Molecular Imaging of Fibrosis Figure 5. In vivo scintigraphy, ex vivo myocardial autoradiography and histology using 99mTc-collagelin. A: Planar thoracic scintigraphy of a rat with fibrotic myocardial infarction: a clear hot-spot (arrows) xcan be seen in the left ventricular myocardial area. B: From left to right, corresponding myocardial histology (Masson’s trichrome, picrosirius red) and autoradiography, confirming tracer uptake in the thin, fibrotic (red) myocardial scar (arrow heads). C: Control experiment: very low activity is observed in the myocardial infarction in a rat injected with irrelevant 99mTc- Pc. doi:10.1371/journal.pone.0005585.g005 directly labeled collagelin, which looks very promising on the basis of our data. The circulation time of the peptide could be prolonged by a moderate increase in its size, such as, for example, by PEGylation or by constructing a dimeric peptide with increased avidity for collagen. Radiolabeling with positron emitters could also be considered for the absolute PET quantification of fibrosis in vivo, which would allow the non invasive therapeutic follow-up of fibrotic diseases. Collagelin is thus a new and original collagen-specific probe with a very wide field of possible applications as a tracer of fibrotic tissues, in both non-vascular and vascular disorders. Material and Methods Screening the FliTrxTM peptide library against the anti- GPVI antibody Anti-GPVI 9O12.2 IgGs and soluble recombinant GPVI-Fc were obtained as previously described [13]. The cyclic dodecapep- tide bacterial FliTrxTM Random Peptide Display Library was from Invitrogen (San Diego, CA). Bacterial cultures and general panning methods were conducted according to the manufacturer’s protocol. Briefly, the pFliTrxTM vector with the PL promoter from the bacteriophage that drives expression was propagated in E. coli strain GI826. after induction of the expression of the thioredoxin– flagellin fusion proteins containing the peptide inserts, bacterial culture in NaCl (150 mM) containing a-methyl mannoside (10 mg.mL21) and non-fat dry milk (10 mg.mL21) were incubated in tissue culture plates coated with 9O12.2 IgG. After several washing steps, bound bacteria were detached and amplified before being subjected to a new round of panning. After five rounds, bacterial colonies plated on solid medium were randomly taken, amplified and induced for further identification. Characterization of the clones Identification of positive clones was done by Western blotting using 9O12 IgG. Briefly, proteins of selected bacterial clones were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with the 9O12. IgG and a phosphatase alkaline- coupled secondary antibody to mouse IgG [13]. Plasmid DNA was isolated using standard protocols, and the nucleotide sequences were determined using the FLITrxTM forward sequencing primer (59-ATT CAC CTG ACT GAC GA-39). The peptide sequences were deduced from DNA sequencing. Peptide synthesis One peptide base on the sequence of the selected clone (underlined) [SGSGCPGRVMHGLHLGDDEGPC] was synthe- sized. The peptide was or was not coupled to biotin at its N- terminus via a short flexible spacer (SGSG), and made cyclic by disulfide bridging of the flanking Cys. The carboxyl function at the C-terminus was substituted by an amide. Peptides were prepared by NeoMPS (Strasbourg, France), and their quality controlled by HPLC and mass spectrometry. The irrelevant, non-cyclic, biotinylated peptide P1 (SGSGVNVYAVTKENTIINPSENGD) and the cyclic peptide PLoS ONE | www.plosone.org 7 May 2009 | Volume 4 | Issue 5 | e5585