultured epithelial autografts in severely burned children by the specific incorporation of monodansylcadaverine into cryostat sections from skin biopsies obtained between 5 d to 17 mo after grafting. The dansyl label was subsequently immunolocalized in the epidermis, dermal connective tissue, and along the basement membrane. Incubation of cryosections of normal and regenerating skin with purified tissue transglutaminase confirmed the dermo-epidermal junction and the papillary dermis as targets for this enzyme and revealed that in regenerating skin transamidation of the basement membrane zone was completed only 4-5 mo after grafting. Immunoelectron microscopy revealed that three distinct regions on the central portion of anchoring fibrils were positive for monodansylcadaverine in normal skin which were negative during the initial phase of de novo formation of anchoring fibrils in regenerating skin. Biochemically, we identified collagen VII as potential substrate for tissue transglutaminase. Thus, tissue transglutaminase appears to play an important role not only in cross-linking of the papillary dermis but also of the dermo-epidermal junction in particular.
... The activity of tTG requires calcium; factor XIII also requires activation by the serine protease thrombin. 1,2 tTG activity has been demonstrated to play a role in stabilization of the basement membrane and adhesion of cells, 3,4 which are important processes in wound healing, angiogenesis, and bone remodeling. [5][6][7] In particular, tTG has been shown to cross-link components of the extracellular matrix (ECM), including fibronectin, vitronectin, laminin, and collagen. ...
Atherosclerosis is characterized by thickening of the vessel wall, smooth muscle cell proliferation, macrophage infiltration, and deposition of a fibrin network. Transglutaminases are a family of enzymes catalyzing the formation of stable covalent cross-links between proteins. Here, we show that tissue transglutaminase (tTG) synthesis by human umbilical vein endothelial cells is upregulated by thrombin, the serine protease that causes fibrin formation and many cellular inflammatory effects. Thrombin upregulated tTG 2-fold at the mRNA and protein level. Cellular cross-linking activity was increased to an even greater extent; antibody to tTG neutralized the increased activity. The effect on tTG expression required active thrombin and was mediated mainly through protease-activated receptor-1, a thrombin receptor. Increased tTG antigen and activity were evident in human umbilical vein endothelial cells and extracellular matrix in situ. Thrombin treatment also led to a cellular redistribution of tTG. Normal vessel wall stained positively for tTG in the smooth muscle cells and in the subendothelium. The intensity of staining increased in vessel walls with plaque, where there was a striking increase in tTG in the smooth muscle cells immediately below the plaque. These studies indicate a role for tTG in the stabilization of atherosclerotic plaques and suggest that its local expression can be controlled by thrombin.
... Despite the fact that type I collagen was not evaluated in this study, thi s delayed overproduction of collagen might suggest that tretinoin cream (0. 1 % ) has a more significant effect on the synthesis of type I collagen [21][22][23][24] which plays an important role on the later stages of wound healing [25]. This suggestion might be supported with the dominance of thick collagen bundles indicating type I collagen versııs indefınite reticular fibers which mostly contain collagen type III [26,27]. It has also been reported that the effect of vitamin A was most pronounced in the final epithelial phase of the healing period [12]. in the study of P'.utkin, vitamin A derivatives directly applied to open cırcular wounds, were found to stimulate wound healing [l]. ...
Adapalene is a new naphthoic acid derivative with strong retinoid agonistic pharmacological properties. We propose that adapalene might contribute to the wound repairing process as is detected with retinoids. In this controlled study, the effects of topical adapalene, tretinoin and collagenase on full-thickness wound healing were compared in an animal model. Thirty-two adult male Wistar-Albino rats were used in the study. Two circular, full-thickness wounds were made for each animal with a standard 8-mm punch biopsy, on both sides of the midline on the back. No treatment was given to Group I rats (n:8) which comprised the control group. Tretinoin cream (0.1%) was applied topically in Group II (n:8), adapalene gel (0.1%) in Group III (n:8), and collagenase ointment in Group IV (n:8) once daily. On day 7, the wounds were photographed to measure the wound surface area. The wounds on the left side of each animal were excised on day 7, for histopathologic and biochemical examination. The treatments were continued for the right side wounds up to 14 days when the same procedure was repeated. In Group II, a significant decrease in hydroxylproline (HP) levels was detected at day 7 (p = 0.018), and an increase at day 14 (p = 0.002) compared to the control group. HP results revealed no difference either in Group III nor in Group IV versus control at day 7 or 14. However, findings of improved healing were more prominent in Groups II and III than the other groups in histopathologic examination. In conclusion, tretinoin and adapalene contributed to the wound healing process resulting in an enhancement of collagen production, angiogenesis and granulation tissue formation.
