Table 1. (A): Amino-acid alignment (Fasta format) of the 20 clones sequenced after screening of the FliTrx random peptide display library against immobilized 9O12 IgG. clones 1–10 clone11 clones12–13 clone14 clone15 clone16 clone17 clone18 clone19 clone20 RFMHGLQLWADE RVMHGLQLWADE RVMHGLQLWADE RVMHGLHLGDDE RVMHGLHLWDDE RVMHGLQLWDDE RVMHGLHLWADE FVMHGLHLGDDE PVMHGLHLWDDE RVMHGLLLGADE The sequence of the peptide that has been selected for synthesis is underlined. doi:10.1371/journal.pone.0005585.t001 acquired two hours after injection, which reveal a significant uptake of the probe in the cardiac area of all but one of the rats with MI scars (n = 8). No tracer uptake was observed in the cardiac area of sham-operated rats (n = 6). Heart-to-lung ratios calculated on planar images were 2.0860.17 and 1.6160.23 in MI and sham-operated rats respectively (p,0.01). Comparison of autora- diography and histology findings for frozen myocardial sections showed that the 99mTc-collagelin coincided with the collagen fibers of the scars (Figure 5B). There was little or no uptake of Pc by the scar (Figure 5C). Mean infarct-to-remote myocardium activity ratios were 2.9260.53 and 1.8360.3 for collagelin and Pc respectively (p,0.008). Any fibrotic adhesions in contact with the myocardial scar (n = 3) also concentrated high levels of 99mTc- B-collagelin. In order to confirm the specificity of imaging with collagelin, the tracer was tested in a mouse model of lung fibrosis. First, an histochemical analysis was performed on sections of healthy and fibrotic lungs (fig 6A). Staining by collagelin coincided with the collagen rich lesions. In vivo imaging was next realized 14 days after instillation of bleomycin. One hour after injection of 99mTc-B- collagelin, scintigraphic imaging showed the uptake of the tracer in the pulmonary area of the mice that received bleomycin as compared to control mice (fig 6B) with lung to muscle ratios of 3.4260.35 and 1.860;14 respectively (p,0.005, n = 5). Autora- diographic studies confirmed higher 99mTc-B-collagelin uptake on sections of lung of mice with pulmonary fibrosis as compared to controls with heterogeneous distribution matched with that of picrosirius staining (fig 6C). Discussion We have developed a specific probe for collagen which makes possible to obtain in vivo, specific SPECT imaging of fibrosis, using a new approach combining the advantages of the specificity of a collagen receptor, and the diffusing properties of a peptide. Combinatorial peptide libraries are usually screened for epitope mapping such as, for example, the epitopes of antibodies directed towards vWF and to its platelet receptor GPIb [17,18]. In the case of the anti–GPVI 9O12.2 moAb, the use of a linear phage display library [19], and of a constrained bacterial library (this study) indicated that the epitope is conformational, but cannot be used to identify a linear or discontinuous motif. Structural studies; such as X-ray analysis and/or modeling, will help to determine the epitope structure. Molecular Imaging of Fibrosis Another increasing application of peptide libraries is to identify peptides with specific biological activities [20,21]. Peptides mimicking proteins of interest obtained by screening libraries versus blocking antibodies, designated mimotopes, are proposed as potential targets, with either therapeutic or imaging applications. The peptide we are describing here reproduces the specificity of GPVI and thus. mimics, at least to some degree, the collagen- binding site of GPVI. It binds in vitro to type-I collagen with high affinity (1027 M) and also to type III collagen. Ex vivo collagen staining on sections of rat tail tendon, which is composed of type I collagen, and of the vascular wall containing high levels of types I and III collagens, indicates that collagelin acts as a collagen probe. A GPVI-based method of isotopic imaging has been proposed [22]. I125/I123-labeled soluble recombinant GPVI-Fc was used for non-invasive imaging of vulnerable atherosclerotic plaques in ApoE2/2 mice in vivo by SPECT, and ex vivo by autoradiography. However, slow blood clearance leading to high background activity and low signal-to-noise ratio was observed. GPVI-Fc, which consists of the extracellular part of GPVI fused to the IgG Fc domain [12], has a high molecular mass that prevents it from diffusing into interstitial tissues, and also has antithrombotic properties [23]. In contrast, collagelin has the advantages of being small, which facilitates its diffusion to its target, and of having no effect on platelet function. Peptides with collagen-binding properties have previously been reported: cyclic peptides mimicking the collagen-binding site of vWF bound to rat tail collagen, and inhibited vWF binding to calfskin and human collagen, but only at high concentrations [24]; phage display also made it possible to identify the collagen-binding protein rNecH1 from Necator americanus [25], but no further use of these peptides or proteins has been reported. Recently, CNA35, the collagen binding part of a bacterial protein, has been successfully used as a fluorescent probe for collagen [26]. However, it cannot be used for in vivo imaging of organs since it does not cross the endothelial barrier [27]. Cy5.5-RGD which targets integrins on myofibroblasts and a sequence in procollagen is an indirect probe for fibrinogenesis but it does not bind to mature collagen type I or III fibers [8]. Since collagelin is a low molecular mass collagen-specific probe, we postulated that it could be used as a collagen tracer in vivo. The results reported here demonstrate that radiolabeled collagelin does not accumulate to any significant degree in healthy tissues or in blood, but that it clearly accumulates in post-infarct myocardial scars. This experimental model yields a clearly identifiable, dense but limited area of fibrosis, generally located at the apex of the heart. Specific accumulation of radiolabeled collagelin provided good contrast molecular imaging of the fibrotic scar, clearly distinguishable from the healthy myocardium. Images obtained by planar scintigraphy in this model, in which the scar is often thin and small in volume, indicate that collagelin represents a powerful probe for detecting fibrosis. This was confirmed by the specific accumulation of the radiotracer in lung fibrotic lesions. In this model, fibrosis that is looser than in the MI scar, was nevertheless identified by the probe. At the time of the observation, we did not notice any accumulation of the tracer in the skin of the animals, that would have interfered with the signal of interest. We however have observed a low level of collagelin uptake in bone marrow. It thus appears there is a preferential uptake of the tracer by the fibrous collagen as compared to constitutive collagen in organs. Indeed, GPVI does not bind to monomeric collagen but interacts with a structural motif present on the cross-linked helicoidal structure of collagen [10]. The lower uptake of collagelin by the organs normally rich in collagen as compared to fibrosis, could be PLoS ONE | www.plosone.org 4 May 2009 | Volume 4 | Issue 5 | e5585