Collagen fibril formation. A new target to limit fibrosis

Abstract

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.

FIGURE 1.

Analysis of specificity of epitope recognition by the anti-α1Cp and anti-α2Ct antibodies. Panel a depicts intact procollagen I and products of its digestion by BMP-1, Lys-C, or pepsin separated in polyacrylamide gels and stained with Coomassie Blue. Specific epitopes present in proteins represented in panel a were analyzed by Western blot with the anti-α1Cp antibody (panel b) or with the anti-α2Ct antibody (panel c). In panel d schematics of domains in intact procollagen I or in products of its digestion by BMP-1, Lys-C, or pepsin are presented. In addition, molecular mass markers are presented. Pro, intact procollagen I; pN, procollagen variant in which the C-terminal propeptide have been cleaved by BMP-1; Col-Lys-C, collagen I variant, includes telopeptides, generated by enzymatic cleavage of procollagen with Lys-C; Col-Peps, collagen I variant, with telopeptides absent, generated by enzymatic cleavage of procollagen I with pepsin; Pro-α1, pro-α1 chain of procollagen I; Pro-α2, pro-α2 chain of procollagen I; dPro-α1 and dPro-α2, partially processed pro-α chains of procollagen I; pN-α1 and pN-α2, procollagen α chains in which the C-terminal propeptides were cleaved by BMP-1; α1 and α2, collagen I α chains; a-α1Cp and a-α2Ct, antibodies against the C-terminal propeptide of the α1 chain and the C-terminal telopeptide of the α2 chain of procollagen I, respectively; α1Cp and dα1Cp, C propeptide derived by cleavage of the procollagen α1 chain with BMP-1 or Lys-C, respectively; Np, Nt, Ct, and Cp, N-terminal propeptides, N-terminal telopeptides, C-terminal telopeptides, and C-terminal propeptides of procollagen I, respectively.

FIGURE 2.

Slot 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.

FIGURE 3.

Kinetics of cleavage of procollagen I by BMP-1 in the presence of the anti-α1Cp or the anti-α2Ct antibody. Panel a illustrates kinetics of cleavage of procollagen I by BMP-1 in the absence of antibodies, whereas panels b and c show kinetics of cleavage of procollagen I in the presence of 1.2 μg/ml anti-α1Cp or the anti-α2Ct antibody, respectively. Time points at which samples were analyzed are indicated. In panels b and c, protein bands corresponding to BMP-1 and to antibodies employed in this study are also indicated. In addition, molecular mass markers are presented in the left lanes of all panels. Panel d presents analysis of the specificity of cleavage of procollagen I with BMP-1. Note that in the presence of EDTA there was no cleavage of a procollagen substrate by BMP-1, thereby indicating the absence of other active enzymes. Pro-α1, pro-α1 chain of procollagen I; Pro-α2, pro-α2 chain of procollagen I; pN-α1 and pN-α2, procollagen α chains in which the C-terminal propeptides were cleaved by BMP-1; Cα1 and Cα2, C propeptides cleaved off respective procollagen chains by BMP-1; a-α1Cp and a-α2Ct, antibodies against the C-terminal propeptide of the α1 chain and the C-terminal telopeptide of the α2 chain of procollagen I, respectively; H-a-α1Cp and L-a-α1Cp, heavy and light chains of an anti-α1Cp immunoglobulin, respectively; H-a-α2Ct and L-a-α2Ct, heavy and light chains of an anti-α2Ct immunoglobulin, respectively; + and – indicate the presence or the absence of selected compounds.

FIGURE 4.

Kinetics of de novo formation of collagen fibrils. Panel a represents analysis of collagen present in fibril and monomer fractions at indicated time points. The upper row depicts control samples, whereas the middle and lower rows show kinetics of fibril formation in the presence of 180 μg/ml anti-α1Cp and anti-α2Ct antibodies, respectively. In the left lane molecular mass markers are indicated. Panel b demonstrates morphology of individual fibrils formed in the presence or absence of tested antibodies. P, pellet fractions that represents collagen fibrils; S, supernatant fractions that represents collagen monomers; α1 and α2, collagen I α chains; Ctrl, fibril formation in the absence of antibodies; a-α1Cp and a-α2Ct, fibril formation in the presence of antibodies against the C-terminal propeptide of the α1 chain or the C-terminal telopeptide of the α2 chain of procollagen I, respectively.

FIGURE 5.

Analysis of morphology of keloid-like constructs formed subcutaneously in nude mice. Panel a shows keloid-like constructs (arrow) implanted under skin of a mouse. Panel b presents a sponge-like scaffold that was employed to host human keloid-derived fibroblasts. Panel c represents overall morphology of a keloid-like construct harvested after 1 month of culture under skin of a nude mouse. Panel d demonstrates immunostaining for human-specific vimentin to confirm human origin of cells cultured in subcutaneous scaffolds, whereas panels e and f present Sirius red staining of collagen deposits. Panels g and h show electron microscopy images of collagen fibrils present in sections of keloid-like constructs. Individual panels show morphology of collagenous matrices formed in the absence (e and g) or presence (f and h) of inhibitory antibodies. Ctrl, keloid-like constructs cultured in the absence of antibodies; a-α2Ct, keloid-like constructs cultured in the presence of inhibitory antibodies against the C-terminal telopeptide of the α2 chain of procollagen I. Scale bars are included for specific sets of panels.