Procollagen Alpha Research Articles

Interstitial fibrosis in mice with overload proteinuria: Deficiency of TIMP-1 is not protective

Progressive renal interstitial fibrosis is characterized by up-regulated expression of the gene that encodes the tissue inhibitor of metalloproteinases-1 (TIMP-1), a regulator of extracellular matrix remodeling, suggesting that impaired matrix turnover contributes to the fibrogenic process. The present study was designed to develop a murine model of renal interstitial fibrosis, and to determine the functional significance of up-regulated Timp-1 expression by comparing the severity of this renal disease in wild-type mice and mice genetically deficient in Timp-1. Initial pilot studies developed and characterized a murine model of bovine serum albumin (BSA)-induced protein-overload proteinuria with respect to the degree of proteinuria, severity of interstitial fibrosis, and renal mRNA levels for genes encoding matrix proteins, transforming growth factor-beta1 (TGF-beta1), and TIMP-1, -2, -3, and -4. In the final study, the severity of interstitial fibrosis was compared in wild-type and Timp-1-deficient mice after six weeks of proteinuria. Mice injected with large daily intraperitoneal doses of BSA developed proteinuria, interstitial inflammation, and progressive interstitial fibrosis. A time course study based on measurements after one, two, and six weeks of BSA injections showed increased renal mRNA levels for the matrix genes procollagens alpha1(I), alpha1(III), and alpha2(IV) and TGF-beta1 and Timp-1. Timp-2 and Timp-3 genes were constitutively expressed at high levels in the normal kidneys and showed little change in the proteinuric kidneys. Timp-4 transcripts were not detected in any of the kidneys. After six weeks of BSA overload-proteinuria, the groups (N = 8 per group) of wild-type and Timp-1-deficient mice developed significant interstitial fibrosis compared with the control saline-injected groups. The severity of the interstitial fibrosis was similar in both proteinuric groups based on an assessment of the final kidney weight, total kidney collagen content, and the number of interstitial fields with increased fibronectin staining. Results of the present study indicate that TIMP-1 deficiency does not alter the degree of interstitial fibrosis in the murine overload proteinuria model. Potential explanations include Timp-1 genetic redundancy, as suggested by the observation that, despite significant intrarenal induction of the Timp-1 gene expression, net renal metalloproteinase-9 (MMP-9) activity was not significantly altered. TIMP-1 is a multifunctional protein that may play a metalloproteinase-independent role in response to renal injury.

Pentoxifylline inhibits human peritoneal mesothelial cell growth and collagen synthesis: Effects on TGF-β

Prevention or treatment of peritoneal fibrosing syndrome has become an important issue in patients on continuous ambulatory peritoneal dialysis (CAPD). Recent evidence has suggested that mesothelial stem cell proliferation and matrix over-production predispose the development of peritoneal fibrosis. We investigated whether pentoxifylline (PTX) affects human peritoneal mesothelial cell (HPMC) growth and collagen synthesis. HPMC was cultured from human omentum by an enzymic disaggregation method. Cell proliferation was assayed using a methyltetrazolium uptake method. Cell cycle analysis was performed by flow cytometry. Collagen synthesis was measured by 3H-proline incorporation into pepsin-resistant, salt-precipitated collagen. Prostaglandins and cAMP were determined by enzyme immunoassay. Northern blot analysis was used to determine mRNA expression. Our data show that PTX inhibited serum-stimulated HPMC growth and collagen synthesis in a dose-dependent manner. Cell cycle analysis showed that PTX arrested the HPMCs in the G1 phase. PTX decreased the procollagen alpha1 (I) mRNA expression either stimulated by serum or transforming growth factor-beta (TGF-beta). PTX did not alter prostaglandins synthesis but dose-dependently increased intracellular cAMP level. PTX, the same as 3-isobutyl-l-methylxanthine, could potentiate prostaglandin E1 (PGE1) increased cAMP levels of HPMC. The antimitogenic and antifibrogenic effects of PTX on HPMC were reversed by N-[2]-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89). Therefore, the mechanism of these effects may be due to the phospodiesterase inhibitory property of PTX. These data suggest that PTX may have a role in treating peritoneal fibrosing syndrome.