... Ethanol has been shown to decrease putrescine levels in the liver, and the administration of putrescine or its precursors, alanine or glutamine, abrogates this inhibition of liver regeneration by ethanol administration [16][17][18]. The mechanism by which putrescine abolishes this effect of ethanol on regeneration may be that putrescine acts as a competitive substrate inhibitor of tTGase activity [33]. This action of putrescine is important because tTG has been shown to inhibit cell proliferation in other systems. ...
Ubiquitous tissue transglutaminase (tTG) is one member of the large transglutaminase (TG) family, which catalyze posttranslational modification of proteins by establishing epsilon(gamma-glutamyl)lysine cross-linking and/or covalent incorporation of polyamines. The unique characteristics of tTG include: (1) possessing both cross-linking activity and GTPase activity; (2) functioning as a G protein; and (3) participating in the signal transduction of alpha1-adrenergic receptor coupling. A growing body of literature suggests that increased tTG levels in the cytosolic or nuclear compartments contribute to the apoptotic process, and lines of evidence exist that nuclear translocation and cross-linking of transcriptional factor Sp1 may represent the underlying mechanisms of these proapoptotic effects of tTG. Our studies indicate that tTG GTPase activation may be responsible for enhanced hepatocyte proliferation, whereas, its tTGase activity may cause increased apoptosis. Moreover, it appears that tTG cross-linking activity contributes to hepatic fibrogenesis in animal models and in human liver disease. Understanding the roles of tTG in the pathogenesis of liver disease could facilitate the development of new treatment regimens.
... TG2 is the most widely distributed form of TG in tissues and accumulates in cells undergoing terminal differentiation and at sites of tissue injury and inflammation (Aeschlimann and Thomazy, 2000). In skin, TG2 is an essential component of the wound healing program, and transamidation of extracellular components is required for the de novo formation of a functional dermo-epidermal cohesion apparatus (Dolynchuk et al., 1994;Raghunath et al., 1996;Haroon et al., 1999;Mearns et al., 2002). However, excessive crosslinking by TG2 has also been implicated in the pathogenesis of diseases related to wound healing and neovascularization, most notably fibrogenic reactions in various organs (Aeschlimann and Thomazy, 2000). ...
Tissue transglutaminase (TG2) affects cell-matrix interactions in cell spreading, migration and extracellular matrix (ECM) reorganisation. Using fibroblasts deficient in TG2 or overexpressing normal or crosslinking-deficient enzyme, we show that the extracellular crosslinking activity and intracellular G-protein function in signal transduction contribute differentially to regulation of cell-matrix interactions. TG2-deficient cells displayed normal attachment but delayed spreading on ECM substrata and defects in motility unrelated to crosslinking. Blocking antibodies to TG2 failed to induce similar defects in normal fibroblasts. TG2-deficient fibroblasts had defects in focal adhesion turnover and stress fibre formation, showed changes in focal adhesion kinase (FAK) phosphorylation and failed to activate protein kinase C alpha (PKCalpha). Phospholipase C (PLC) and PKCalpha inhibitors blocked spreading of normal fibroblasts whilst PKC activators induced spreading in TG2-deficient cells. In contrast, ECM remodelling was not only compromised by TG2 deficiency but also by overexpression of dominant negative enzyme and TG inhibitors. TG2 activity increased matrix tension and was required for membrane type 1-MMP (MT1-MMP)-dependent activation of MMP-2. Our results demonstrate that TG2 is involved in the control of dynamic adhesion formation in cell spreading and migration via regulation of phospholipase C activity. By virtue of its crosslinking activity, the enzyme plays a central role in regulating ECM remodelling.
