Molecular Imaging of Fibrosis Figure 2. Interaction of collagelin with collagen. A: B-collagelin was immobilized on a streptavidin-coated sensorchip (,20 RU). Collagen (10 mg/ml) was injected over the sensorchip. A representative sensorgram (dark line) and interaction fit (gray line) are shown after subtracting the non-specific background signal from a control flow cell coated with an irrelevant peptide. B: B-collagelin (250, 500 mg.mL21) was injected over a collagen-coated sensorchip. Sensorgrams (black) and interaction fits (gray) are shown. Representative sensorgrams are shown after subtracting the non-specific response from the irrelevant peptide. C: B-collagelin or control peptide (50 mg. mL21) were incubated with immobilized, fibrillar, type-I collagen in microtitration plates, and detected using HRP-coupled extravidin. In competition experiments, collagelin was mixed with GPVI-Fc (50 mg.mL21), 9012.2 IgGs (50 mg.mL21) or 3J24.2 IgGs (50 mg.mL21) before being added to collagen-coated wells. Means6SD (n = 3) are presented; *** p,0.01. D: B-collagelin (50 mg.mL21, black) or B-Pc (gray) were incubated with immobilized collagen I or III, CRP, fibrinogen, fibronectin, vitronectin and laminin in microtitration plates, and detected as above. Means6SD (n = 3) are shown. doi:10.1371/journal.pone.0005585.g002 explained by a lower number of accessible GPVI binding sites due to fiber structure and interaction with other proteins of the matrix in this normal tissue. Specific tracers for collagen represent a challenge and considerable interest in various imaging methods, including radionucleide imaging and MRI, is currently developing. A gadolinium-based, collagen-targeting contrast agent, EP-3533, has recently been reported to make it possible to carry out in vivo molecular MR imaging of fibrosis in another model of scarred infarction, in the mouse [28]. Collagelin appears to have greater affinity for collagen than EP-3533 [9]. In addition, compared to MRI, specific radionucleide imaging by SPECT or PET presents the advantages of having much higher sensitivity, and no toxicity, since a very low tracer dose (pico- or nanomolar) is sufficient to obtain an accurate target-to-background ratio. In a recent study, direct comparison of signals obtained with nanoparticles labeled with both iron oxide and Cu-64 showed that the sensitivity of PET is 50-fold higher than that of MRI [29]. This, explains why SPECT and PET imaging are undergoing considerable develop- ment worldwide, especially in malignant and cardiovascular diseases. Similar developments are also expected in other medical fields, in which the targeting of relevant processes is potentially clinically useful. This is the first time, to our knowledge, that a mimotope of GPVI has been produced with proven in vitro and in vivo collagen- specific targeting properties. Optimizing collagelin should now be considered to improve the in vivo signal to noise ratio. Improved derivatives of collagelin should be developed by further increasing the affinity of the molecule for collagen, and improving the labeling procedures. Indeed, the labeled streptavidin-B-collagelin complex has the disadvantages of hepatic accumulation and a long half life. However, these difficulties could be avoided by using PLoS ONE | www.plosone.org 5 May 2009 | Volume 4 | Issue 5 | e5585