Birth associated changes in pulmonary arterial connective tissue gene expression in the normal and hypertensive lung

To determine the temporal and spatial expression of the connective tissue precursors, procollagen and tropoelastin mRNA in normal and pulmonary hypertensive porcine pulmonary arteries from birth onwards. Using in situ hybridisation, connective tissue gene expression for procollagen alpha1(I) and alpha1(III) and tropoelastin was studied in intrapulmonary arteries from normal piglets, 5 min-16 weeks, and from piglets made pulmonary hypertensive by exposure to hypobaric hypoxia for 3 days, from birth, 3 or 14 days of age. In addition, Type III pN-procollagen, tropoelastin and collagen I and III were studied by immunohistochemistry. Quantitative or semi-quantitative techniques were applied to both in situ and immunohistochemical studies. Procollagen alpha1(I) and alpha1(III) mRNA expression increased rapidly in the media and adventitia between birth and 3 days of age (P<0.05). The increase was transient and the number of cells expressing procollagen mRNA decreased to the low newborn number after 6 days of age. Type III pN-procollagen immunostaining was greatest in newborn elastic and muscular arteries and then decreased. Collagen I and III increased mainly after 6 days of age. In animals exposed to chronic hypobaric hypoxia from birth, the increase in procollagens I and III mRNA was prevented. Exposure to hypoxia from 3 or 14 days led to little change in either gene expression or in procollagen and mature collagen from the normal. Tropoelastin gene expression was high at birth in the endothelium and media for the first 6 days, and then decreased. Normally, tropoelastin decreased in the media and increased in the adventitia after 16 days of age. Hypoxia had no effect on the mRNA but led to increased tropoelastin. We demonstrated marked, rapid changes in temporal and cell specific connective tissue gene expression in normal pulmonary arteries immediately after birth as the vasculature remodels. Each gene appeared to have its own timetable of expression and responded differently to hypoxia-induced hypertension.

Repetitive self-limited acute pancreatitis induces pancreatic fibrogenesis in the mouse.

The role of repetitive acute injury in the pathogenesis of chronic pancreatitis remains unknown. To determine if repetitive injury induced by pancreatic hyperstimulation would reproduce the characteristic features of human chronic pancreatitis, acute reversible pancreatic injury was induced in mice by twice weekly cerulein treatment, 50 microg/kg/hr x 6 hr, for 10 weeks. Procollagen alpha1(I) mRNA was markedly increased by week 2. Sirius red staining of interstitial collagen demonstrated progressive accumulation of extracellular matrix surrounding acinar units and in interlobular spaces. Atrophy, transdifferentiation of acinar units to ductlike tubular complexes, and dilatation of intraacinar lumina also developed. Electron microscopy demonstrated the presence of stromal cells in areas of fibrosis with morphologic characteristics of pancreatic stellate cells. These findings demonstrate that, in a murine model, repetitive acute injury to the pancreas by hyperstimulation can reproduce the major morphological characteristics of human chronic pancreatitis.

Tissue responses around polymethylmethacrylate particles implanted into bone: analysis of expression of bone matrix protein mRNAs by in situ hybridization.

Tissue responses around implanted polymethylmethacrylate (PMMA) particles were analyzed by in situ hybridization with digoxigenin-labeled procollagen alpha1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) mRNA probes. PMMA particles (150-300 microm in diameter) were implanted into rat tibiae, and specimens were collected at 3, 5, 7, and 10 days after operation. New bone was formed centripetally, and bone-forming osteoblasts expressed all four kinds of mRNAs. A COL signal was expressed most strongly and widely. In the early stage, COL-positive cells were detected on and among particles sporadically. A COL signal was rarely detected in cells on the surfaces of the particles, suggesting that PMMA particles may suppress osteoblast differentiation. Osteonectin and osteocalcin mRNAs were expressed in bone-forming osteoblasts in a similar pattern by day 7. By contrast, an OPN signal was detected mainly on the particles, not only in COL-positive osteoblasts but also in COL-negative round cells. The latter cells had acid phosphatase activity, suggesting that they might be macrophages responding to a foreign body. At day 10, an OPN signal was detected continuously in multinucleated cells on PMMA particles, whereas new bone was formed away from particles. Our approach helped us to understand the initial cellular reaction to materials, which may determine their biocompatibility.