... The stabilization of newly formed ECM is thought to be largely accomplished through the actions of tissue transglutaminase (or transglutaminase II) (Ricard-Blum et al, 1996;Grenard et al, 2001). The activity of this enzyme is markedly increased during wound healing, substantially effecting both the breaking strength of wounds and the solubility of ECM proteins (Bowness et al, 1987b(Bowness et al, , 1988Dolynchuk et al, 1994;Haroon et al, 1999). Tissue transglutaminase cross-links proteins through the formation of isopeptide bonds between reactive g-glutaminyl groups in certain specific proteins and the e-amino group of lysine in other proteins. ...
Failure of apoptosis has been postulated to cause the hypercellularity and thus excess scar-tissue formation of hypertrophic scars (HTS). Here, we have examined the susceptibility of fibroblasts derived from normal or HTS to apoptosis induced during collagen-gel contraction, a wound-healing model. Normal scar (NS) fibroblasts underwent significant apoptosis (>40% total) in contractile collagen, whereas apoptosis was not detected in HTS cells. This inability was specific to apoptosis induced by contractile collagen because apoptosis could be induced using diverse modalities. Since chronic fibrotic tissue is known to be excessively cross-linked, we next examined whether collagen matrix that had been conditioned by HTS fibroblasts became refractory to enzymatic breakdown and indeed, found that it is resistant to breakdown by both collagenase D and matrix metalloproteinase-2. Newly formed extracellular matrix is stabilized by the enzyme, tissue transglutaminase, which we demonstrated to be overexpressed by HTS fibroblasts in vivo and in vitro. Reducing tissue transglutaminase activity in collagen gels containing HTS fibroblasts permitted induction of apoptosis on gel contraction, whereas increasing enzymic activity in NS cell-containing gels completely abrogated collagen-contraction-induced-apoptosis. Together, these observations show that HTS fibroblasts exhibit resistance to a specific form of apoptosis elicited by contraction of collagen gels, and that this phenomenon is dependent on excess activity of cell surface tissue transglutaminase.
The presence of antibiotic resistance has increased the urgency for more effective treatments of bacterial infections. Biofilm formation has complicated this issue as biofilm bacteria become tolerant to antibiotics due to environmental factors such as nutrient deprivation and adhesion. In septic arthritis, a disease with an 11% mortality rate, bacteria in synovial fluid organize into floating, protein-rich, bacterial aggregates (mm-cm) that display depressed metabolism and antibiotic tolerance. In this study, Staphylococcus aureus (S. aureus), which is the most common pathogen in septic arthritis, was tested against different inhibitors that modulate bacterial surface protein availability and that should decrease bacterial aggregation. One of these, berberine, a quaternary ammonium compound, was found to reduce bacterial counts by 3-7 logs in human synovial fluid (aggregating medium) with no effect in tryptic soy broth (TSB, non-aggregating). Unlike traditional antibiotics, the bactericidal activity of berberine appeared to be independent of bacterial metabolism. To elucidate the mechanism, we used synovial fluid fractionation, targeted MRSA transposon insertion mutants, dyes to assess changes in membrane potential (DiSC3(5)) and membrane permeability (propidium iodide (PI)), colony counting, and fluorescence spectroscopy. We showed that berberine's activity was dependent on an alkaline pH and berberine killed both methicillin-sensitive S. aureus and MRSA in alkaline media (pH 8.5-9.0; p < 0.0001 vs. same pH controls). Under these alkaline conditions, berberine localized to S. aureus where berberine was isolated in cytoplasmic (∼95%) and DNA (∼5%) fractions. Importantly, berberine increased bacterial cell membrane permeability, and disrupted the proton motive force, suggesting a mechanism whereby it may be able to synergize with other antibacterial compounds under less harsh conditions. We suggest that berberine, which is cheap and readily available, can be made into an effective treatment.
Arginase (ARG) represents an important evolutionarily conserved enzyme that is expressed by multiple cell types in the skin. Arg acts as the mediator of the last step of the urea cycle, thus providing protection against excessive ammonia under homeostatic conditions through the production of L-ornithine and urea. L-ornithine represents the intersection point between the ARG-dependent pathways and the urea cycle, therefore contributing to cell detoxification, proliferation and collagen production. The ARG pathways help balance pro- and anti-inflammatory responses in the context of wound healing. However, local and systemic dysfunctionalities of the ARG pathways have been shown to contribute to the hindrance of the healing process and the occurrence of chronic wounds. This review discusses the functions of ARG in macrophages and fibroblasts while detailing the deleterious implications of a malfunctioning ARG enzyme in chronic skin conditions such as leg ulcers. The review also highlights how ARG links with the microbiota and how this impacts on infected chronic wounds. Lastly, the review depicts chronic wound treatments targeting the ARG pathway, alongside future diagnosis and treatment perspectives.
