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

Transforming growth factor β exerts opposite effects from interleukin-1β on cultured rabbit articular chondrocytes through reduction of interleukin-1 receptor expression

Objective: The aim of the study was to determine whether transforming growth factor beta (TGF beta) modulates the effects and the receptor expression of interleukin-1 (IL-1) in rabbit articular chondrocytes (RAC).Methods: Collagen, glycosaminoglycan, and collagenase production, together with 125I-labeled IL-1 binding, were analyzed in RAC cultures.Results: TGF beta reduced both IL-1 effects on matrix metabolism and IL-1 receptor expression.Conclusion: TGF beta acts as an antagonist of the effects of IL-1 through down-regulation of its receptor expression

Anticorps recombinants antithrombotiques

Les accidents coronariens et cérébrovasculaires aigus sont la première cause de mortalité dans le monde et vont le rester au moins jusqu'en 2020. Les maladies cardiovasculaires (ischémie myocardique et cérébrale) tuent environ 17 millions de personnes par an avec un accroissement prévu à 20 millions par an en 2020 et 24 millions par an en 2030. L'incidence globale des récidives et décès dans les 6 mois suivant un syndrome coronarien aigu reste de 8 à 15 % en l'état actuel de la pratique médicale. Les accidents cardiovasculaires sont dus à la formation d'un thrombus en regard d'une lésion athéromateuse érodée. Les traitements de la thrombose sont médicaux, associant fibrinolytiques, anticoagulants et anti-plaquettaires, et mécanique par angioplastie coronaire pour recanaliser l'artère coronaire. Mais ces traitements n'évitent pas une morbi-mortalité de l'ordre de 15 % à 6 mois. Enfin, le traitement des accidents vasculaires cérébraux ischémiques reste très limité. Il y a donc, malgré les progrès des traitements antithrombotiques, un vrai besoin clinique d'amélioration et de découverte de nouvelles molécules en raison des limites des drogues existantes. L'activation des plaquettes a un rôle critique dans l'étiologie des maladies cardiovasculaires ischémiantes. De ce fait les molécles antiplaquettaires sont le plus fréquemment prescrites dans ces indications. Un seul anticorps recombinant est actuellement utilisé en thérapie antithrombique. Il s'agit d'un Fab chimérique, c7E3 ou abciximab, qui inhibe la phase finale de l'agrégation plaquettaire. Les indications de l'abciximab sont limitées aux accidents coronariens aigus traités par angioplastie et il présente des effets secondaires, hémorragies et thrombopénies, qui peuvent être sévères. D'autres cibles dont le rôle est plus précoce dans l'activation plaquettaire ont été identifiées. Il s'agit des phases de contact des plaquettes avec le sous-endothélium et de la phase d'activation des plaquettes par le collagène. Des anticorps monoclonaux actifs sur ces voies sont en développement et sont décrits dans cette revue

Absence of bleeding upon dual antiplatelet therapy in a patient with a immune GPVI deficiency

Acquired deficiencies in platelet glycoprotein VI are rare and have not been found associated with other defects. Here we report the case of a 64-year old male patient presenting an immune GPVI deficiency associated to a mutation in the alpha-actinin gene and who has been treated with dual anti platelet therapy without bleeding. Introduction: Glycoprotein (GP) VI, a pluripotent receptor interacting with collagen and fibrin(ogen) is responsible for thrombus formation, growth and stability (). It is co-expressed with the Fc receptor γ (FcRγ) chain (). GPVI is not critical for haemostasis since subjects with a GPVI deficiency usually present low or even no bleeding tendency (, ). Acquired GPVI deficiency due to antibody-induced GPVI depletion is the most frequent finding. At least 10 patients have been described with an acquired GPVI deficiency, most often associated to immune thrombocytopenia, moderate bleeding and impaired collagen-induced platelet aggregation (). Several mechanisms leading to the GPVI deficiency are proposed including antibody-triggered GPVI internalization and/or shedding of the extracellular domain (, ). We report the case of a patient presenting an acquired GPVI deficiency different from those previously described: (i) he is male whereas all previous cases were female, (ii) he is heterozygous for a mutation in α (alpha)-actinin-1 gene and (iii) he was treated with dual antiplatelet therapy with no haemorrhagic manifestation

Transforming growth factor-β1 (TGF-β1) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing

International audienc

The mouse dorsal skinfold chamber as a model for the study of thrombolysis by intravital microscopy.: Thrombolysis imaging in the mouse skinfold chamber

International audienceAlthough intravital microscopy models of thrombosis in mice have contributed to dissect the mechanisms of thrombus formation and stability, they have not been well adapted to study long-term evolution of occlusive thrombi. Here, we assessed the suitability of the dorsal skinfold chamber (DSC) for the study of thrombolysis and testing of thrombolytic agents by intravital microscopy. We show that induction of FeCl3-induced occlusive thrombosis is achievable in microvessels of DSCs, and that thrombi formed in DSCs can be visualised by intravital microscopy using brightfield transmitted light, or fluorescent staining of thrombus components such as fibrinogen, platelets, leukocytes, and von Willebrand factor. Direct application of control saline or recombinant tissue-plasminogen activator (rtPA) to FeCl3-produced thrombi in DSCs did not affect thrombus size or induce recanalisation. However, in the presence of hirudin, rtPA treatment caused a rapid dose-dependent lysis of occlusive thrombi, resulting in recanalisation within 1 hour after treatment. Skin haemorrhage originating from vessels located inside and outside the FeCl3-injured area was also observed in DSCs of rtPA-treated mice. We further show that rtPA-induced thrombolysis was enhanced in plasminogen activator inhibitor-1-deficient (PAI-1-/-) mice, and dropped considerably as the time between occlusion and treatment application increased. Together, our results show that by allowing visualization and measurement of thrombus lysis and potential bleeding complications of thrombolytic treatments, the DSC provides a model for studying endogenous fibrinolysis and for first-line screening of thrombolytic agents. Furthermore, using this system, we found that thrombin and clot aging impair the thrombolytic action of rtPA towards FeCl3-produced thrombi

