Collagen Fibril Formation
2008; Elsevier BV; Volume: 283; Issue: 38 Linguagem: Inglês
10.1074/jbc.m804272200
ISSN1083-351X
AutoresHye Jin Chung, Andrzej Steplewski, Kee Yang Chung, Jouni Uitto, Andrzej Fertala,
Tópico(s)Cell Adhesion Molecules Research
ResumoWe present a concept for reducing formation of fibrotic deposits by inhibiting self-assembly of collagen molecules into fibrils, a main component of fibrotic lesions. Employing monoclonal antibodies that bind to the telopeptide region of a collagen molecule, we found that blocking telopeptide-mediated collagen/collagen interactions reduces the amount of collagen fibrils accumulated in vitro and in keloid-like organotypic constructs. We conclude that inhibiting extracellular steps of the fibrotic process provides a novel approach to limit fibrosis in a number of tissues and organs. We present a concept for reducing formation of fibrotic deposits by inhibiting self-assembly of collagen molecules into fibrils, a main component of fibrotic lesions. Employing monoclonal antibodies that bind to the telopeptide region of a collagen molecule, we found that blocking telopeptide-mediated collagen/collagen interactions reduces the amount of collagen fibrils accumulated in vitro and in keloid-like organotypic constructs. We conclude that inhibiting extracellular steps of the fibrotic process provides a novel approach to limit fibrosis in a number of tissues and organs. Collagen I is the most abundant structural protein of connective tissues such as skin, bone, and tendon. This protein is first synthesized as a precursor molecule, procollagen, that is characterized by the presence of a rod-like central triple-helical domain flanked by short linear telopeptides and globular N-terminal and C-terminal propeptides (1Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1388) Google Scholar). Single procollagen molecules are the building blocks for the biologically and mechanically relevant collagen fibrils. Formation of collagen fibrils is initiated by enzymatic cleavage of the N-terminal and the C-terminal propeptides. The N-terminal propeptides are cleaved by a group of enzymes that includes a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-2, -3, and -14, whereas the C-terminal propeptides are cleaved by the metalloprotease bone morphogenetic protein 1 (BMP-1) 2The abbreviations used are: BMP-1bone morphogenetic protein 1PBSphosphate-buffered saline. and by the other members of a closely related family of mammalian tolloid-like metalloproteases (2Colige A. Vandenberghe I. Thiry M. Lambert C.A. Van Beeumen J. Li S.W. Prockop D.J. Lapiere C.M. Nusgens B.V. J. Biol. Chem. 2002; 277: 5756-5766Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (458) Google Scholar, 4Hopkins D.R. Keles S. Greenspan D.S. Matrix Biol. 2007; 26: 508-523Crossref PubMed Scopus (202) Google Scholar). Such a removal of procollagen propeptides exposes telopeptides, which by engaging in site-specific intermolecular interactions drive collagen self-assembly. bone morphogenetic protein 1 phosphate-buffered saline. In native tissues a precise balance between the processes of biosynthesis and degradation maintains the physiological homeostasis of tissue collagens. At the same time, accelerated biosynthesis is required for proper wound healing, whereas excessive accumulation of collagen is the hallmark of a number of localized fibrotic diseases, such as keloids and hypertrophic scars, and systemic fibrosis, such as systemic scleroderma. Localized fibrotic reactions are quite common and frequently develop as a consequence of surgical procedures. For instance, after surgery of the abdomen, the formation of excessive scar tissue around abdominal organs, such as the intestines, can interfere with the functionality of such organs and may cause severe pain and even death. Another situation where excessive scar formation presents a major complication is in the eye after glaucoma surgery performed to create a pressure-maintenance valve. Frequently, however, excessive scar formation closes this pressure-reducing valve, thereby forcing the intraocular pressure to rise (5Addicks E.M. Quigley H.A. Green W.R. Robin A.L. Arch. Ophthalmol. 