The expression of bone matrix protein mRNAs around beta-TCP particles implanted into bone.

Tissue response around beta-tricalcium phosphate (beta-TCP) particles (150-300 microm in diameter) implanted into rat tibiae was analyzed by in situ hybridization with digoxigenin-labeled procollagen alpha1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) RNA probes. Specimens were collected at 3, 5, 7, and 10 days after the operation. Holes without implantation were used as control. In both the beta-TCP implanted and control groups, new bone was formed centripetally and all four kinds of mRNA were expressed in activated osteoblasts. A COL signal was expressed most strongly and widely, and was detected at the peripheral region of the hole at day 3. The other three mRNAs were also expressed in bone forming osteoblasts by day 7. However, in the earlier cell reaction stage, OPN expression in the beta-TCP implanted group was different than that in the control group: OPN mRNA was seen exclusively in the cells on the particles, and an OPN signal was detected not only in COL-positive cells, but also in COL-negative cells. The former cells may be osteoblasts and reflect the early process of bone formation on biomaterials. The latter cells may be macrophages and reflect foreign body reactions. Expression of these OPN mRNAs induced by implantation of beta-TCP may play a role in bone formation on the materials and in determining their biocompatibility.

Analysis of isoaspartate in peptides by electrospray tandem mass spectrometry.

In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic L-Asp/L-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of L-Asp by L-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (L-Asp/L-isoAsp)-X bond and of the X-(L-Asp/L-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b-ion formation on both its N- and C-terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either L-Asp or L-isoAsp, these identify one or the other form. Characterization and relative quantification of L-Asp/L-isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG-D/isoD-AAAAK (deamidated peptide 1-7 of protein kinase A catalytic subunit) and VQ-D/isoD-GLR (deamidated peptide 41-46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.

Differential regulation of extracellular matrix synthesis during liver regeneration after partial hepatectomy in rats.

Little is known about the modulation of the extracellular matrix (ECM) during liver regeneration. We studied the temporospatial expression of procollagens and of matrix metalloproteinases (MMPs) and their physiological antagonists, the tissue inhibitors of metalloproteinases (TIMPs) after two-thirds partial hepatectomy (PH) by Northern blot analysis and in situ hybridization. The entry of hepatocytes into the S-phase at 24 hours after PH was accompanied by a peak (sixfold induction) of hepatic TIMP-1 RNA levels that steadily declined thereafter to reach normal levels 144 hours after PH. Moderate MMP-2 and TIMP-2 RNA levels remained constant up to 144 hours after PH, and MMP-1 and -13 RNA were always undetectable. In situ hybridization showed a dramatic upregulation of TIMP-1 RNA transcripts in mesenchymal cells of portal, perisinusoidal and, to a lesser extent, pericentral areas. In contrast, scattered hepatocytes represented only a minor fraction (below 10%) of TIMP-1 RNA positive cells. When hepatocytes stopped DNA synthesis at 72 hours after PH, an upregulation of procollagen alpha1(I) and alpha2(III) transcripts was observed paralleled by threefold increased PIIINP levels in the sera. Our data reveal a tightly regulated program of de novo matrix synthesis after PH. Whereas interstitial procollagens appear to participate in the induction and maintenance of the quiescent hepatocyte phenotype, the early and localized expression of TIMP-1 indicates a role unrelated to its function as a general MMP-antagonist, e.g., as a growth promoting agent for hepatocytes.

Effect of integrin on procollagen synthesis by fibroblasts from scleroderma.