In cardiac myocytes, stimulation of the alpha(1)-adrenoreceptor(AR) leads to a hypertrophic phenotype. The Gh protein(transglutaminase II, TGII) is tissue type transglutaminase that transmits the alpha(1B)-AR signal with GTPase activity. Recent evidence suggests that integrin activation, in concert with G protein activation, might be essential for cardiomyocyte growth. In this study, our objectives were to evaluate how Gh and integrin interact to promote cardiomyocyte hypertrophy and to identify Gh molecular determinants and downstream signals involved. First, we observed the selectivity of alpha-adrenoceptors in norepinephrine(NE)-induced cardiac hypertrophy by protein synthesis and estimation of the constitutive genes MLC-2 and ß-MHC. NE-mediated hypertrophic responses activated ERK1, 2 and their upstream regulators MEK1, 2, which were inhibited by siRNA for Gh. We also identified significant activation of ß1-integrin in NE-induced cardiomyocyte hypertrophy. In addition, FAK and Shc, a regulator of FAK, were directly related with integrin and Gh protein in NE-induced cardiomyocyte hypertrophy. The activation of FAK and Shc were suppressed by siRNA for Gh. In conclusion, the hypertrophic response induced by norepinephrine in cardiomyocytes occurs through ß1-Integrin/Shc/FAK/MEK1, 2/ERKs signal pathways via α1-AR/Gh.
Transglutaminases (Tg) are a family of calcium-dependent enzymes that catalyse the crosslinking of polypeptide chains, including those of extracellular matrix (ECM) proteins, through the formation of e(v-glutamyl) lysine bonds. This leads to the formation of protein polymers that are highly resistant to degradation. Consequently, tissue Tg (tTg) has been associated with ECM protein deposition in fibrotic diseases such as pulmonary fibrosis and atherosclerosis. hi this study, we have investigated the involvement of tTg in the development of kidney fibrosis in adult male Wistar rats submitted to subtotal nephrectomy (SNx). Groups of 4 to 6 rats were sacrified on days 7, 30, 90, and 120 after SNx. As previously described these rats developed progressive glomerulosclerosis and tubulo-interstitial fibrosis. The tissue level of e(y-glutamyi) lysine crosslink (as determined by exhaustive proteolytic digestion followed by cation exchange HPLC) increased from 347 + 94 pmol/mg protein (M.+S.E.M.) in controls to 1324 + 143 pmol/mg 90 days after SNx (p<0.01). Levels of crosslink correlated well with the renal fibrosis score throughout the 120 observation days (r=0.78, pO.Ol). Tissue homogenates showed no significant change in overall tTg activity (14C-putrescine incorporation assay) during this period. Immunohistochemistry demonstrated that this large increase in crosslink was predominantly in the cytoplasm of tubular cells and that these cells also showed a large increase in tTg antigen. Some of the cells high in crosslink and tTg displayed the morphology of a cell undergoing apoptosis, although these cells did not frequently show DNA cleavage (in-situ end labelling) typically associated with apoptosis. We noted an association between tTg, crosslink and renal scarring in rats submitted to SNx. We would like to postulate a role for tissue transglutamrnase in the development of experimental renal fibrosis.
Introduction:
Keloids are fibroproliferative disorders that are characterized by histological accumulation of collagens and fibroblasts, refractory clinical symptoms such as itching, topical invasiveness, and frequent postsurgical recurrence. At present, to treat or prevent keloids, new drugs are currently being designed and the pharmaceutical indications of known drugs are being expanded.
Areas covered:
The current pharmacological interventions for keloids are mainly described on the basis of the various hypotheses on keloid etiology and the keloid ingredients that are targeted. These interventions include angiotension-converting enzyme inhibitors and calcium-channel blockers (based on hypertension hypothesis), selective estrogen receptor modulator (based on endocrinological hypothesis), vitamins and essential fatty acids (based on immunonutritional hypothesis), and transglutaminase inhibitor (based on metabolic hypothesis). Drugs that directly target the reduction or destruction of the major extracellular matrix or cellular constituents of keloids are also included. Besides, drugs that indirectly modulate the biochemical microenvironment are described. These include growth factors, immunomodulators, and anti-inflammation and anti-allergy drugs.