1983; 101: 795-798Crossref PubMed Scopus (419) Google Scholar). Moreover, excessive scarring of the vocal folds may severely alter their ability to vibrate, thereby causing a number of voice disorders (6Lim X. Tateya I. Tateya T. Munoz-Del-Rio A. Bless D.M. Ann. Otol. Rhinol. Laryngol. 2006; 115: 921-929Crossref PubMed Scopus (78) Google Scholar). At present, several biological processes critical for development of fibrotic lesions are considered potential targets for inhibitors of fibrosis. These inhibitors aim at (i) reducing inflammatory processes associated with fibrosis, (ii) inhibiting biological functions of cytokines and growth factors that promote fibrosis, (iii) reducing cell proliferation, and (iv) decreasing biosynthesis and processing of procollagens. Because most of those potential targets are involved not only in pathological fibrosis but also in a number of physiological processes, their inhibition is frequently associated with significant adverse effects (7Roseborough I.E. Grevious M.A. Lee R.C. J. Natl. Med. Assoc. 2004; 96: 108-116PubMed Google Scholar, 8Sanders K.W. Gage-White L. Stucker F.J. Arch. Facial Plast. Surg. 2005; 7: 172-175Crossref PubMed Scopus (51) Google Scholar, 9Shah M. Foreman D.M. Ferguson M.W. J. Cell Sci. 1995; 108: 985-1002Crossref PubMed Google Scholar, 10Ovens A. Joule J.A. Kadler K.E. J. Pept. Sci. 2000; 6: 489-495Crossref PubMed Scopus (20) Google Scholar, 11Riley D.J. Berg R.A. Edelman N.H. Prockop D.J. J. Clin. Investig. 1980; 65: 643-651Crossref PubMed Scopus (43) Google Scholar). Here, we tested a new approach to reduce excessive scarring by specifically targeting the extracellular process of formation of collagen fibrils, a main component of fibrotic scars. By employing custom-designed antibodies that specifically bind to the C-terminal telopeptide of the α2-chain of collagen I, we demonstrated that blocking telopeptide-mediated collagen/collagen interaction limits accumulation of collagen fibrils in vitro and in organotypic constructs formed by keloid-derived fibroblasts. Because excessive deposition of collagen fibrils is characteristic of all fibrotic processes, we predict that the basic design for the inhibitors of collagen fibril formation we tested in a skin-based keloid model will be applicable for reducing a number of localized and systemic fibrotic changes in other tissues and organs as well. Purification of Human Procollagen I—Procollagen I was purified from cell culture media of normal human dermal fibroblasts, as described (12Fiedler-Nagy C. Bruckner P. Hayashi T. Prockop D.J. Arch. Biochem. Biophys. 1981; 212: 668-677Crossref PubMed Scopus (23) Google Scholar, 13Romanic A.M. Adachi E. Kadler K.E. Hojima Y. Prockop D.J. J. Biol. Chem. 1991; 266: 12703-12709Abstract Full Text PDF PubMed Google Scholar). Monoclonal Antibodies—Hybridomas were prepared by a commercial company (Abgent, Inc. San Diego, CA). Inhibitory antibodies were raised against a synthetic peptide (GGGYDFGYDGDFYRA) whose sequence corresponds to that of the C-terminal telopeptide of the α2 chain (α2Ct). Initial screening of these antibodies produced by various hybridoma clones was done with synthetic α2Ct. Finally, the specificity of selected antibodies to bind a native epitope was tested against intact human procollagen I and against enzymatically processed procollagen I. In brief, purified procollagen I was processed by BMP-1 (R&D Systems), lysyl endopeptidase (Lys-C; Roche Applied Science) or pepsin (Sigma-Aldrich), as described (14Fertala A. Sieron A.L. Hojima Y. Ganguly A. Prockop D.J. J. Biol. Chem. 1994; 269: 11584-11589Abstract Full Text PDF PubMed Google Scholar, 15Steplewski A. Ito H. Rucker E. Brittingham R.J. Alabyeva T. Gandhi M. Ko F.K. Birk D.E. Jimenez S.A. Fertala A. J. Struct. Biol. 2004; 148: 326-337Crossref PubMed Scopus (39) Google Scholar). Note that enzymatic cleavage of procollagen I with BMP-1 or Lys-C leaves telopeptide regions intact, whereas digestion of procollagen with pepsin degrades the telopeptides. At the same time, digestion with all enzymes employed here preserves the triple-helical region of collagen I. Intact procollagen I and products of its enzymatic cleavage were electrophoresed in polyacrylamide gels in denaturing