Koninklijke Bibliotheek, National Library of the Netherlands
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Effect of Posaconazole in an in vitro model of cardiac fibrosis induced by Trypanosoma cruzi
Nisimura, Lindice Mitie
Ferrão, Patrícia Mello
Nogueira, Alanderson da Rocha
Waghabi, Mariana Caldas
Meuser-Batista, Marcelo
Moreira, Otacílio C.
Urbina, Julio A.
Garzoni, Luciana Ribeiro
text
article
monographic
Molecular and biochemical parasitology
continuing
01666851
0000000010408
238
C
text
Born digital tijdschriften
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Elektronische Wetenschappelijke Tijdschriften
EWTIJ
10.1016/j.molbiopara.2020.111283
urn:nbn:nl:kb-1604323634688
Automatisch gegenereerd op basis van de EWTIJ XML in release 1.5 van het digitaal magazijn.
MOLBIO
111283
111283
S0166-6851(20)30047-5
10.1016/j.molbiopara.2020.111283
Fig. 1
Effect of 10 nM POS on the intracellular cycle of T. cruzi. Primary cardiomyocytes were cultured in monolayers and infected with trypomastigotes of T. cruzi (Y strain). After 2 h of infection, cultures were treated with 10 nM of POS. At 72 h post infection, not treated cultures (A) showed intracellular amastigotes (*). Not treated cultures exhibit trypomastigote forms (#) after 96 h of infection (C). After 144 h of infection, intense culture parasitism was observed. At 96 h post infection (D), treatment with 10 nM of POS drastically reduced the parasitism of the monolayers cultures. After 144 h of T. cruzi infection and 142 h of POS treatment (F), the compound promoted parasite clearance. Evaluation of treatment on inhibition of T. cruzi infection by light microscopy. An important effect of the POS in the reduction of the percentage of infected cells compared to infected and not treated cultures (G). Bar =50 μm. ** p < 0.01; ***p < 0.001. One-way ANOVA test.
Fig. 1
Fig. 2
Time and dose response analysis in T. cruzi. infected cardiomyocytes treated with POS after 48 h of infection. Quantification of the percentage of T. cruzi infection inhibition in cardiac monolayer culture was evaluated after 24, 48, 72 and 96 h of POS treatment. POS induced significant inhibition of T. cruzi infection. IC50 after 144 h post infection was 1.58 nM. *p < 0.05; **p<0.01; ***p < 0.001. One-way ANOVA test.
Fig. 2
Fig. 3
Evaluation of T. cruzi infection in cardiac microtissues by DNA staining with DAPI and qPCR. DAPI staining shows the nuclei of cardiac cells with nucleoli (A-E) and the nuclei and kinetoplast of intracellular amastigote forms (B-E). (A) A non-infected cardiac microtissue showing a regular morphology and distribution of nucleus. (B) Cardiac microtissues after 240 h of T. cruzi infection presenting a high number of amastigote forms and the sparse distribution of nucleus. (C) Cardiac microtissue after 240 h of T. cruzi infection and 96 h of treatment with POS showing reduction of intact parasites. (D) Increased image of an insert area of microtissue presented in B, showing the kinetoplasts of intracellular amastigotes (*) and cardiomyocyte nucleus (N). (E) Increased image of an insert area of microtissue presented in C, showing remnants of parasites (#) after the treatment with POS and cardiomyocyte nucleus (N). (F) Quantification of parasite load evaluated by qPCR. Bar graph shows the reduction in parasite load after 96 h of treatment with POS. *p < 0.05. t test. Bar =20 μm.
Fig. 3
Fig. 4
Immunofluorescence analysis of laminin (A-C) and fibronectin (D-F) after 240 h of T. cruzi infection and 96 h of POS treatment. Protein distribution labeled with AlexaFluor 488 conjugated secondary antibody (green) and DAPI staining (blue) in control non-infected (NI), T. cruzi infected (Y) and T. cruzi infected and treated with POS (Y + POS) 3D culture. T. cruzi infection induces intense deposition of laminin and fibronectin after 240 h of infection. POS treatment initiated 144 h post infection and continued for 96 h inhibited fibrosis. Bar =20 μm.