Expert opinion:
Due to the unclear etiology of keloids and the lack of animal models, efficient, reliable, and specific pharmaceutical interventions for keloids continue to be lacking. The reliability of current data and clinical observations must be strengthened by large-scale, randomized, controlled clinical trials.
Angiogenesis is vital to tumor growth and metastasis. The scope of this grant was to study in detail the role of Tissue transgiutaminase (tTG) during wound healing and tumorigenesis. In the first year of the grant proposal, we have described the expression, localization, molecular form and tTG's association with other major determinants of wound healing and tumorigenesis. Our findings clearly show that tTG is readily unregulated in wound healing and rat mammary adenocarcinoma and is associated with endothelial and inflammatory cells. Hypoxia, Vascular endothelial growth factor, Transforming growth factor heta and Tumor necrosisi factor alpha are also unregulated alongside tTG in those cells. tTG is quickly proteolysed in the tissues and that may have important consequences as tTG can hydrolyze ATP/GTP in its fragmented form. We are submitting these findings to Journal of Clinical investigation Proceedings of National Academy of Sciences and American Journal of Pathology where we have detailed the potential significance of these observations. We have also addressed most of the tasks detailed in the statement of work and are now set to proceed with the rest of the project.
The mechanisms by which ethanol inhibits hepatocyte proliferation have been a source of some considerable investigation. Our
studies have suggested a possible role for tissue transglutaminase (tTG) in this process. Others have shown that tTG has two
distinctly different functions: it catalyzes protein cross-linking, which can lead to apoptosis and enhancement of extracellular
matrix stability, and it can function as a G protein (Gαh). Under that circumstance, we speculated that the cross-linking activity would be decreased and that it would function to
enhance hepatocyte proliferation in response to adrenergic stimulation. Ethanol treatment inhibited hepatocyte proliferation
and led to enhanced tTG cross-linking activity, whereas treatment of hepatocytes with an α1 adrenergic agonist, phenylephrine,
enhanced hepatocyte proliferation while decreasing tTG cross-linking. However, phenylephrine treatment of several hepatoma
cell lines had no effect on cellular proliferation or tTG cross-linking activity, and of note, Northern blot analysis demonstrated
that whereas primary hepatocytes had high levels of the α1β adrenergic receptor (α1BAR) mRNA, the hepatoma cell lines did
not have this mRNA. When the Hep G2 cell line was stably transduced with an expression vector containing the α1BR cDNA, the cell line responded to phenylephrine
treatment with enhanced proliferation and with decreased tTG cross-linking activity. Ethanol treatment of the α1BAR-transfected
cells suppressed the phospholipase C-mediated signaling pathways, as detected in the phenylephrine-induced Ca2+ response. These results suggest that phenylephrine stimulation of hepatocyte proliferation appears to be occurring through
the α1BAR, which is known to be coupled with the tTG G protein moiety, Gαh, and that tTG appears to play a significant role in either enhancing or inhibiting hepatocyte proliferation, depending on
its cellular location and on whether it functions as a cross-linking enzyme or a G protein.
Previous studies indicated that tissue transglutaminase plays a role in the cross-linking of type III procollagen in wound matrices and that this may be inhibited by 50 mM putrescine in vitro. For this reason, the clinical effect of 50 mM putrescine in a eutectic vehicle (Fibrostat) was studied in this phase II double-blind crossover study in 43 patients. Twenty of the patients had had recent surgery and were studied for product safety rather than efficacy. No toxic effects were observed in this group of patients, and only 1 of the 23 unoperated patients had a rash during treatment. The observed effect of Fibrostat versus sham treatment of 1 month's duration in active hypertrophic scar was a significant improvement of hypertrophy in 23 patients during the Fibrostat treatment arm, regardless of the order in which treatment was received. It is suggested that Fibrostat is a safe therapeutic agent for treatment of hypertrophic scar. Clinical examples to illustrate its use are given.