conditions. Subsequently, protein bands were transferred onto nitrocellulose membranes and detected with primary anti-α2Ct antibody and secondary antibodies conjugated with horseradish peroxidase (Sigma-Aldrich). Finally, the protein bands were visualized by chemiluminescence. The ability of the anti-α2Ct antibody to interact with a native antigen was also tested by applying unprocessed procollagen I and products of its enzymatic digestion directly onto nitrocellulose membranes and detecting these proteins as described above. Moreover, to ensure the lack of any cross-reactivity of analyzed antibodies with employed enzymes, BMP-1, Lys-C, and pepsin alone were also loaded onto nitrocellulose membranes. Monoclonal antibody, described by Fisher et al. (16Fisher L.W. Stubbs III, J.T. Young M.F. Acta Orthop. Scand. 1995; 66: 61-65Crossref Scopus (420) Google Scholar) as LF-41, was raised against a peptide (PLDVGAPDQEFGFDVGPVCFL) that corresponds to the C-terminal propeptide of the pro-α1 chain of procollagen I (α1Cp). Specificity of the anti-α1Cp antibody to recognize the native epitopes was tested in a way similar to that described for the anti-α2Ct antibody. Purification of Monoclonal Antibodies—Isotyping of antibodies was performed by Rockland Immunochemicals, Inc., Gilbertsville, PA. The anti-α2Ct monoclonal antibody (an IgA isoform with kappa light chains) produced by a selected hybridoma clone was purified from cell culture media. In brief, cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum with reduced IgG content (Ultra-Low IgG fetal bovine serum, Invitrogen). About 10 liters of cell culture medium was collected and filtered through glass fiber filters (Millipore, Inc.) to remove cell debris. Subsequently, a protein fraction that included immunoglobulins was precipitated with a 50% saturation of ammonium sulfate. A protein pellet was collected by centrifugation and then resuspended in a buffer consisting of 0.1 m Tris-HCl, pH 7.8, 120 mm NaCl, and 0.02% NaN3. The anti-α2Ct monoclonal antibody was finally purified by affinity chromatography on a Protein-L column (Pierce). Column-bound IgA was eluted with 0.1 m glycine, pH 3.0. Collected fractions were immediately neutralized with a buffer consisting of 1.5 m Tris-HCl, pH 8.0, 120 mm NaCl, and 25 mm EDTA. The purity of the antibody was determined by electrophoresis in polyacrylamide gels. The anti-α1Cp antibody (an IgG type) was prepared in a similar way, but instead of Protein-L, Protein-G was employed for purification (Pierce). Purified antibodies were dialyzed against phosphate-buffered saline (PBS) and stored at –80 °C. Cleavage of Procollagen I by BMP-1 in the Presence of Anti-α2Ct Antibody—Because the anti-α2Ct antibody was designed to bind the C-terminal α2 telopeptide, we tested whether its binding would interfere with the enzymatic activity of BMP-1, an enzyme that specifically cleaves procollagen I at the C telopeptide/C propeptide junction (17Hojima Y. van der Rest M. Prockop D.J. J. Biol. Chem. 1985; 260: 15996-16003Abstract Full Text PDF PubMed Google Scholar). Samples containing 50 μg/ml procollagen I and 1.2 or 0.2 μg/ml purified anti-α2Ct antibody solubilized in a buffer supplemented with 5 mm CaCl2 were incubated for 0.5 h at room temperature. Subsequently, recombinant BMP-1 at a concentration of 25 μg/ml was added to the procollagen I-antibody mixtures, and the samples were incubated at 37 °C. At defined time points aliquots were withdrawn and boiled with a protein-loading buffer. After collecting all samples, the products of cleavage of procollagen I by BMP-1 were separated in 7.5% polyacrylamide gels. Proteins were visualized by staining with Coomassie Blue (BioSafe Coomassie, Bio-Rad). Subsequently, employing a computer program, the pixel intensities of bands corresponding to substrates and products of enzymatic digestion were determined (ImageQuant, GE Healthcare). Data from three independent measurements were used to derive rates of cleavage of procollagen I. In addition, samples that contained the anti-α1Cp antibody and those with no antibodies were prepared and analyzed in a similar way. To exclude the possibility of the presence of any nonspecific proteolytic activities derived from enzymes