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

Dendritic cells and pulmonary fibrosis : study on an animal model of bleomycin-induced fibrosis in mice

Les cellules dendritiques, puissantes cellules présentatrices d'antigène, jouent un rôle-clé dans l'initiation et la régulation des réponses immunitaires et inflammatoires (Lipscomb et al., 2002). Elles sont présentes à l'état de cellules immatures dans la plupart des muqueuses, disséminées dans le tissu interstitiel et les épithéliums. Elles sont chargées de capter les antigènes exogènes, de les apprêter et de migrer vers les organes lymphoïdes pour les présenter aux lymphocytes T spécifiques. Au cours de cette migration, elles acquièrent les caractéristiques des cellules dendritiques matures présentes dans les organes lymphoïdes (expression forte des molécules HLA de classe II et des molécules de costimulation CD40, CD80, CD83, CD86). Les cellules dendritiques sont présentes dans le poumon normal (Vermaelen et al., 2005). Elles sont impliquées dans la physiopathologie de différentes maladies pulmonaires et jouent un rôle pathogénique essentiel dans certaines d'entre elles comme l'histiocytose langerhansienne ou l'asthme (Tazi et al ., 2000; Lambrecht et Hammad, 2003). Leur rôle au cours des processus fibrosants n'a cependant jamais été évalué. En effet, contrairement à d'autres cellules présentatrices d'antigènes que sont les macrophages, dont le rôle dans la réaction fibrosante est clairement établi (Agostini et al ., 1997), le rôle des cellules dendritiques au cours des processus de réparation alvéolaire n'a pas été étudié. Notre travail visait à étudier in vivo le rôle des cellules dendritiques au cours de la fibrose pulmonaire sur un modèle animal par induction d’une fibrose à la bléomycine chez la souris. Les premières étapes du projet ont consisté à caratériser par cytométrie en flux la présence de cellules dendritiques dans le poumon des animaux 3, 7 et 14 jours après instillation intratrachéale de bléomycine. L'étude s'est poursuivie par une caractérisation du phénotype de surface des cellules dendritiques en cherchant à préciser leur état d’activation et de maturation. L'étape suivante a consisté à rechercher par RT-QPCR les principales chimiokines responsables du recrutement des cellules dendritiques. Les résultats montrent une augmentation du nombre des cellules dendritiques CD11c+ / CMH II+ infiltrant le poumon dès J7, avec une sous-population de cellules activées exprimant fortement les molécules CMH II. Ces résultats sont corroborés par une augmentation de la population cellulaire totale du broyat de poumon dès J3, ainsi que par une augmentation de la cellularité totale et du nombre de cellules inflammatoires dans les lavages bronchoalvéolaires (LBA) à J7 et J14. L'étude a été complétée par une caractérisation plus approfondie du phénotype de surface des cellules dendritiques en cherchant à préciser leur état d’activation/maturation. [...]Dendritic cells (DCs), potent antigen-presenting cells, play a key role in the initiation and regulation of immune and inflammatory responses (Lipscomb et al., 2002). Immature DCs are present in the most of mucous membranes, disseminated in the interstitial tissue and the epithelia. They are able to deal with exogenous antigens, to process them and to migrate to lymphoid organs and to present them to T lymphocytes. During their migration, they have the characteristics of mature DCs in lymphoid organs (higher expression of MHC class-II molecules and costimulation-molecules (CD40, CD80, CD83, CD86). Dendritic cells are present in the normal lung (Vermarlen et al., 2005). They are implicated in the pathophysiology of different pulmonary diseases and play a crucial pathogenic role in some of them like Langerhan’s cell Histiocytosis or asthma (Tazi et al., 2000; Lambrecht et Hammad, 2003). However, their role in the fibrosing process has never been studied. In fact, the role of macrophages (other antigen presenting cells (APCs)) in fibrosing reaction has been clearly established (Agostini et al., 1997), but DCs function during the alveolar healing process has not been studied. The purpose of this thesis is to study the in vivo role of DCs in bleomycin-induced pulmonary fibrosis in mice. The first step of this project is to demonstrate by flow cytometry the presence of DCs in the lung of these animals 3, 7 and 14 days after intratracheal bleomycin instillation. We continued our research with the surface phenotypic characterization of DCs identifying their activation state and maturation. The next step consists to search the main chemokines which are responsive for dendritic cells recruitment by RT-qPCR. Our results show an increase of CD11c+ / MHC class II+ DCs number infiltrating the lung after D7, with an activated cell subpopulation which strongly express MHC class II molecules. The results are corroborated by an increase of the total cell population in the lung homogenate after D3 and by an increase of the total cellularity and inflammatory cells number in broncho-alveolar lavages (BAL) at D7 and at D14. This study was completed by the surface phenotypic characterization of DCs identifying their activation state and maturation. [...

