Molecular Imaging of Fibrosis Figure 1. Identification of collagelin. A: Identification of 9O12.2 binding bacterial clones. Proteins from bacterial clones were separated by electrophoresis under non-reducing conditions, and analysed by immunoblot using the 9O12.2 IgG. The band at ,63 kDa corresponds to the FliTrx fusion protein containing a peptide recognized by 9O12.2. Results are from six selected clones (10, 12, 14, 15, 16, 18), and from one clone selected from the same library but using an irrelevant antibody (2). The sequence of clone 14 was retained for peptide synthesis. B–D Surface plasmon resonance (SPR) analysis of collagelin binding to 9O12.2. In B, increasing concentrations of the 9O12.2 IgGs were passed over the sensorchip (4, 6, 8, 10 mg/ml from bottom to top). In C: 9O12.2 IgG (8 mg/ml) was injected over immobilized B-collagelin that was either non-reduced (black) or reduced by DTT (gray) on the sensorchip. In D, 9O12.2 IgG (5 mg/ml) was injected over immobilized B-collagelin in the absence (black) or presence of recombinant soluble GPVI (25 mg/ml) (gray). Representative sensorgrams are shown after subtracting the non-specific response from a control flow cell coated with an irrelevant peptide. doi:10.1371/journal.pone.0005585.g001 operated rats respectively (p,0.01). Histological analysis was performed on frozen sections of the myocardium obtained 6– 8 hours after the injection. Autoradiography showed that the areas labeled by 99mTc-streptavidin-collagelin superimposed on the collagen fibers of the infarct scars stained by picrosirius red (Figure 4C). In contrast, there was little or no uptake of the non- binding peptide Pc (Figure 4D). Mean infarct-to-remote myocar- dium radioactivity ratios were of 2.5260.20 and 1.8260.32 with collagelin and Pc respectively (p,0.006). Any fibrotic adhesions in contact with the myocardial scar (n = 1), concentrated high levels of the radiotracer. In a second set of experiments, collagelin and Pc were directly labeled with Tc-99m to avoid the drawbacks of high, non-specific liver uptake and slow blood clearance which could be attributed to streptavidin, and impaired the target-to-background ratio of the images. Radiolabeling efficiency exceeded 96% at 30 min, and exceeded 90% at 6 h, indicating high labeling stability. Ex vivo incubation of myocardial sections in the presence of radiotracers demonstrated that 99mTc-collagelin accumulated in the scarred tissue, whereas the uptake of 99mTc-Pc was significantly lower (p,0.05). After intravenous injection (1 nmol, 70 MBq) in rats, 99mTc-collagelin demonstrated fast blood clearance (half life: 560.5 min), very early (,5 min) biliary excretion, low liver uptake (0.36% of injected dose per gram of tissue), fast digestive excretion occurring as soon as 10 min post-injection, and low renal uptake (n = 6). No thyroid or gastric uptake was observed until 6 h post- injection, suggesting that the radiolabeling was very stable in vivo. Figure 5A shows representative planar and SPECT images PLoS ONE | www.plosone.org 3 May 2009 | Volume 4 | Issue 5 | e5585