molecular imaging of cardiac fibrosis was reported using radiotracers specific for targets co-expressed or co-located with fibrosis in patients and mice with post-infarction cardiomyopathy: 18F-fluorobenzoyl-lisinopril specific for angiotensin-converting enzyme [5], Tc-99m losartan specific for angiotensinII receptors [6], 99mTc-Cy5.5 RGD imaging peptide targeting proliferating myofibroblats [7,8]. However such indirect tracers are not adapted to all clinical situations involving fibrosis, because of different physiopathology and the need to detect fibrosis as well as fibrogenesis. Specific and direct tracers for the molecular imaging of fibrosis, especially collagen-targeting molecules, constitute a challenge and a potentially wide field of interest for imaging methods, including radionucleide imaging and MRI [9]. The inherent collagen binding properties of the collagen receptors should make them good models for developing collagen probes. Collagen receptors interact with the triple helical structures of collagen fibrils [10]. Several members of the integrin family, including the alpha1beta1, alpha2beta1 and alpha1beta1 integrins, are widely expressed collagen receptors, but since they also bind to other matrix proteins, they are not suitable for specifically targeting collagen. The immunoadhesin glycoprotein VI [11,12] has good affinity and high specificity for type-I and type-III collagens and has been extensively characterized. GPVI seems to be an attractive target for the development of collagen probes. Soluble recombinant GPVI has even been proposed as a tool for in vivo imaging of collagen exposed by unstable atherosclerotic plaques. However, an efficient collagen probe must be small enough to gain access to the interstitial space. We therefore decided to focus on peptides that mimicGPVI, and have taken advantage of a monoclonal antibody, 9O12.2, which binds GPVI with a high affinity, and neutralizes the interaction between GPVI and collagen in vitro and in vivo [13,14]. We hypothesized that the 9O12.2 epitope must, at least in part, overlap with the collagen binding-site on GPVI. Using a bacterial display approach, a peptidomimetic of GPVI has been identified. This peptide, designated collagelin, exhibits collagen-binding properties both in vitro and ex vivo. The suitability of collagelin as a probe for the molecular imaging of fibrosis is assessed in vivo by isotopic imaging of scars in a rat model of healed myocardial infarction and a mouse model of lung fibrosis. Results Identification of 9O12.2-binding peptides After five rounds of panning the combinatorial bacterial peptide library using 9O12.2 IgG, 20 clones were selected that produced a flagellar fusion protein recognized by 9O12.2 on immunoblots (Figure 1A). DNA sequencing of all the inserts showed some redundancy and identified 9 peptide sequences (Table 1) with 7 common residues and differing from each other by one to 5 residues. None of these sequences was registered in any database. Sequence 14, RVMHGLHLGDDE (single letter amino acid code), was selected for synthesis. A constrained peptide, designated collagelin, was synthesized and conjugated with biotin (B– collagelin). The molecular masses of the peptide and the conjugate (2155 and 2404 Da respectively) and their purity (.95%) were determined by mass spectrometry. Surface plasmon resonance experiments showed that 9O12.2 IgG bound to immobilized B–collagelin in a dose-dependent manner (Figure 1B) with a KD of 1026 M. The 9O12.2 IgG did not bind to reduced collagelin (Figure 1C), confirming that the 9O12.2 epitope is conformational [13]. Binding of 9O12.2 IgG to collagelin was inhibited in the presence of soluble GPVI (Figure 1D), indicating that GPVI and collagelin competed for binding to 9O12.2. Molecular Imaging of Fibrosis Binding to collagen Since collagelin mimicked the 9O12.2epitope, at least in part, it was assumed that it also mimicked the collagen binding site of GPVI. Using SPR, type I collagen was found to bind to a B- collagelin-coated sensorchip (Figure 2A). Furthermore, B-collage- lin bound to a sensorchip coated with fibrillar type I collagen in a dose-dependent manner with a KD of 1.1027 M. (Figure 2B). In contrast, a non-relevant biotinylated peptide (B-Pc), did not bind to collagen. B–collagelin also bound to collagen-coated plates, and this binding was completely inhibited in the presence of GPVI-Fc or of 9O12.2 IgGs (Figure 2C), whereas the anti-GPVI monoclonal antibody 3J24.2, which binds to an epitope distinct from 9O12.2 [15], did not inhibit B-collagelin interaction with collagen. These results demonstrate that collagelin and GPVI bind to sites on collagen that must overlap or be identical. The binding specificity of collagelin was determined using the GPVI specific ligand, collagen related peptide (CRP), and various different macromolecules from the extracellular matrix. The non- relevant peptide (Pc) did not bind to any of these proteins. Collagelin bound to type III collagen and to CRP, confirming its GPVI-like specificity. The binding of collagelin to vitronectin fibrinogen or fibronectin was found either to be weak or not to occur at all (Figure 2D). In contrast, collagelin consistently bound to laminin, previously identified as a GPVI accessory ligand [16]; the sites of GPVI that interact with laminin and collagen thus share a common structure that is mimicked by collagelin. Nevertheless, SPR analysis indicated that the affinity of collagelin for laminin was two orders lower than for collagen (KD of 1.83 1025 M). Ex vivo labeling of collagen with collagelin The capacity of collagelin to interact with collagen was analyzed histochemically on frozen sections of rat aorta and tail tendon. Positive and specific brown staining developed when sections were treated with B-collagelin, but not with B-Pc. The staining produced by collagelin coincided with that of collagen stained with picrosirius red in serial sections (Figure 3). The intensity of the staining by collagelin decreased in the presence of 9O12.2 IgG, confirming the specificity of the interaction. In vivo isotopic molecular imaging of fibrosis Collagelin was evaluated as a potential probe for fibrosis in vivo in rats. A model of healed myocardial infarction was used so that the uptake of the probe in healthy tissues could be compared to that in the collagen-rich fibrotic scar formed after myocardial remodeling. Imaging was performed at least three weeks after MI after the acute inflammatory phase and when the scar is well formed. First, B-collagelin and B-Pc were mixed with 99mTc-streptavi- din. Ex vivo, the uptake of 99mTc-streptavidin-coupled-collagelin by rat aortic collagen was three times greater than the uptake of the control peptide Pc (p,0.001), and the uptake of labeled collagelin was inhibited by 9012.2 IgG. After intravenous injection of 1 nmol (70 MBq) to rats, SPECT imaging revealed high uptake by the liver, and slow blood clearance. Visual analysis of planar and SPECT images 4 h–6 h post injection revealed significant uptake of 99mTc-streptavidin-collagelin in the cardiac area of all rats with an MI scar (n = 6), whereas no tracer uptake was observed in the cardiac area of sham-operated rats (n = 6) (Figures 4A–4B). Significant tracer uptake was also observed in the thoracotomy scar in 2 out of 6 rats with MI, and in 2 of 6 sham-operated rats. Mean heart-to-lung ratios which represent the contrast between the target and the surrounding tissues, were calculated on planar images as being of 2.7660.36 and 1.9560.28 for MI and sham- PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5585