Transglutaminase (TGase) activities were measured in rat tissues 1-7 days after intraperitoneal injection of saline or lipopolysaccharide (LPS) and in the cells and media from pre-confluent human fibroblasts cultured for two days in the presence or absence of LPS. epsilon (gamma-Glutamyl)lysine and [3H]putrescine-labelled gamma-glutamyl derivatives in extracellular and cellular fibroblast proteins were also measured. Three effects of LPS were observed. Firstly, total TGase activity is greater in the tissues from the LPS-injected animals, with the maximum increase occurring at 1 day in dermis, epidermis and liver, at 5 days in the aorta and, after a decrease at 2-5 days, at 7 days in the panniculus muscle. Secondly, the fraction of the total activity which is buffer-extractable is greater on days 1 and/or 2 in all the tissues from the LPS-injected rats. Thirdly, in cultures of human fibroblasts, LPS increases that fraction of bound [3H]putrescine and of TGase and its gamma-glutamylamine products which occurs in the extracellular medium. In addition, a higher concentration of TGase-derived crosslinks was found in extracellular as opposed to intracellular proteins. In conjunction with previous findings in skin wound healing and in atherosclerosis these results support the concept of an extracellular function for tissue TGase and indicate that there is a widespread association of increases in TGase and its extracellular products with inflammation and the healing or fibrotic processes which follow it.
Tissue transglutaminase is a calcium-dependent enzyme that catalyzes the cross-linking of polypeptide chains, including those of extracellular matrix (ECM) proteins, through the formation of epsilon-(gamma-glutamyl) lysine bonds. This crosslinking leads to the formation of protein polymers that are highly resistant to degradation. As a consequence, the enzyme has been implicated in the deposition of ECM protein in fibrotic diseases such as pulmonary fibrosis and atherosclerosis. In this study, we have investigated the involvement of tissue transglutaminase in the development of kidney fibrosis in adult male Wistar rats submitted to subtotal nephrectomy (SNx). Groups of six rats were killed on days 7, 30, 90, and 120 after SNx. As previously described, these rats developed progressive glomerulosclerosis and tubulo-interstitial fibrosis. The tissue level of epsilon-(gamma-glutamyl) lysine cross-link (as determined by exhaustive proteolytic digestion followed by cation exchange chromatography) increased from 3.47+/- 0.94 (mean+/-SEM) in controls to 13.24+/-1.43 nmol/g protein 90 d after SNx, P
Tissue transglutaminase (TG) is an enzyme that stabilizes the structure of tissues by covalently ligating extracellular matrix molecules. Expression and localization of TG are not well established during wound healing. We performed punch biopsy wounds on anesthetized rats and monitored the wound healing process by histological and immunohistochemical methods. The TG antigen and activity are expressed at sites of neovascularization in the provisional fibrin matrix within 24 h of wounding. Endothelial cells, macrophages, and skeletal muscle cells expressed TG throughout the healing process. The TG antigen within the wound was active in vivo based on the detection of isopeptide bonds. The TG antigen increased four- to fivefold by day 3 postwounding and was proteolytically degraded. TG expression occurred in association with TGF-beta, TNF-alpha, IL-6, and VEGF production in the wound. Recombinant TG increased vessel length density (a measure of angiogenesis) when applied topically in rat dorsal skin flap window chambers. We have established that TG is an important tissue stabilizing enzyme that is active during wound healing and can function to promote angiogenesis.
Supplemental arginine has been shown to enhance wound healing, in particular collagen synthesis. Ornithine is the main metabolite of arginine in the urea cycle and shares many of the biopharmacologic effects of arginine. The present study examines the effect of ornithine supplementation on wound healing and attempts to describe its possible mechanism.
Wild type (WT) and iNOS knockout (KO) mice were randomized to receive either normal chow and tap water or chow and water each supplemented with 0.5% ornithine (w/w). All animals underwent a midline dorsal skin incision with implantation of polyvinyl alcohol sponges into subcutaneous pockets. On postoperative day 14 the animals were sacrificed. The dorsal wound was harvested for breaking strength determination while the wound sponges were assayed for hydroxyproline content, total wound fluid amino acid, and nitrite/nitrate (NOx) concentration.