To investigate the effect of integrin on fibroblasts from scleroderma in the production of procollagen. Phosphorothioate modified antisense oligonucleotides were used to interfere with the expression of integrin alpha 5 or beta 1 subunit on fibroblasts from 10 cases of scleroderma, and then the changes of procollagen mRNA due to the decline in level of integrin were observed by using RT-PCR. Expression of integrin alpha 5 or beta 1 subunit was specifically inhibited by their corresponding antisense oligonucleotides. Fibroblasts with decreased expression of alpha 5 or beta 1 subunit produced lower level of procollagen alpha 1 (I), alpha 1 (III) mRNA in comparison to the control. Overproduction of procollagen may be inhibited at the level of transcription by lowering the expression of integrin alpha 5 or beta 1 subunit on fibroblasts in scleroderma.

Activation of Pancreatic Stellate Cells in Human and Experimental Pancreatic Fibrosis

The mechanisms of pancreatic fibrosis are poorly understood. In the liver, stellate cells play an important role in fibrogenesis. Similar cells have recently been isolated from the pancreas and are termed pancreatic stellate cells. The aim of this study was to determine whether pancreatic stellate cell activation occurs during experimental and human pancreatic fibrosis. Pancreatic fibrosis was induced in rats (n = 24) by infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct. Surgical specimens were obtained from patients with chronic pancreatitis (n = 6). Pancreatic fibrosis was assessed using the Sirius Red stain and immunohistochemistry for collagen type I. Pancreatic stellate cell activation was assessed by staining for alpha-smooth muscle actin (alphaSMA), desmin, and platelet-derived growth factor receptor type beta (PDGFRbeta). The relationship of fibrosis to stellate cell activation was studied by staining of serial sections for alphaSMA, desmin, PDGFRbeta, and collagen, and by dual-staining for alphaSMA plus either Sirius Red or in situ hybridization for procollagen alpha(1) (I) mRNA. The cellular source of TGFbeta was examined by immunohistochemistry. The histological appearances in the TNBS model resembled those found in human chronic pancreatitis. Areas of pancreatic fibrosis stained positively for Sirius Red and collagen type I. Sirius Red staining was associated with alphaSMA-positive cells. alphaSMA staining colocalized with procollagen alpha(1) (I) mRNA expression. In the rat model, desmin staining was associated with PDGFRbeta in areas of fibrosis. TGFbeta was maximal in acinar cells adjacent to areas of fibrosis and spindle cells within fibrotic bands. Pancreatic stellate cell activation is associated with fibrosis in both human pancreas and in an animal model. These cells appear to play an important role in pancreatic fibrogenesis.

Fibrogenesis and Fibrolysis in Collagenous Colitis: Patterns of Procollagen Types I and IV, Matrix-Metalloproteinase-1 and -13, and TIMP-1 Gene Expression

Collagenous colitis is characterized by the deposition of a superficial subepithelial collagenous layer, the pathogenesis of which is unknown. Because the excess matrix deposition is potentially reversible, a labile imbalance between fibrogenesis and fibrolysis may be suspected. Expression of procollagen alpha1(I) and alpha1(IV), matrix-metalloproteinase (MMP)-1 and -13, and tissue inhibitor of metalloproteinase (TIMP)-1 genes was semiquantitated by in situ hybridization on serial biopsies of 12 patients with collagenous colitis and compared to controls. Collagen types I, III, IV, and VI, tenascin, undulin/collagen XIV, and alpha-actin were localized by immunohistology. The superficial collagen layer stained strongly for collagen types I, III, and VI, and particularly for tenascin, but not for undulin. Elevated procollagen alpha1(I), procollagen alpha1(IV), and TIMP-1 transcript levels were found in alpha-actin-positive cells with linear distribution underneath the superficial collagenous layer, whereas MMP-1 RNA expression was variable and restricted to cell clusters. MMP-13 expression was undetectable. The patterns of procollagen alpha1(I)- and alpha1(IV)-specific labeling, combined with an intense tenascin- but absent undulin-specific staining, indicate deposition of an immature interstitial matrix that may be susceptible to degradation. The restricted MMP-1 RNA expression, counteracted by increased TIMP-1 expression, suggests locally impaired fibrolysis as a relevant factor in the pathogenesis of collagenous colitis.