that could co-purify with procollagen itself or with antibodies, in one set of experiments we employed EDTA, an inhibitor of BMP-1. In brief, samples containing procollagen I, BMP-1, and antibodies were prepared as described above. EDTA was added to these samples to a final concentration of 10 mm. Subsequently, the samples were incubated for 4 h at 37°C and then they were analyzed by gel electrophoresis as described above. Fibril Formation Assays in Vitro—De novo collagen fibril formation assays were employed to analyze the inhibitory effect of the anti-α2Ct antibody on collagen fibril formation in vitro, as described (15Steplewski A. Ito H. Rucker E. Brittingham R.J. Alabyeva T. Gandhi M. Ko F.K. Birk D.E. Jimenez S.A. Fertala A. J. Struct. Biol. 2004; 148: 326-337Crossref PubMed Scopus (39) Google Scholar, 18Fertala A. Holmes D.F. Kadler K.E. Sieron A.L. Prockop D.J. J. Biol. Chem. 1996; 271: 14864-14869Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 19Ito H. Rucker E. Steplewski A. McAdams E. Brittingham R.J. Alabyeva T. Fertala A. J. Mol. Biol. 2005; 352: 382-395Crossref PubMed Scopus (32) Google Scholar, 20Cabral W.A. Makareeva E. Letocha A.D. Scribanu N. Fertala A. Steplewski A. Keene D.R. Persikov A.V. Leikin S. Marini J.C. Hum. Mutat. 2007; 28: 396-405Crossref PubMed Scopus (57) Google Scholar). In these assays self-assembly of collagen monomers into fibrils is initiated by enzymatic digestion of procollagen propeptides. In brief, procollagen I at a concentration 230 μg/ml was digested with Lys-C (1 μg/ml) for 0.5 h at 25 °C, experimental conditions in which formation of collagen fibrils does not occur (21Kadler K.E. Hojima Y. Prockop D.J. J. Biol. Chem. 1987; 262: 15696-15701Abstract Full Text PDF PubMed Google Scholar). After initial incubation, a possible enzymatic digestion of Lys-C-sensitive antibodies was inhibited by Nα-p-tosyl-l-lysine chloromethyl ketone (TPCK, Sigma-Aldrich) added to a final concentration of 1 mm (19Ito H. Rucker E. Steplewski A. McAdams E. Brittingham R.J. Alabyeva T. Fertala A. J. Mol. Biol. 2005; 352: 382-395Crossref PubMed Scopus (32) Google Scholar, 22Elliott Jr., B.W. Cohen C. J. Biol. Chem. 1986; 261: 11259-11265Abstract Full Text PDF PubMed Google Scholar). Subsequently, the anti-α2Ct antibody was added to the Lys-C-processed collagen samples at concentrations of 180 or 10 μg/ml to achieve collagen/antibody molar ratios of about 1:2 or 10:1, respectively. Samples were incubated for 0.5 h at room temperature followed by incubation at 37 °C. To determine the kinetics of fibril formation, samples were analyzed at defined time points, as described (14Fertala A. Sieron A.L. Hojima Y. Ganguly A. Prockop D.J. J. Biol. Chem. 1994; 269: 11584-11589Abstract Full Text PDF PubMed Google Scholar). In brief, fibril incorporated collagen was separated from the soluble collagen monomers by centrifugation. Supernatant was transferred to a fresh tube, and a fibril-containing pellet was resuspended in 20 μl of a buffer containing 0.1 m Tris-HCl, pH 7.4, 0.4 m NaCl, 25 mm EDTA, and 0.02% NaN3. After boiling all samples with a protein loading buffer, pellet and supernatant collagen fractions were electrophoresed in 7.5% polyacrylamide gels. Subsequently, protein bands were visualized by staining with Coomassie Blue, and the pixel intensities of bands corresponding to the collagen α-chains present in a pellet or in a supernatant fraction were determined by densitometry. Data from independent studies were plotted to determine rates of fibril formation, as described (23Gelman R.A. Williams B.R. Piez K.A. J. Biol. Chem. 1979; 254: 180-186Abstract Full Text PDF PubMed Google Scholar). Fibril formation studies in the presence of the anti-α1Cp antibody and those with no antibodies were carried out in a similar way. Microscopic Analyses of Morphology of Individual Collagen Fibrils—For ultrastructural analysis by transmission electron microscopy, the collagen fibrils were processed as described (18Fertala A. Holmes D.F. Kadler K.E. Sieron A.L. Prockop D.J. J. Biol. Chem. 1996; 271: 14864-14869Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In brief, 10-μl samples containing fibrils formed for 24 h were transferred onto 200-mesh Formvar-supported