Dietary ornithine supplementation enhanced wound breaking strength and collagen deposition in both WT and KO mice. This was accompanied by increased wound fluid proline and ornithine levels but not arginine, citrulline, or NOx levels.
The results from this study demonstrate that ornithine supplementation enhances wound healing in both WT and KO mice. This suggests that ornithine's effect on wound healing is independent of the iNOS pathway.
Transglutaminase (TGase) has been reported to be involved in the regulation of cell growth. We examined the effects of polyamines on TGase activity. The polymerization of casein was inhibited by putrescine (PUT) and spermidine (SPD). On the other hand, polymerization of N,N-dimethylcasein was increased by spermine (SPM) and SPD. These results suggested polyamines played two distinct roles as inhibitor and promoter for TGase-catalyzed protein polymerization.
A complex series of events involving inflammation, cell migration and proliferation, ECM stabilisation and remodelling, neovascularisation and apoptosis are crucial to the tissue response to injury. Wound healing involves the dynamic interactions of multiple cells types with components of the extracellular matrix (ECM) and growth factors. Impaired wound healing as a consequence of aging, injury or disease may lead to serious disabilities and poor quality of life. Abnormal wound healing may also lead to inflammatory and fibrotic conditions (such as renal and pulmonary fibrosis). Therefore identification of the molecular events underlying wound repair is essential to develop new effective treatments in support to patients and the wound care sector.
Recent advances in the understating of the physiological functions of tissue transglutaminase a multi functional protein cross-linking enzyme which stabilises tissues have demonstrated that its biological activities interrelate with wound healing phases at multiple levels. This review describes our view of the function of tissue transglutaminase in wound repair under normal and pathological situations and highlights its potential as a strategic therapeutic target in the development of new treatments to improve wound healing and prevent scarring.
Several experimental studies have shown the importance of arginine in wound healing. However, little is known about its role in human wound healing. In this study, we investigated arginine metabolism in impaired wound healing.
Twenty patients with chronic wounds and 10 patients with acute wounds were included in a prospective study. Amino acids, nitrate/nitrite, and arginase concentrations were determined in plasma and wound fluid using high-performance liquid chromatography and enzyme-linked immunosorbent assay. Chronic wounds were divided into two groups: noninfected chronic wounds (n = 11) and infected chronic wounds (n = 9), based on quantitative bacterial analysis of wound fluid samples.
Plasma arginine levels, next to total plasma amino acid levels, were significantly decreased in patients with infected chronic wounds compared with patients having acute or noninfected wounds. Citrulline and ornithine levels were significantly increased in infected chronic wounds and related to decreased nitrate/nitrite levels, whereas wound fluid arginine levels were similar in all groups. In addition, wound fluid arginase levels of infected chronic wounds were significantly enhanced.
This study demonstrates that patients with infected chronic wounds have decreased plasma arginine levels and suggests enhanced arginine conversion in the wound. In contrast to noninfected chronic wounds, arginine seems to be mainly metabolized by arginase in infected chronic wounds. In conclusion, our hypothesis is that impaired wound healing is related to an altered arginine usage.
Scarring of the filtering bleb site is the main cause of failure in glaucoma filtration surgery. In the present study, the role of tissue transglutaminase (tTgase) in the accumulation of extracellular matrix (ECM) proteins in these scars was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes.
Expression of tTgase, its reaction product epsilon-(gamma-glutamyl)-lysine, and fibronectin and their colocalization were investigated immunohistochemically in failed blebs and in an in vitro trabeculectomy model. Failed blebs were analyzed by RT-PCR for the presence of tTgase mRNA. Human Tenon fibroblasts (HTFs) were treated with transforming growth factor-beta2 (TGF-beta2). The effect was studied with immunohistochemistry, Northern blot analysis, and Western blot analysis. tTgase activity was assayed by incorporation of biotinylated cadaverine into fibronectin.
Expression of tTgase and epsilon-(gamma-glutamyl)-lysine was present in all failed blebs. Staining was most prominent at the rim of the Tenon cyst. In the in vitro trabeculectomy model, tTgase and epsilon-(gamma-glutamyl)-lysine were barely present at the incision side of the flap but were perspicuously increased by TGF-beta2 treatment. Enzyme and its reaction product were colocalized with fibronectin. Cultured HTFs contained a basal level of tTgase mRNA. After treatment with TGF-beta2, expression and activity of tTgase significantly increased.