Chondrocalcin is internalized by chondrocytes and triggers cartilage destruction via an interleukin-1β-dependent pathway

International audienceChondrocalcin is among the most highly synthesized polypeptides in cartilage. This protein is released from its parent molecule, type II pro-collagen, after secretion by chondrocytes. A participation of extracellular, isolated chondrocalcin in mineralization was proposed more than 25 years ago, but never demonstrated. Here, exogenous chondrocalcin was found to trigger MMP13 secretion and cartilage destruction ex vivo in human cartilage explants and did so by modulating the expression of interleukin-1 beta in primary chondrocyte cultures in vitro. Chondrocalcin was found internalized by chondrocytes. Uptake was found mediated by a single 18-mer peptide of chondrocalcin, which does not exhibit homology to any known cell-penetrating peptide. The isolated peptide, when artificially linked as a tetramer, inhibited gene expression regulation by chondrocalcin, suggesting a functional link between uptake and gene expression regulation. At the same time, the tetrameric peptide potentiated chondrocalcin uptake by chondrocytes, suggesting a cooperative mechanism of entry. The corresponding peptide from type I pro-collagen supported identical cell-penetration, suggesting that this property may be conserved among C-propeptides of fibrillar pro-collagens. Structural modeling localized this peptide to the tips of procollagen C-propeptide trimers. Our findings shed light on unexpected function and mechanism of action of these highly expressed proteins from vertebrates

Role of CXCL13 in cigarette smoke-induced lymphoid follicle formation and chronic obstructive pulmonary disease

Rationale: The B cell-attracting chemokine CXCL13 is an important mediator in the formation of tertiary lymphoid organs (TLOs). Increased numbers of ectopic lymphoid follicles have been observed in lungs of patients with severe chronic obstructive pulmonary disease (COPD). However, the role of these TLOs in the pathogenesis of COPD remains unknown. Objectives: By neutralizing CXCL13 in a mouse model of chronic cigarette smoke (CS) exposure, we aimed at interrogating the link between lymphoid follicles and development of pulmonary inflammation, emphysema, and airway wall remodeling. Methods: We first quantified and localized CXCL13 in lungs of air-or CS-exposed mice and in lungs of never smokers, smokers without airflow obstruction, and patients with COPD by reverse transcriptase-polymerase chain reaction, ELISA, and immunohistochemistry. Next, CXCL13 signaling was blocked by prophylactic or therapeutic administration of anti-CXCL13 antibodies in mice exposed to air or CS for 24 weeks, and several hallmarks of COPD were evaluated. Measurements and Main Results: Both mRNA and protein levels of CXCL13 were increased in lungs of CS-exposed mice and patients with COPD. Importantly, expression of CXCL13 was observed within B-cell areas of lymphoid follicles. Prophylactic and therapeutic administration of anti-CXCL13 antibodies completely prevented the CS-induced formation of pulmonary lymphoid follicles in mice. Interestingly, absence of TLOs attenuated destruction of alveolar walls and inflammation in bronchoalveolar lavage but did not affect airway wall remodeling. Conclusions: CXCL13 is produced within lymphoid follicles of patients with COPD and is crucial for the formation of TLOs. Neutralization of CXCL13 partially protects mice against CS-induced inflammation in bronchoalveolar lavage and alveolar wall destruction

Role of B cell-activating factor in chronic obstructive pulmonary disease

Rationale: B cell-activating factor (BAFF) plays a major role in activation of B cells and in adaptive humoral immune responses. In chronic obstructive pulmonary disease (COPD), lymphoid follicles have been associated with disease severity, and overexpression of BAFF has been demonstrated within lymphoid follicles of patients with severe COPD. Objectives: To investigate expression and localization of BAFF in the lungs of patients with COPD and to study the role of BAFF in COPD by antagonizing BAFF in a mouse model of chronic cigarette smoke (CS) exposure. Methods: We quantified and localized BAFF expression in lungs of never-smokers, smokers without COPD, and patients with COPD and in lungs of air- or CS-exposed mice by reverse-transcriptase polymerase chain reaction, ELISA, immunohistochemistry, and confocal imaging. Next, to investigate the role of BAFF in COPD, we antagonized BAFF by prophylactic or therapeutic administration of a soluble fusion protein of the BAFF-receptor, BAFFR-Fc, in mice exposed to air or CS for 24 weeks and evaluated several hallmarks of COPD and polarization of lung macrophages. Measurements and Main Results: BAFF expression was significantly increased in lungs of patients with COPD and CS-exposed mice. BAFF staining in lymphoid follicles was observed around B cells, CD4(+) cells, dendritic cells, follicular dendritic cells, and fibroblastic reticular cells. Prophylactic and therapeutic administration of BAFFR-Fc in mice reduced pulmonary B-cell numbers and prevented CS-induced formation of lymphoid follicles and increases in immunoglobulin levels. Interestingly, prophylactic BAFFR-Fc administration significantly attenuated pulmonary inflarnmation and destruction of alveolar walls. Moreover, antagonizing BAFF altered the phenotype of alveolar and interstitial macrophages. Conclusions: BAFF is significantly increased in lungs of patients with COPD and is present around both immune and stromal cells within lymphoid follicles. Antagonizing BAFF in CS-exposed mice attenuates pulmonary inflammation and alveolar destruction

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

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

<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