and carbon-coated copper grids (Ted Pella, Inc.). Collagen fibrils deposited on the grids were stained negatively with 1% phosphotungstate (pH 7.0) and visualized by a transmission electron microscope operating at 75 kV (model H-7000, Hitashi Ltd., Japan). Organotypic Cultures of Keloid-derived Fibroblasts—To test the ability of the anti-α2Ct antibody to limit collagen fibril formation in vivo, we employed a system that utilizes keloid-derived fibroblasts cultured in a three-dimensional environment. Keloid fibroblasts from patients undergoing elective surgery were obtained from the Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, South Korea. The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, a mixture of penicillin and streptomycin, and l-ascorbic acid phosphate magnesium salt n-hydrate at a concentration of 40 μg/ml (WAKO Inc., Japan). The organotypic constructs were prepared by employing three-dimensional sponge-like scaffolds fabricated from polylactic acid (OPLA, BD Biosciences). Cells were seeded dynamically into scaffolds by a method adopted from Wang et al. (24Wang H.J. Bertrand-de Haas M. van Blitterswijk C.A. Lamme E.N. Tissue Eng. 2003; 9: 909-917Crossref PubMed Scopus (27) Google Scholar). In brief, the scaffolds were placed into cell culture chambers of a bioreactor (Sythecon, Inc.) filled with cell suspension containing 2 × 105 cells/ml, and the cells were allowed to populate scaffolds for 7 days with the culture chambers rotating at 20 rpm. For fibril formation assay, in vitro individual scaffolds were transferred into wells of low-cell adhesion culture plates (Corning, Inc.) and placed into a cell culture incubator onto a platform rotating at 80 rpm. Each day cell culture media supplemented with the tested antibody at a concentration of 70 μg/ml was exchanged in each well. After 2 weeks of culture, samples were collected and prepared for analysis. Inhibition of collagen deposition was also tested in organotypic constructs implanted subcutaneously into nude mice (Nude-Foxn1nu, HARLAN). After 7 days of incubation in cell culture chambers, four constructs seeded with keloid fibroblasts were implanted subcutaneously into an athymic nude mouse. Subsequently, two of those constructs received a 100-μl dose of an inhibitory antibody present at a concentration of 1.5 mg/ml, injected every 3 days directly into the scaffold and the surrounding area. Two remaining constructs received injections of equal amounts of PBS. After 1 month, the mice were sacrificed, and the scaffolds were prepared for biochemical and morphological analyses. In the first set of experiments, in which the organotypic cultures were grown in cell culture conditions, one control group received the anti-α1Cp antibody treatment, and the second one was treated with PBS only. Because of the limits in supply of the anti-α1Cp antibody in studies with the subcutaneous implants, the only control group was the one receiving PBS injections. Analysis of the Total Collagen Content in Keloid-like Constructs—Scaffolds were rinsed with PBS, cut into two portions, and then lyophilized. Subsequently, the dry mass of lyophilized samples was measured. Next, one portion of a scaffold was hydrolyzed in 6 n HCl, and the hydroxyproline concentration was analyzed to determine collagen content, as described (25Woessner J.F. Archiv. Biochem. Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3388) Google Scholar). The remaining portion of a scaffold was utilized to determine the total protein content. Microscopic Analyses of Organotypic Keloid-like Constructs—Keloid-like constructs were harvested and then processed for histology and electron microscopy analyses. The samples were stained for collagen deposits with Sirius red. In addition, the human origin of cells present in the constructs grown subcutaneously in mice was confirmed by immunostaining with anti-human vimentin-specific antibody (Santa Cruz Biotechnology, Inc.). The stained samples were observed under a microscope equipped with a digital camera (model Eclipse E600, Nikon, Japan). For electron microscopy, the samples were fixed in 2% glutaraldehyde and 1% tannic acid in 0.1 m phosphate buffer (Electron Microscopy Sciences, Inc.). Subsequently, the