Fig. 4
Fig. 5
Western blot analysis of laminin (A), fibronectin (B) and α-SMA (C). Anti-GAPDH (36 kDa) antibody was used as a loading control. Expression in control non-infected (NI), non-infected and treated with POS (POS), T. cruzi infected (Y) and infected and treated with POS (Y + POS) 3D culture. 3D cultures infected by T. cruzi exhibit higher laminin, fibronectin and α-SMA protein levels compared to non-infected cultures. Reduction of laminin and fibronectin was observed after 96 h of POS treatment in infected cultures. Results are shown as index of variation of the mean (IV)±SEM. *p < 0.05; **p < 0.01. One-way ANOVA test.
Fig. 5
Fig. 6
Western blot analysis of MMP-9 (A) and TIMP-4 (B). Anti-GAPDH (36 kDa) antibody was used as a loading control. T. cruzi infected cardiomyocytes 3D culture have higher TIMP-4 levels compared to control group. POS treatment of infected culture induces reduction of this protein. TGF-β levels in the supernatant (C) of 3D cultures non-infected (NI), non-infected and treated with POS (POS), T. cruzi infected (Y) and 3D culture infected and treated with POS (Y + POS). T. cruzi infection induces TGF-β secretion when compared to non-infected cultures. In addition, infected and POS treated cultures present even higher TGF-β levels in the supernatant. Results are showed as index of variation of the mean (IV)±SEM *p < 0.05; **p < 0.01; ***p < 0.001. One-way ANOVA test.
Fig. 6
Effect of Posaconazole in an in vitro model of cardiac fibrosis induced by Trypanosoma cruzi
Lindice Mitie
Nisimura
Conceptualization
Investigation
Formal analysis
Writing - original draft
Writing - review & editing
a
Patrícia Mello
Ferrão
Investigation
Formal analysis
a
b
Alanderson da Rocha
Nogueira
Investigation
c
Mariana Caldas
Waghabi
Supervision
Formal analysis
b
Marcelo
Meuser-Batista
Investigation
Formal analysis
a
Otacílio C.
Moreira
Formal analysis
Investigation
d
Julio A.
Urbina
Writing - review & editing
Funding acquisition
e
Luciana Ribeiro
Garzoni
Supervision
Investigation
Formal analysis
Writing - original draft
Writing - review & editing
Funding acquisition
a
*
largarz@ioc.fiocruz.br
a
Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Laboratório de Inovações em Terapias
Ensino e Bioprodutos
Instituto Oswaldo Cruz
Fundação Oswaldo Cruz
Rio de Janeiro
RJ
Brazil
Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
b
Laboratório de Genômica Funcional e Bioinformática, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Laboratório de Genômica Funcional e Bioinformática
Instituto Oswaldo Cruz
Fundação Oswaldo Cruz
Rio de Janeiro
RJ
Brazil
Laboratório de Genômica Funcional e Bioinformática, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
c
Laboratório de Ultra-estrutura Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Laboratório de Ultra-estrutura Celular
Instituto Oswaldo Cruz
Fundação Oswaldo Cruz
Rio de Janeiro
RJ
Brazil
Laboratório de Ultra-estrutura Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
d
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Laboratório de Biologia Molecular e Doenças Endêmicas
Instituto Oswaldo Cruz
Fundação Oswaldo Cruz
Rio de Janeiro
RJ
Brazil
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
e
Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela
Instituto Venezolano de Investigaciones Cientificas
Caracas
Venezuela
Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela
⁎
Corresponding author at: Av. Brasil, 4365 - CEP. 21045-900, Rio de Janeiro, RJ, Brazil.
Av. Brasil
4365 - CEP. 21045-900
Rio de Janeiro
RJ
Brazil
Graphical abstract
Highlights
•
Fibrosis in heart tissue and cardiac hypertrophy are features in Chagas disease.