A regulatory role of fibroblast growth factor in the expression of decorin, biglycan, betaglycan and syndecan in osteoblasts from patients with Crouzon's syndrome

Bone development is controlled by the autocrine and/or paracrine effects of regulatory molecules. We previously showed that the phenotype of fibroblasts obtained from patients affected by Crouzon's syndrome, an autosomal dominant disease characterized by pathological skull bone development, differed from that of normal cells and was regulated by interleukin treatments. The changes in the relative concentrations of extracellular macromolecules (glycosaminoglycans-GAG, collagen and fibronectin) were associated with abnormal interleukin secretion that affected the microenvironment where the osteogenic processes take place. Mutations in human fibroblast growth factor receptors are now thought to be involved in Crouzon's syndrome. Since coactivation of interleukins and basic fibroblast growth factor (bFGF) is probably implicated in morphogenetic and osteogenic processes and heparan sulphate proteoglycans have a critical role in regulating bFGF activity, the phenotypes of normal and Crouzon osteoblasts were studied and the effects of bFGF on the expression of bFGF, procollagen alpha1 (I), and proteoglycan (PG) genes for biglycan, decorin, betaglycan and syndecan analyzed. Specific human cDNA probes were used to screen the relative levels of mRNA by Northern analysis. Spontaneous or bFGF-modulated release of interleukins was also assayed. The bFGF gene transcript was detected only in Crouzon osteoblasts. We showed for the first time that Crouzon osteoblasts, despite a mutation in the FGF receptor, still responded to exogenous bFGE In fact, the growth factor induced changes in the GAG profile and in the levels of mRNA coding for PG and procollagen alpha1 (I) and down-regulated heparan sulfate GAG chains. ELISA showed that bFGF-induced interleukin secretion differed in normal and Crouzon osteoblasts. The observed differences in PG core protein, procollagen alpha1 (I) and bFGF could be associated with the Crouzon bone phenotype and also should provide further understanding on the molecular basis of the diseased state of bone.

Palmitoyl ascorbate: Selective augmentation of procollagen mRNA expression compared withL-ascorbate in human intestinal smooth muscle cells

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 microM. By contrast, L-ascorbate was required at 4-5 times the concentration for the same response. However, at 20 microM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen alpha2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro.

Individual or combined effects of enalapril and verapamil on chronic cyclosporine nephrotoxicity in rats.

Previous studies have demonstrated that enalapril and verapamil seem to attenuate the cyclosporine nephrotoxicity. However, the mechanisms have not been completely understood, especially on molecular events. The aim of this study was to examine the effect of individual or combined treatment on osteopontin, TGF-beta, endothelin-1 and procollagen alpha 1(I) mRNA expressions. Enalapril (50 mg/L in drinking water) and verapamil (0.5 mg/kg/day, subcutaneously), alone or in combination, were administered to rats with chronic cyclosporine nephrotoxicity (cyclosporine, 25 mg/kg/day, subcutaneously) (n = 5 each). Five rats treated with olive oil vehicle were used as control. After 4 weeks, biochemical parameters were measured, and renal cortical mRNA levels were evaluated by Northern blot analysis. Cyclosporine reduced renal creatinine clearance significantly and induced renal cortical osteopontin, TGF-beta, endothelin-1 and procollagen alpha 1(I) gene expressions around 13.5 +/- 1.3, 2.4 +/- 0.2, 1.5 +/- 0.1, 1.9 +/- 0.1 folds, respectively. Individual treatment with enalapril or verapamil significantly suppressed the osteopontin and TGF-beta mRNA expression, but not endothelin-1 and procollagen alpha 1(I). Combined treatment also inhibited the osteopontin and TGF-beta mRNA expression but there was no difference between combined and individual treatment. In conclusion, enalapril or verapamil significantly blunted the cyclosporine-induced osteopontin and TGF-beta gene expressions. However, combined treatment did not show any additive effect.