samples were post-fixed in 2% osmium tetraoxide in 0.1 m phosphate buffer, rinsed in distilled water, and then stained with 1% uranyl acetate (Electron Microscopy Sciences). After that, the samples were embedded in 3% agarose followed by dehydration in graded concentrations of acetone. Next, the samples were embedded in Spurr's resin (Electron Microscopy Sciences). After polymerization, blocks of samples were sectioned to 70-nm sections with a diamond knife (Diatome, AG, Switzerland) mounted on a Leica Ultracut UCT microtome (Leica Microsystems, Germany). Finally, the sections were post-stained with 2.5% uranyl acetate in 50% ethanol and sodium bismuth (Electron Microscopy Sciences) and then observed with a transmission electron microscope (model H-7000, Hitashi Ltd., Japan). Statistics—Collected data were analyzed, and results are presented as the mean ± S.E. for each sample. Comparisons between control groups and other groups were performed by an unpaired, two-tailed t test. In experiments with keloid-like constructs grown subcutaneously in mice, a paired t test was employed. In all tests the α level was set to 0.05. Statistical analyses were performed with GraphPad Prism Version 5.0 (GraphPad Software Inc.). Epitope Specificity of Monoclonal Antibodies—Monoclonal anti-α1Cp and anti-α2Ct antibodies were purified from cell culture media of selected hybridomas. The monoclonal anti-α2Ct antibodies were an IgA type with the kappa light chains, whereas the anti-α1Cp antibody belonged to the IgG class. As determined by Western blot assays, monoclonal antibodies produced by selected hybridoma clones specifically recognized their respective epitopes (Figs. 1 and 2). In particular, the anti-α2Ct antibody-specific signal was apparent only in collagen variants in which the telopeptides were intact. Specifically, a group of collagen variants interacting with the anti-α2Ct antibody included intact procollagen I and procollagen processed with BMP-1 or with Lys-C (Fig. 1c). Moreover, we observed that the α2Ct-specific signal was stronger for a partially processed pro-α2 chain than the signal for the intact pro-α2 chain, suggesting more unrestricted access of the tested antibody to the target telopeptide in the absence of the C-terminal propeptide (Fig. 1c). There was no apparent signal from the pepsin-digested collagen I in which telopeptides were enzymatically degraded (Fig. 1c).FIGURE 2Slot blot analysis of the specificity of recognition of native epitopes by the anti-α1Cp and anti-α2Ct antibodies. Panel a shows results of assays of binding of the anti-α1Cp antibody to native procollagen I and to the products of its cleavage with BMP-1, Lys-C, or pepsin. Panel b depicts results of analyses of binding of the anti-α2Ct antibody to the same proteins. In addition, both panels show the lack of binding of analyzed antibodies to BMP-1, Lys-C, and pepsin. Pro, procollagen I; + indicates samples digested with BMP-1, Lys-C, or pepsin; asterisks indicate positions of proteins present on nitrocellulose membranes but not detected by analyzed antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Similarly, the anti-α1Cp antibody specifically recognized the C propeptide of the α1 chain of procollagen I. As indicated in Fig. 1b, the α1 C propeptides present in the intact procollagen as well as C propeptides cleaved by BMP-1 or Lys-C were recognized by the anti-α1Cp antibody. There was an apparent difference in molecular masses of products generated by digestion of the C propeptides with Lys-C and BMP-1, most likely due to the differences in location of the specific cleavage sites recognized by these enzymes (Fig. 1b). The lack of binding of the anti-α1Cp antibody to the pepsin-treated procollagen in which the C propeptides were degraded provides even more evidence for its epitope-specific binding (Fig. 1b). As determined by slot-blot assays, both the anti-α1Cp and anti-α2Ct antibodies were able to interact with native epitopes (Fig. 2). Specifically, the antibodies interacted with the intact procollagen I and with products generated by cleavage of procollagen I with BMP-1 or Lys-C. In contrast, the product generated by digestion with pepsin was not recognized by analyzed