•
Trypanosoma cruzi fibrotic cardiac microtissue is a three-dimensional culture.
•
Posaconazole has potent and selective anti-T. cruzi activity.
•
Posaconazole induced reorganization of the myocardial tissue.
Abstract
Posaconazole (POS) is an inhibitor of ergosterol biosynthesis in clinical use for treating invasive fungal infections. POS has potent and selective anti-Trypanosoma cruzi activity and has been evaluated as a possible treatment for Chagas disease. Microtissues are a 3D culture system that has been shown to reproduce better tissue architecture and functionality than cell cultures in monolayer (2D). It has been used to evaluate chemotropic response as in vitro disease models. We previously developed an in vitro model that reproduces aspects of cardiac fibrosis observed in Chagas cardiomyopathy, using microtissues formed by primary cardiac cells infected by the T. cruzi, here called T. cruzi fibrotic cardiac microtissue (TCFCM). We also showed that the treatment of TCFCM with a TGF-β pathway inhibitor reduces fibrosis. Here, we aimed to evaluate the effect of POS in TCFCM, observing parasite load and molecules involved in fibrosis. To choose the concentration of POS to be used in TCFCM we first performed experiments in a monolayer of primary cardiac cell cultures and, based on the results, TCFCM was treated with 5 nM of POS for 96 h, starting at 144 h post-infection. Our previous studies showed that at this time the TCFCM had established fibrosis, resulting from T. cruzi infection. Treatment with POS of TCFCM reduced 50 % of parasite load as observed by real-time PCR and reduced markedly the fibrosis as observed by western blot and immunofluorescence, associated with a strong reduction in the expression of fibronectin and laminin (45 % and 54 %, respectively). POS treatment also changed the expression of proteins involved in the regulation of extracellular matrix proteins (TGF-β and TIMP-4, increased by 50 % and decreased by 58 %, respectively) in TCFCM. In conclusion, POS presented a potent trypanocidal effect both in 2D and in TCFCM, and the reduction of the parasite load was associated with a reduction of fibrosis in the absence of external immunological effectors.
Keywords
Three-dimensional culture
Cardiac microtissues
Chagas disease
Posaconazole
Cardiac fibrosis
Extracellular matrix protein
1
Introduction
Chagas disease (CD) is a chronic systemic parasitosis caused by the Kinetoplastid protozoan Trypanosoma cruzi and is the main cause of cardiomyopathy in endemic areas of Latin America [1]. The disease afflicts 6 to 7 million people worldwide and approximately 30 % of infected people develop cardiac alterations [2]. These alterations include fibrosis and hypertrophy culminating in heart electrical abnormalities and failure [2].
Currently, available chemotherapy for CD is based on two nitroheterocyclic drugs benznidazole (BZ) and nifurtimox (NF). Treatment with BZ can result in 80 % of cure when started during the acute phase and may also be indicated to chronic asymptomatic patients during the chronic phase [3]. However, the treatment of patients presenting severe chronic Chagas cardiomyopathy (CCC) is not mandatory due to the low rates of cure, despite its contribution to the reduction of parasite detection [4].
Posaconazole (POS) is an inhibitor of ergosterol biosynthesis and has been used for treating fungal infections in humans [5]. Ergosterol and other 24 alkyl-sterols are the main endogenous sterols in fungi and Kinetoplastid protozoan parasites such as T. cruzi [6]. Studies with POS demonstrated potent activity of this compound against T. cruzi both in vitro and in vivo, in experimental models of the acute and chronic CD [7,8]. In humans, the trypanocidal effect of POS was reported [9,10], however, a treatment protocol with POS that results in Chagas disease parasitological cure in the chronic phase in humans still needs to be elaborated [3].
The persistence of T. cruzi contributes to chronic inflammation and consequently fibrosis in heart tissue, cardiac hypertrophy and heart failure [11]. In this context, the treatment of CCC patients should consider a therapeutic strategy focused not only on the parasite elimination but also on the inhibition and reversion of heart tissue damage. Studies in experimental models of acute and chronic CD using trypanocidal and anti-inflammatory drugs emerge as a new strategy to eliminate the parasite and to reverse the heart damage [12–14].
