Non-Invasive Molecular Imaging of Fibrosis Using a Collagen-Targeted Peptidomimetic of the Platelet Collagen Receptor Glycoprotein VI

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 10−7 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.Psycholog

Interaction of collagelin with collagen.

<p>A: B-collagelin was immobilized on a streptavidin-coated sensorchip (∼20 RU). Collagen (10 µg/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 µg.mL⁻¹) 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 µg. mL⁻¹) 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 µg.mL⁻¹), 9012.2 IgGs (50 µg.mL⁻¹) or 3J24.2 IgGs (50 µg.mL⁻¹) before being added to collagen-coated wells. Means±SD (n = 3) are presented; *** p<0.01. D: B-collagelin (50 µg.mL⁻¹, 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. Means±SD (n = 3) are shown.</p

<i>In vivo</i> scintigraphy, <i>ex vivo</i> myocardial autoradiography and histology using 99mTc-collagelin.

<p>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.</p

Identification of collagelin.

<p>A: <i>Identification of 9O12.2 binding bacterial clones</i>. 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 (−). The sequence of clone 14 was retained for peptide synthesis. B–D <i>Surface plasmon resonance (SPR) analysis of collagelin binding to 9O12.2</i>. In B, increasing concentrations of the 9O12.2 IgGs were passed over the sensorchip (4, 6, 8, 10 µg/ml from bottom to top). In C: 9O12.2 IgG (8 µg/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 µg/ml) was injected over immobilized B-collagelin in the absence (black) or presence of recombinant soluble GPVI (25 µg/ml) (gray). Representative sensorgrams are shown after subtracting the non-specific response from a control flow cell coated with an irrelevant peptide.</p

<i>In- vivo</i> scintigraphy, <i>ex vivo</i> myocardial autoradiography and histology using ^99mTc-streptavidin-B-collagelin.

<p>A: Planar thoracic scintigraphy of a control rat (sham). B: Planar and tomographic (frontal and sagittal views) thoracic images of a rat with a fibrotic myocardial infarction: a hot-spot (arrows) can be seen in the left ventricular myocardial area. C: Corresponding myocardial autoradiography and histology (collagen-specific picrosirius red staining,), confirming tracer uptake in the thin, fibrotic (red) myocardial scar (arrows). D: Control experiment: no activity can be seen in the myocardial scar of a rat injected with irrelevant ^99mTc-streptavidin-B-Pc.</p

(A): Amino-acid alignment (Fasta format) of the 20 clones sequenced after screening of the FliTrx random peptide display library against immobilized 9O12 IgG.

<p>The sequence of the peptide that has been selected for synthesis is underlined.</p

Histochemical analysis of peptide binding to tissue collagen.

<p>A: Frozen sections of rat aorta were incubated with B-collagelin or control peptide (200 µg/mL) and detected using HRP-coupled streptavidin. Sections were counter-stained with hematoxylin. Contiguous serial sections were stained with picrosirius red. In a competition experiment, the peptide was mixed with anti-GPVI IgG 9O121.2 (300 µg/mL). B: Paraffin embedded sections of rat tail tendon were treated as above.</p