Inhibition of Glycoprotein VI Clustering by Collagen as a Mechanism of Inhibiting Collagen-Induced Platelet Responses: The Example of Losartan

<div><p>Exposure of platelets to collagen triggers the formation of a platelet clot. Pharmacological agents capable of inhibiting platelet activation by collagen are thus of potential therapeutic interest. Thrombus formation is initiated by the interaction of the GPIb-V-IX complex with collagen-bound vWF, while GPVI interaction with collagen triggers platelet activation that is reinforced by ADP and thromboxane A2. Losartan is an angiotensin II (Ang II) type I receptor (AT1R) antagonist proposed to have an antiplatelet activity via the inhibition of both the thromboxane A2 (TXA2) receptor (TP) and the glycoprotein VI (GPVI). Here, we characterized <i>in vitro</i> the effects of losartan at different doses on platelet responses: losartan inhibited platelet aggregation and secretion induced by 1 μg.mL^-1 and 10 μg.mL^-1 of collagen with an IC50 of ~ 6 μM. Losartan inhibited platelet responses induced by the GPVI specific collagen related peptide but not by the α2β1 specific peptide. However, losartan did not inhibit the binding of recombinant GPVI to collagen, which is not in favor of a simple competition. Indeed, the clustering of GPVI observed in flow cytometry and using the Duolink methodology, was inhibited by losartan. The impact of a therapeutic dose of losartan (100 mg/day) on platelet responses was analyzed <i>ex vivo</i> in a double blind study. No statistically significant differences were observed between losartan-treated (n=25) and non-treated (n=30) patients in terms of collagen and U46619-induced platelet activation. These data indicate that in treated patients, losartan does not achieve a measurable antiplatelet effect but provide the proof of concept that inhibiting collagen-induced GPVI clustering is of pharmacological interest to obtain an antithrombotic efficacy.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT00763893" target="_blank">NCT00763893</a></p></div

Duolink imaging of collagen-induced GPVI clustering and its inhibition by losartan.

<p>(A) Washed platelets pretreated with PBS or losartan (22 μM) were incubated with 5 μg.mL^-1 9E18-MINUS and 5 μg.mL^-1 9E18-PLUS and added to collagen-coated Lab-Tek chambers for 45 min at 37°C. Bound platelets were fixed with 4% PFA and stained with 40 μg.mL^-1 anti-CD41 alexa fluo 488 (Green) before ligation and the MINUS and PLUS oligonucleotides and amplification. PLA dots (Orange) are only generated if 9E18-MINUS and—PLUS are in close proximity (<40 nm). Numerous and large GPVI clusters are observed in control conditions while only few spots are visible in the presence of losartan. (B) Quantitative analysis of adherent platelets (green). Data are expressed as the area covered by platelets and the mean + SEM of n = 30 random fields from 3 independent experiments. Scale bars: 5 μm. *<i>P</i><0.05. (C) Quantitative analysis of platelets GPVI clustering as determined by measuring the proportion of Duolink positive platelets on the same fields.</p

Losartan inhibits collagen induced clustering of GPVI but not ADP- or TRAP-induced GPVI dimerization.

<p>Washed platelets preincubated with PBS or losartan (22 μM), were stimulated with collagen (10 μg.mL^-1), TRAP (20 μM) or ADP (10 μM) for 20 min. (A) GPVI expression and (B) GPVI dimerization were measured by flow cytometry using FITC-coupled 3J24 and 9E18, respectively. Results (mean + SEM) are from 5 independent experiments. (C) Washed platelets pre-treated with PBS or 22 μM losartan and pre-stimulated or not with TRAP (20 μM) were incubated with FITC-coupled collagen (10 μg.mL^-1). Bound collagen was analyzed by flow cytometry. PBS: open bars; losartan: black bars. Data are mean + SEM from 7 independent experiments. * <i>P</i><0.05, *** <i>P</i><0.001.</p

Losartan inhibits collagen induced platelet activation even when GPVI dimers are preformed.

<p>Washed platelets were preincubated with PBS or losartan (22 μM) for 10 min at 37°C. Buffer or TRAP (10 μM) were added to the samples and the incubation continued without stirring for 10 min. Collagen (1 μg.mL^-1) was then added, stirring started and aggregation recorded. (A) Representative aggregation curves. (B) When the aggregation reached 25% in control condition (preincubation with PBS, no losartan), the extent of aggregation in other conditions was measured. Data are represented as bar charts. (Mean + SEM of 3 experiments)</p