The fibrosis in CCC is characterized by the deposition of extracellular matrix proteins including fibronectin, laminin and collagen in the heart tissue [15,16]. Cytokines, matrix metalloproteases (MMPs) and tissue inhibitors of matrix metalloproteases (TIMPs) are regulators of extracellular matrix (ECM) and consequently of fibrosis in many tissues [17]. MMPs 2 and 9 are differentially expressed in patients with indeterminate and clinical cardiac forms of Chagas disease [18]. Interestingly, it was previously demonstrated that POS inhibited the production of fibronectin in the heart tissue of T. cruzi-infected mice [19]. However, the effect of this compound in the reduction of ECM proteins in cardiac tissue infected by T.cruzi is unknown.
Two-dimensional (2D) culture systems (or monolayers) are limited in reproducing aspects observed in vivo. In which, tissues are organized in a three-dimensional (3D) network constituted by cells and extracellular matrix associated with signaling molecules. Cell-cell and cell-ECM interactions regulate the biochemical and molecular cell responses and are directly influenced by 3D tissue architecture. For that reason, 3D cell culture is an advanced in vitro method, since it reproduces with more accuracy the tissues’ architecture and the responses observed in vivo [20].
Our group developed a functional cardiac microtissue model, also called cardiac microtissues, based on a 3D culture system, that when infected by T. cruzi, presents fibrosis and hypertrophy, thus mimicking aspects of CCC [21,22]. Here we will call it T. cruzi fibrotic cardiac microtissue (TCFCM). Recently, we also demonstrated that the sera from patients with chronic CD induced ECM components increase in TCFCM [18]. These results using a 3D culture system showed that both T. cruzi alone or soluble immunological regulators contribute to cardiac fibrosis in the context of CD [18,21]. Moreover, we also recently demonstrated that the treatment of TCFCM with a TGF-β inhibitor reduced fibrosis [23].
In the present study, we evaluated the trypanocidal effect of POS and also the impact of treatment on the expression of ECM in TCFCM.
2
Materials and methods
2.1
Ethics statement
All procedures were approved by the Oswaldo Cruz Foundation Animal Welfare Committee (License number LW-40/13) and were consistent with the USA National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).
2.2
Drug
Posaconazole (SCH56592; (−)-4-[4-[4-[4-[[(2R-cis)-5-(2,4-difluorophenyl)-tetrahydro-5-(1H-1,2,4-triazol-1-ylmethyl)-3-furanyl]methoxy]phenyl]-1-piperazinyl]phenyl]-2,4-dihydro-2-[(S)-1-ethyl-2(S)-hydroxypropyl]-3H-1,2,4-triazol-3-one) (Noxafil®, Merck, Sharp & Dohme Corp., USA) was suspended in DMSO and utilized at 1.25, 2.5, 5 and 10 nM final concentrations in 2D cardiac cell cultures and at 5 nM in 3D cultures.
2.3
Parasites
The Y strain (MHOM/BR/1950/Y) of T. cruzi was used in this study. Trypomastigote forms of T. cruzi were obtained from the supernatant of infected heart muscle cells grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1 mM CaCl2, 1 mM L-glutamine, 2% chick embryo extract, 1000 U/mL penicillin and 50 μg/mL streptomycin. After 96 h of infection, the parasites were collected, centrifuged and resuspended in supplemented DMEM. Handling of live T. cruzi was performed according to established guidelines [24].
2.4
Cardiac cell cultures
Hearts of 18-day old Swiss Webster mouse fetuses were submitted to mechanical and enzymatic dissociation as previously described [25]. Briefly, cells were harvested using 0.05 % trypsin and 0.01 % collagenase in phosphate buffered saline (PBS) at 37 °C. Ventricular heart muscle cells (HMCs) were plated at a density of 1.5 × 105 cells/well on 0.02 % gelatin-coated glass coverslips in 24-well plates (monolayer). Cardiac microtissues (3D culture) were produced plating cardiac cells at a density of 2.5 × 104 cells/well on 1% (w/v) agarose coated 96-well plates. Cells were maintained at 37 °C in a 5% CO2 atmosphere in supplemented DMEM.