Transforming Growth Factor-β1 Fails to Stimulate Wound Healing and Impairs Its Signal Transduction in an Aged Ischemic Ulcer Model: Importance of Oxygen and Age

Clinical trials of exogenous growth factors in treating chronic wounds have been less successful than expected. One possible explanation is that most studies used animal models of acute wounds in young animals, whereas most chronic wounds occur in elderly patients with tissue ischemia. We described an animal model of age- and ischemia-impaired wound healing and analyzed the wound-healing response as well as the transforming growth factor (TGF)-beta1 effect in this model. Rabbits of increasing ages were made ischemic in the ear where dermal ulcers were created. Histological analysis showed that epithelium ingrowth and granulation tissue deposition were significantly impaired with increased age under ischemia. TGF-beta1 stimulated wound repair under both ischemic and non-ischemic conditions in young animals, although it showed no statistical difference in aged animals. Procollagen mRNA expression decreased under ischemic conditions and with aging. Neither TGF-beta1 nor procollagen alpha1(I) mRNA expression increased in response to TGF-beta1 treatment under ischemia in aged animals. Therefore, the wound-healing process is impaired additively by aging and ischemia. The lack of a wound-healing response to TGF-beta1 in aged ischemic wounds may play a role in the chronic wounds.

Increased Expression of Heat-Shock Protein 47 is Associated with Overproduction of Type I Procollagen in Systemic Sclerosis Skin Fibroblasts

Heat-shock protein 47 (HSP47) is a collagen-binding stress protein that is thought to act as a collagen-specific molecular chaperon during the biosynthesis and secretion of procollagen. In this study we examined the expression of HSP47 mRNA and protein in systemic sclerosis (SSc) skin fibroblasts. HSP47 mRNA and protein levels were significantly higher in fibroblast cultures from SSc patient-involved skin samples than in fibroblasts from normal skin from healthy individuals, as assessed by northern blot and immunoblot analyses, respectively. SSc cultured fibroblasts with increased levels of HSP47 mRNA and protein showed high expression of type I procollagen. By in situ hybridization, SSc skin had a higher number of fibroblasts with high HSP47 and procollagen alpha1(I) mRNA levels than normal skin, and the distribution of HSP47 mRNA was similar to that of procollagen alpha1(I) mRNA. We also investigated the effects of cytokines on the expression of HSP47 in normal cultured fibroblasts. Transforming growth factor-beta1 and interleukin-4 increased HSP47 mRNA and protein levels, whereas interferon-gamma reduced HSP47 expression. The same pattern of cytokine-regulated expression was observed for type I procollagen levels. These results indicate that HSP47 expression is closely associated with that of type I procollagen in skin fibroblasts, and that increased expression of HSP47 may be involved in the abundant production of type I procollagen by SSc fibroblasts.

Epitope-specific monoclonal antibodies against human C-terminal procollagen alpha1(III)-propeptide.

We have generated monoclonal antibodies against recombinant C-terminal human procollagen alpha1(III) propeptide (PIIICP), produced in E. coli in high yields. The monoclonal antibodies were screened for epitope specificity using recombinant truncated PIIICP. Several antibodies were identified which recognized different regions of the PIIICP molecule. The ability of the antibodies to detect PIIICP antigens in human cell line lysates and supernatants was demonstrated. As PIIICP antigens are a key marker of extracellular matrix metabolism, the monoclonal antibodies described here should be of value for clinical and basic research.

The effect of tissue expansion on the expression of collagen type I and type III mRNA in distinct areas of skin in the dog as an animal model.