antibodies. As demonstrated in Fig. 2, the enzymes themselves did not react with tested antibodies. Processing of the C Propeptide by BMP-1 in Vitro in the Presence of the Anti-α2Ct Antibody—Because the anti-α2Ct antibody binds to the C-terminal telopeptide, we tested whether its binding could hinder BMP-1 cleavage of the Ala-Asp bond present at the C telopeptide/C propeptide junction (Fig. 3). As demonstrated in Table 1, the presence of the anti-α2Ct antibody decreased the rate of cleavage of procollagen chains in a concentration-dependent fashion (Fig. 3 and Table 1). Under the same conditions, there was no statistically significant difference between the rates of cleavage of procollagen α chains by BMP-1 in control samples containing the anti-α1Cp antibody and those to which no antibody was added (Fig. 3 and Table 1).TABLE 1Summary of results on inhibitory effects of the anti-α2Ct antibody on BMP-1 cleavage, kinetics of fibril formation, and accumulation of collagen in keloid-like constructsControl(+) Anti-α2Ct(+) Anti-α1CpMean ± S.E. (% of control)Mean ± S.E. (% of control)Mean ± S.E. (% of control)Rate of cleavage of procollagen I with BMP-1 in vitro (μg/min/ml)0.22 ± 0.01; n = 3 (100%)a0.2 μg/ml anti-α2Ct was used.0.04 ± 0.02; n = 3; p = 0.001 (18%)b1.2 μg/ml anti-α2Ct or the anti-α1Cp was used.0.18 ± 0.009; n = 3; p = 0.07 (82%)b1.2 μg/ml anti-α2Ct or the anti-α1Cp was used.0.03 ± 0.003; n = 3; p = 0.0001 (14%)Rate of fibril formation in vitro (μg/min/ml)0.27 ± 0.046; n = 4 (100%)cThe collagen/antibody molar ratio was 1:2.0.053 ± 0.02; n = 4; p = 0.005 (20%)dThe collagen/antibody molar ratio was 10:1; NA, not assayed.0.25 ± 0.054; n = 4; p = 0.8 (93%)dThe collagen/antibody molar ratio was 10:1; NA, not assayed.0.132 ± 0.003; n = 4; p = 0.02 (49%)Total accumulated fibrils in vitro (μg/ml)72.08 ± 8.3; n = 4 (100%)cThe collagen/antibody molar ratio was 1:2.21.87 ± 7.04; n = 3; p = 0.007 (30%)dThe collagen/antibody molar ratio was 10:1; NA, not assayed.73.38 ± 10.01; n = 4; p = 0.9 (100%)dThe collagen/antibody molar ratio was 10:1; NA, not assayed.40.77 ± 6.11; n = 4; p = 0.02 (57%)Accumulation of collagen in keloid-like constructs grown in cell culture conditions (collagen (mg)/total protein (mg))0.104 ± 0.016; n = 3 (100%)0.026 ± 0.004; n = 5; p = 0.0008 (25%)0.117 ± 0.01; n = 2; p = 0.6 (113%)Accumulation of collagen in keloid-like constructs grown subcutaneously in nude mice (collagen (mg)/total protein (mg))0.495 ± 0.07; n = 7 (100%)0.209 ± 0.03; n = 8; p = 0.001 (42%)NAa 0.2 μg/ml anti-α2Ct was used.b 1.2 μg/ml anti-α2Ct or the anti-α1Cp was used.c The collagen/antibody molar ratio was 1:2.d The collagen/antibody molar ratio was 10:1; NA, not assayed. Open table in a new tab Employing EDTA, we determined that there was no proteolytic activity other than that derived from BMP-1. In particular, in the presence of EDTA there was not any detectable cleavage of procollagen I (Fig. 3d). Kinetics of Self-assembly of Collagen and Morphology of Fibrils Formed in Vitro in the Presence of the Anti-α2Ct Antibody—In a number of in vitro assays it has been established that de novo fibril formation, i.e. where individual collagen molecules derived from enzyme-processed procollagen self-assemble into fibrils, is characterized by distinct phases of nucleation, propagation, and equilibrium (21Kadler K.E. Hojima Y. Prockop D.J. J. Biol. Chem. 1987; 262: 15696-15701Abstract Full Text PDF PubMed Google Scholar). Here, in vitro fibril formation assays demonstrated that binding of the anti-α2Ct antibody to the C-terminal α2 telopeptide decreased both the rate of fibril formation and the amount of accumulated collagen fibrils (Fig. 4a, Table 1). By employing various concentrations of inhibitory antibodies, we demonstrated that the decrease in the rates of fibril formation and in the amounts of accumulated fibrils at equilibrium was concentration-dependent (Table 1). In contrast, the presence of the anti-α1Cp antibody did not have any significant effects on the kinetics of fibril assembly or on the total amount of accumulated fibrils (Fig. 4a, Table 1). In addition to electrophoretic assays of the kinetics of formation of fi
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