The findings demonstrated that tTgase is present and functionally active in failed blebs. Expression and activity of tTgase appeared to be stimulated by TGF-beta2, a growth factor known to be increased in primary open angle glaucoma. Intervention at this pathway might open a new approach to prevent scarring after glaucoma filtration surgery.
Accelerated wound healing should be possible if one could eliminate the lag phase, a period of time when the wound is quite weak while the building blocks of collagen are being brought into the wound. In the present experiments injection of autogenous blood into the skin and fascia of rats initiated a response characterized by vasodilatation, accumulation of inflammatory cells, and proliferation of capillaries and fibroblasts. During the first week of healing, wounds made in areas injected with blood 2 days prior to wounding were up to twice as strong as controls.
The problem of the biochemical quantification of long term human wound healing was approached by measuring collagen synthesis in reincisions using specific radioimmunoassays for the wound fluid concentrations of the carboxyterminal propeptide of type I procollagen (PICP) and the aminoterminal propeptide of type III procollagen (PIIINP). First-day wound fluid PICP concentration after reincising a 3 week old scar was 25 times higher than the mean value in 20 reference standard incisions but scars older than 3 months did not show this difference. Wound fluid taken in subsequent days demonstrated that the initial acceleration of synthesis disappeared by the fourth day. When wound fluid PIIINP was assessed, high concentrations were found in reincisions of wounds for up to 5 months after the previous operation. The acceleration was also lost more slowly during the first postoperative week. The duration of a high rate of type I collagen synthesis compares well with studies in experimental wounds which show increased gain of strength if they are made not more than 6 weeks after previous surgery. The longer activity of the metabolism of type III collagen related antigens could reflect their function in the regulation of collagen fibril formation.
To study the glycoproteins and transglutaminase substrates involved in healing, wounds were made in the skin of anesthetized rats with a biopsy punch. Explants made 1-5 days later were incubated with [3H]-labelled putrescine, fucose or proline. As compared with unwounded skin there was an increased incorporation of label which was greatest at 3 days. Incubation for various times suggests that the incorporation of fucose and proline is dependent on protein synthesis, whereas putrescine is incorporated into preformed proteins. Putrescine and fucose label polypeptides with an Mr of about 45,000 before and 14,000 after reduction. These correspond in size with the aminopropeptide of type III collagen. Other labelled material of higher molecular weight is partly degraded to similar polypeptides on collagenase digestion. Much of the [3H]putrescine in the polypeptides is in the form of gamma-glutamyl putrescine. It is hypothesized that isopeptide linkage of the aminopropeptide III occurs in wound healing.
One of the most important and least understood problems in medicine is that of what variables govern the rate and the quality
of the wound healing process. It is highly desirable to know the effects of external or internal agents such as drugs, radiation,
and physical irritants on the healing process with a view toward accelerating and improving it. Since mechanical strength
is a basic criterion, it is important that standardized methods for the physical testing, analysis and interpretation of results
be devised. This paper presents a basic technique for the determination of the mechanical properties of wounded and unwounded
skin.
Experimental wounds in rats were tested for breaking strength at 4, 7, 11, 14, and 21 days to determine whether incisions treated with homogenous fibronectin differed from control incisions. At 7, 11, 14, and 21 days after wounding, the fibronectin-treated incisions were significantly stronger than the control incisions.
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.
The interaction of fibronectin with native collagen during collagen fibril formation was investigated. Fibronectin prepared from serum, or from the cell surface, bound to the forming collagen fibrils while less fibronectin bound to preformed fibers. Denatured collagen competed with native collagen in binding fibronectin. Fibronectin delayed the precipitation of collagen fibrils but did not alter the total amount of fibrils formed. Fibronectin which was heated to 30 degrees C for 30 min did not promote cell adhesion but still bound to native collagen and delayed fiber formation. The collagen-binding fragment of fibronectin produced by digestion either with chymotrypsin or with neutrophil elastase had a similar effect in delaying fibril formation, but the cell-binding fragment was not active. These studies indicate that fibronectin can bind to aggregating collagen fibers probably at the same site shown previously to bind to denatured collagen. Since fibronectin inhibits the rate of collagen fibrillogenesis, it may regulate the size of collagen fibers.