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. 2019 Apr 13;9(10):2868-2881.
doi: 10.7150/thno.28892. eCollection 2019.

Imaging fibrosis in inflammatory diseases: targeting the exposed extracellular matrix

Nicolas Beziere  1 , Kerstin Fuchs  1 , Andreas Maurer  1 , Gerald Reischl  1 , Jürgen Brück  2 , Kamran Ghoreschi  2 , Birgit Fehrenbacher  2 , Daniel Carvajal Berrio  3 , Katja Schenke-Layland  3   4   5 , Ursula Kohlhofer  6 , Leticia Quintanilla-Martinez  6 , Meinrad Gawaz  7 , Manfred Kneilling  1   2 , Bernd Pichler  1
Affiliations

Imaging fibrosis in inflammatory diseases: targeting the exposed extracellular matrix

Nicolas Beziere et al. Theranostics. .

Abstract

In a variety of diseases, from benign to life-threatening ones, inflammation plays a major role. Monitoring the intensity and extent of a multifaceted inflammatory process has become a cornerstone in diagnostics and therapy monitoring. However, the current tools lack the ability to provide insight into one of its most crucial aspects, namely, the alteration of the extracellular matrix (ECM). Using a radiolabeled platelet glycoprotein VI-based ECM-targeting fusion protein (GPVI-Fc), we investigated how binding of GPVI-Fc on fibrous tissue could uncover the progression of several inflammatory disease models at different stages (rheumatoid arthritis, cutaneous delayed-type hypersensitivity, lung inflammation and experimental autoimmune encephalomyelitis). Methods: The fusion protein GPVI-Fc was covalently linked to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and subsequently labeled with 64Cu. We analyzed noninvasively in vivo64Cu-GPVI-Fc accumulation in murine cutaneous delayed-type hypersensitivity, anti-glucose-6-phosphate isomerase serum-induced rheumatoid arthritis, lipopolysaccharide-induced lung inflammation and an experimental autoimmune encephalomyelitis model. Static and dynamic Positron Emission Tomography (PET) of the radiotracer distribution was performed in vivo, with ex vivo autoradiography confirmation, yielding quantitative accumulation and a distribution map of 64Cu-GPVI-Fc. Ex vivo tissue histological staining was performed on harvested samples to highlight the fusion protein binding to collagen I, II and III, fibronectin and fibrinogen as well as the morphology of excised tissue. Results:64Cu-GPVI-Fc showed a several-fold increased uptake in inflamed tissue compared to control tissue, particularly in the RA model, with a peak 24 h after radiotracer injection of up to half the injected dose. Blocking and isotype control experiments indicated a target-driven accumulation of the radiotracer in the case of chronic inflammation. Histological analysis confirmed a prolonged accumulation at the inflammation site, with a pronounced colocalization with the different components of the ECM (collagen III and fibronectin notably). Binding of the fusion protein appeared to be specific to the ECM but unspecific to particular components. Conclusion: Imaging of 64Cu-GPVI-Fc accumulation in the ECM matrix appears to be a promising candidate for monitoring chronic inflammation. By binding to exposed fibrous tissue (collagen, fibronectin, etc.) after extravasation, a new insight is provided into the fibrotic events resulting from a prolonged inflammatory state.

Keywords: fibrosis; glycoprotein VI; positron emission tomography.

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Conflict of interest statement

Competing interests: M. Gawaz is a shareholder of advanceCOR GmbH.