To evaluate the transcriptional response of skin to tissue expansion in the dog, the expression of procollagen alpha 1(I) mRNA and procollagen alpha 1(III) mRNA were analyzed by in situ hybridization. This expression was evaluated in distinct skin areas (subepidermal zone, dermis, capsular zone) after 4-85 days of expansion. Within the first 4 days of expansion expression of procollagen alpha 1(I) and alpha 1(III) mRNA was not affected in the subepidermal and dermal zone. Only a slightly elevated level of type III procollagen mRNA was demonstrated in tissue surrounding the implanted silicone expander. After 7 days of expansion an enhanced level of procollagen alpha 1(III) mRNA was observed in the dermis and capsular zone. A slightly enhanced type I collagen mRNA level occurred in all zones of the dermis that was even stronger in the capsular zone after 9 days of expansion. Concurrently, the number of transcriptionally active cells was significantly higher. Return to the basal level of procollagen alpha 1(I) mRNA was attained after 40 days. At this time a significant expression of procollagenase mRNA was observed. Procollagen III mRNA expression reached its basal level on day 85.

Antibody to Transforming Growth Factor Beta Reduces Collagen Production in Injured Peripheral Nerve

Epineurial scarring in peripheral nerve after injury inhibits normal axonal regeneration primarily due to fibroblast deposition of type I collagen. The transforming growth factor beta (TGF-β) family is an important class of signaling molecules that has been shown to stimulate fibroblasts to produce collagen. The aim of this study was to design a prototypic therapeutic system in which the neutralization of TGF-β in crushed rat sciatic nerve would decrease collagen formation. A total of 45 experimental Lewis rats were used. Group 1 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate buffer solution. Group 2 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate-buffered saline and goat, anti-rat, panspecific TGF-β antibody. Group 3 control animals (five rats) underwent only exposure of sciatic nerve with injection of antibody. All animals were killed at 14 days and sciatic nerve specimens were harvested at that time. Slides of experimental tissue were processed using a 35S-labeled oligomer for procollagen alpha-1 mRNA, then dipped in photographic emulsion and examined by darkfield autoradiography. Morphometric analysis of pixel counts was then performed. A significant reduction in total pixel count per high-power field and in total number of fibroblasts per high-power field was found in crushed rat sciatic nerve treated with anti-TGF-β antibody when compared with those treated only with phosphate-buffered saline. These findings are consistent with successful reduction in procollagen induction after a crush injury by topical administration of blocking antibody against transforming growth factor beta. The concept of growth factor blockade for therapeutic collagen reduction is attractive in the context of nerve injury, and the current article provides a model for future clinical application. (Plast. Reconstr. Surg. 102: 1100, 1998.)

Antibody to Transforming Growth Factor Beta Reduces Collagen Production in Injured Peripheral Nerve

Epineurial scarring in peripheral nerve after injury inhibits normal axonal regeneration primarily due to fibroblast deposition of type I collagen. The transforming growth factor beta (TGF-beta) family is an important class of signaling molecules that has been shown to stimulate fibroblasts to produce collagen. The aim of this study was to design a prototypic therapeutic system in which the neutralization of TGF-beta in crushed rat sciatic nerve would decrease collagen formation. A total of 45 experimental Lewis rats were used. Group 1 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate buffer solution. Group 2 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate-buffered saline and goat, anti-rat, panspecific TGF-beta antibody. Group 3 control animals (five rats) underwent only exposure of sciatic nerve with injection of antibody. All animals were killed at 14 days and sciatic nerve specimens were harvested at that time. Slides of experimental tissue were processed using a 35S-labeled oligomer for procollagen alpha-1 mRNA, then dipped in photographic emulsion and examined by darkfield autoradiography. Morphometric analysis of pixel counts was then performed. A significant reduction in total pixel count per high-power field and in total number of fibroblasts per high-power field was found in crushed rat sciatic nerve treated with anti-TGF-beta antibody when compared with those treated only with phosphate-buffered saline. These findings are consistent with successful reduction in procollagen induction after a crush injury by topical administration of blocking antibody against transforming growth factor beta. The concept of growth factor blockade for therapeutic collagen reduction is attractive in the context of nerve injury, and the current article provides a model for future clinical application.