Non-Invasive Molecular Imaging of Fibrosis Using a Collagen-Targeted Peptidomimetic of the Platelet Collagen Receptor Glycoprotein VI Julien Muzard1,5., Laure Sarda-Mantel2,5,6., Ste´ phane Loyau1,5, Alain Meulemans2,5,6, Liliane Louedec1,5, Claudie Bantsimba-Malanda4,5, Florence Hervatin5, Joe¨ lle Marchal-Somme4,5, Jean Baptiste Michel1,5,6, Dominique Le Guludec2,5,6, Philippe Billiald3, Martine Jandrot-Perrus1,5,6* 1 INSERM, U698, Hoˆ pital Bichat, Paris, France, 2 INSERM, U773, CRB3, Faculte´ Xavier Bichat, Paris, France, 3 Museum National d’Histoire Naturelle, CNRS-FRE 3206, Paris, France, 4 INSERM, U700, Faculte´ Xavier Bichat, Paris, France, 5 Universite´ Paris7, Paris, France, 6 AP-HP, H oˆpital Bichat, Paris, France Abstract Background: Fibrosis, which is characterized by the pathological accumulation of collagen, is recognized as an important feature of many chronic diseases, and as such, constitutes an enormous health burden. We need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in various disorders. Collagen targeting molecules are therefore of interest for potential in vivo imaging of fibrosis. In this study, we developed a collagen-specific probe using a new approach that takes advantage of the inherent specificity of Glycoprotein VI (GPVI), the main platelet receptor for collagens I and III. Methodology/Principal Findings: An anti-GPVI antibody that neutralizes collagen-binding was used to screen a bacterial random peptide library. A cyclic motif was identified, and the corresponding peptide (designated collagelin) was synthesized. Solid-phase binding assays and histochemical analysis showed that collagelin specifically bound to collagen (Kd 1027 M) in vitro, and labelled collagen fibers ex vivo on sections of rat aorta and rat tail. Collagelin is therefore a new specific probe for collagen. The suitability of collagelin as an in vivo probe was tested in a rat model of healed myocardial infarctions (MI). Injecting Tc-99m-labelled collagelin and scintigraphic imaging showed that uptake of the probe occurred in the cardiac area of rats with MI, but not in controls. Post mortem autoradiography and histological analysis of heart sections showed that the labeled areas coincided with fibrosis. Scintigraphic molecular imaging with collagelin provides high resolution, and good contrast between the fibrotic scars and healthy tissues. The capacity of collagelin to image fibrosis in vivo was confirmed in a mouse model of lung fibrosis. Conclusion/Significance: Collagelin is a new collagen-targeting agent which may be useful for non-invasive detection of fibrosis in a broad spectrum of diseases. Citation: Muzard J, Sarda-Mantel L, Loyau S, Meulemans A, Louedec L, et al. (2009) Non-Invasive Molecular Imaging of Fibrosis Using a Collagen-Targeted Peptidomimetic of the Platelet Collagen Receptor Glycoprotein VI. PLoS ONE 4(5): e5585. doi:10.1371/journal.pone.0005585 Editor: Annarosa Leri, Harvard Medical School, United States of America Received October 29, 2008; Accepted April 17, 2009; Published May 18, 2009 Copyright: 2009 Muzard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by INSERM, University Paris 7, the Leducq Foundation, grant 2004002917 from the Fondation de France, and ET7-464 from the Fondation de l’Avenir. J. Muzard was supported by Stago (Asnieres, France) and the Fondation pour la Recherche Medicale and M.Jandrot-Perrus by the Bettencourt-Schueller Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: martine.jandrot-perrus@inserm.fr . These authors contributed equally to this work. Introduction Collagen, a major component of the extracellular matrix (ECM), is one of the determinants of tissue structure. Fibrosis is characterized by the pathological accumulation of collagen, and is increasingly recognized as an important feature of many chronic diseases, and as such, represents an enormous health burden [1]. It is estimated that 45% of deaths in the United States can be attributed to conditions associated with fibrosis. In the absence of a non-invasive specific marker, the only method available for quantifying fibrosis is tissue biopsy, which is invasive and carries a risk of complications in a variety of organs and cannot be easily repeated. Functional tests are currently used to assess the degree to which organs are affected, but functional impairment only occurs in the presence of a relatively high degree of fibrosis. This means that we still need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in many disorders in which fibrosis is of major prognostic interest. For this purpose, quantitative imaging methods have the advantage over blood biomarkers of being able both to quantify and localize the fibrotic process. Recent studies have shown that transient echography or MRI elastography provide ways to assess liver fibrosis by non- invasively measuring liver stiffness in adult patients [2,3]. Preliminary experiments have also been performed using diffu- sion-weighted MRI to quantify liver fibrosis [4]. However, these techniques are not specific for fibrosis and may suffer from a lack of sensitivity, high levels of fibrosis being necessary before tissue elasticity and diffusion properties are impaired. Recently, PLoS ONE | www.plosone.org 1 May 2009 | Volume 4 | Issue 5 | e5585