2.5
Infection of cardiac microtissues and treatment with Posaconazole
Cardiac monolayers were infected with culture-derived trypomastigotes (20:1, parasites to host cell ratio) and the treatment of cultures was performed following the protocols: (I) addition of 1.25−10 nM POS after 2 h or 48 h of parasites interaction in 2D cultures. At specific times, coverslips (in triplicate) with cardiac cells in monolayers were collected, fixed with Bouin’s fixative solution and stained in Giemsa solution. The percentage of infection was quantified by counting randomly at least 300 cells in light microscope.
Based in our results in monolayers, the treatment of non-infected cardiac microtissues (NICM) and TCFCM previously infected with T. cruzi for 144 h [21] was performed using 5 nM POS for 96 h. In TCFCM a huge infection level and fibrosis are observed at 144 h post-infection but the microtissues are not yet destroyed; for that reason, we chose this condition to start the antiparasitic treatment. TCFCM and NICM treated or not were collected in a pool of sixty spheroids for each condition for processing and qPCR, immunofluorescence and western blotting experiments.
2.6
Quantitation of parasite load by real-time quantitative PCR
Total DNA was extracted from a pool of sixty cardiac microtissues for each experimental condition, using the High Pure PCR Template Preparation Kit according manufacturer’s instructions (Roche, USA) and DNA from samples was analyzed by real-time quantitative PCR (qPCR). The qPCR experiments were performed using ABI Prism 7500 Fast (Life Technologies, USA), in a final volume of 20 μL containing 750 nM of primers Cruzi 1 (5′-AST CGG CTG ATC GTT TTC GA-3′), Cruzi 2 (5′-AAT TCC TCC AAG CAG CGG ATA-3′) for targeting T. cruzi nuclear satellite DNA and probe (FAM- CACACACTGGACACCAA-NFQ-MGB), 5 μL DNA samples and 10 μl TaqMan Master Mix 2x (Promega, USA) [26,27]. The GAPDH gene (TaqMan probe with a FAM dye label from Applied Biosystems) was used as target to quantify the number of mouse cardiomyocytes. Thus, T. cruzi and cardiomyocytes were estimated by absolute quantification, results were normalized and parasite load was expressed as Par. Eq./106 cells. PCR assays were performed in duplicate for each sample. Three independent experiments were analyzed. T. cruzi DNA extracted from epimastigotes (Y strain) ranging from 106 to 10° parasite equivalents were utilized to build the standard curve. To cardiomyocytes, DNA extracted from cells, ranging from 1.5 × 106 to 1.5 × 101 cells equivalents was used.
2.7
Immunofluorescence and confocal microscopy
TCFCM and NICM treated or not, were fixed in 4% paraformaldehyde (PFA) for 5 min at 4 °C and permeabilized with 0.5 % (v/v) Triton X-100. The blockage was performed in PBS 3% (w/v) bovine serum albumin and cardiac spheroids were incubated overnight at 4 °C with primary antibody against fibronectin and laminin both from Sigma-Aldrich. The samples were then washed with PBS and were incubated with a secondary AlexaFluor 488 conjugated antibody at 25 °C for 5 h. DNA was stained with DAPI diluted in PBS. Three independent experiments were performed and analyzed in Zeiss 510 Meta laser scanning confocal microscope.