Figures

Figure 1
Figure 1
Glycoprotein-VI (GPVI), fusion protein of GPVI with Immunoglobulin 1 Fc domain (GPVI-Fc) and [64Cu]Cu-NOTA-GPVI-Fc (64Cu-GPVI-Fc). (A) The GPVI is expressed by platelets and displays a high collagen affinity. (B) GPVI-Fc binds to extracellular matrix fibers and prevents platelet aggregation. 64Cu-GPVI-Fc is obtained by conjugation of GPVI-Fc to the chelator p-SCN-Bn-NOTA and complexation using a 64CuCl2 solution. GPIb: glycoprotein Ib; GPVI: glycoprotein-VI; IgG: immunoglobulin; vWF: von Willebrand factor.
Figure 2
Figure 2
64Cu-GPVI-Fc in cutaneous delayed-type hypersensitivity reaction (DTHR). (A) Representative section images of 64Cu-GPVI-Fc distribution 24 h after injection in an animal presenting unilateral DTHR with in vivo CT (top), highlighting the inflamed (dashed circle, right) ear and the contralateral unstimulated ear (dotted circle, left), PET (middle) and overlay (bottom). The ears were laid flat on the holder surface during acquisition. (B) Accumulation of 64Cu-GPVI-Fc 24 h after injection in control animal ears (Control, n=3) or after 1 (Acute, Ac., n=3) or 5 inflammatory challenges leading to DTHR (Chronic, Ch., n=6). The results obtained with a coinjection of nonradioactive Cu-GPVI-Fc for each group are indicated (Blocked). They are shown as single values with geometric means and a geometric standard deviation. P values are indicated using * (P ≤ 0.05), ** (P ≤ 0.01) and *** (P ≤ 0.001). % ID/cc: percentage of injected dose per cubic centimeter.
Figure 3
Figure 3
GPVI-Fc distribution at the microscopic scale in a cutaneous delayed-type hypersensitivity reaction (DTHR) model. Movat (top left) and Masson (bottom left) staining of sections of chronically inflamed DTHR ears. Fluorescence images of nuclei (blue), fibronectin (red) and glycoprotein-VI fusion protein (GPVI-Fc) (green) distribution in histological samples (top middle), with selected overlay of fibronectin and GPVI-Fc (yellow) (bottom middle). Bright field image of Picro Sirius stain (top right), along with an image of the same sample observed under polarized light. Scale bar: 150 µm.
Figure 4
Figure 4
64Cu-GPVI-Fc in Rheumatoid Arthritis (RA). (A) Representative in vivo PET images of 64Cu-GPVI-Fc distribution 1 h (top row), 24 h (middle row) and 48 h (bottom row) after injection in a mouse before (left column), 3 days (middle column) and 6 days (right column) after anti-glucose-6-phosphate isomerase (GPI) stimulation. (B) Accumulation kinetics of 64Cu-GPVI-Fc in animals on day 0, 3 and 6 of GPI serum stimulation 1 h, 24 h and 48 h after tracer injection (n=3 animals, both sides). (C) Accumulation profile of 64Cu-GPVI-Fc in RA animals on day 6 after stimulation 1 h, 24 h and 48 h after injection of 64Cu-GPVI-Fc alone (Ref., n=13 animals, both sides), with nonradioactive Cu-GPVI-Fc co-injection (Blck. n=7) and of a labeled isotype antibody (Iso. n=3). The results are shown as single values with a geometric means and geometric standard deviation. P values are indicated using * (P ≤ 0.05), ** (P ≤ 0.01) and *** (P ≤ 0.001). % ID/cc: percentage injected dose per cubic centimeter.
Figure 5
Figure 5
Microscopic view of 64Cu-GPVI-Fc in the ankles of rheumatoid arthritis mice. (A) Histological stains of the ankle of an RA animal on day 6 after stimulation. Left: hematoxylin and eosin (H&E) stain, right: Masson stain (top: 12.5x, bottom 200x magnification). (B) Fluorescence image overlay of nuclei (blue), GPVI-Fc (green) and fibrinogen (left, red) or collagen III (right, red). Bottom: selected overlay of GPVI-Fc and fibrinogen or collagen (yellow) for the fluorescence images.
Figure 6
Figure 6
64Cu-GPVI-Fc in a lipopolysaccharide (LPS)-induced lung inflammation model. (A) Representative in vivo images of the distribution of 64Cu-GPVI-Fc in a lung inflammation model in rats with CT image (left), PET image (middle) and PET overlaid on CT background (right) 48 h after tracer injection. Lungs are highlighted with a dashed white outline. (B) Accumulation kinetics of 64Cu-GPVI-Fc in LPS-induced bilateral lung inflammation injected alone (LPS, n=12) or alongside nonradioactive Cu-GPVI-Fc (n=4) in the lungs observed by PET in vivo. Results shown as single values with a geometric mean and geometric standard deviation. (C) Accumulation kinetics of 64Cu-GPVI-Fc in LPS-induced unilateral lung inflammation in one example animal. % ID/cc: percentage injected dose per cubic centimeter.
Figure 7
Figure 7
64Cu-GPVI-Fc in an experimental autoimmune encephalomyelitis (EAE) model. (A) Representative images of 64Cu-GPVI-Fc accumulation in the spine of an EAE model (top) and control animal (bottom) acquired by PET and overlaid on MRI data. Brain: outlined by a pointed line. Spine location: dashed line. (B) Accumulation kinetics of 64Cu-GPVI-Fc in the spine of control animals (ctrl, n=4) and EAE animal model (n=3). Results shown as single values with a geometric mean and geometric standard deviation. (C) Biodistribution of 64Cu-GPVI-Fc measured ex vivo in an EAE model (EAE, n=3) and in control animals (Ctrl, n=4) in the brain and in the spinal cord. Average values and standard deviations are shown. P values are indicated using * (P ≤ 0.05), ** (P ≤ 0.01) and *** (P ≤ 0.001). % ID/cc: percentage injected dose per cubic centimeter.
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