2.8
Immunoblotting
Proteins were extracted from a pool of sixty TCFCM or NICM for each experimental condition (treated or not) in three independent experiments. Protein extraction was performed using 300 μL of lysis buffer (2 mM PMSF, 5 mM EDTA, 1 mM Na3VO4, 1 mM NaHCO3 and Roche Protease Inhibitor Cocktail) and sonicated. Samples were frozen at −80 °C until used and protein concentration was measured using the BCA Protein Assay Reagent. 20 μg of protein was loaded and resolved in 10 % or 12 % SDS polyacrylamide gels. After resolving, proteins were transferred to nitrocellulose membranes (Bio-Rad) and incubated with primary antibodies rabbit polyclonal anti-fibronectin (220 kDa), rabbit polyclonal anti-laminin (220 kDa), mouse monoclonal anti-alpha smooth muscle actin (α-SMA) (42 kDa), rabbit polyclonal anti TIMP-4 (23 kDa) or rabbit polyclonal anti MMP-9 (92 kDa) – all from Sigma-Aldrich diluted in TBST with 5% skin milk overnight at 4 °C. For loading controls, mouse anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (36 kDa) – from Fitzgerald monoclonal antibody and was used as loading control. Membranes were incubated with secondary goat anti-rabbit IgG or goat anti-mouse IgG HRP-labeled antibody for 1 h at 25 °C, followed by incubation with chemoluminescent substrate kit [SuperSignal West Pico PLUS Chemiluminescent Substrate or SuperSignal West Femto Maximum Sensitivity (both fromThermo Scientific)] and exposition to X-ray film. Biological triplicates were analyzed in a minimum. Densitometric analysis of the X-ray film bands was performed with the software Image Studio Lite version 4.0 and standardized by the respective GAPDH density. The results were showed as index of variation (IV) of the control non-infected that was a normalization of the data by the non-infected mean.
IV group = group density / non-infected and non-treated density mean
2.9
ELISA TGF-β
Measurement of TGF-β in the supernatants of TCFCM or NICM for each experimental condition (treated or not) was performed using TGFβ1 Emax® ImmunoAssay System (Promega) according to manufacturer’s protocol. Measurement of total TGF-β (active + latent) was performed by heat activation (80 °C) of latent TGF-β. Three independent experiments were analyzed. The results were showed as index of variation (IV) of the control non-infected that was a normalization of the data by the non-infected mean.
2.10
Statistical analysis
Unpaired two-sample t-test or One-way ANOVA and Tukey´s post-test was used to analyze statistical significance (p < 0.05) in GraphPad Prism 4.00 software (GraphPad Software Inc., San Diego, CA).
3
Results
3.1
Effect of Posaconazole in monolayers of cardiac cells infected by T.cruzi
We started our experiments treating 2D cultures of cardiomyocytes infected by T. cruzi with 10 nM of POS. The treatment was initiated 2 h post-infection and a time-dependent response was evaluated until 144 h post-infection. Morphological alterations of intracellular amastigotes were observed at 72 h of infection in POS treated cultures (Fig. 1
B). At 96 h of infection, no trypomastigote forms were observed in treated cultures, showing that POS impaired the differentiation of amastigotes to trypomastigote forms (Fig. 1D). At 72 h of infection, we observed 70 % of infection inhibition, while inhibition levels reached 95 % and >99 % after 96 and 144 h of infection in treated cultures, respectively (Fig. 1F, G).
Starting the treatment with POS at 48 h post-infection, when invasive trypomastigote forms already differentiated to amastigote forms, and the proliferation process occurs, we also observed a dose and time-dependent reduction in the percentage of infected cells (Fig. 2
). At 120 h of infection and 72 h of treatment, the lowest tested dose of POS (1.25 nM) inhibited 43 % (p < 0.01) and the highest (5.0 nM) inhibited 63 % of infection (p < 0.001). In the last time point of infection and treatment (144h and 96h respectively), 2.5 nM of POS inhibited 75 % (p < 0.05) and 5 nM inhibited 84 % (p < 0.01) of infection (Fig. 2).
3.2
Effect of Posaconazole in T. cruzi fibrotic cardiac microtissues
Based on our previous results, showing that at 144 h post T. cruzi infection most cardiac cells of TCFCM were infected by the parasite [21], we choose this time point to start the treatment with POS and used 5 nM POS and 96 h of treatment.
Fluorescence images using the DNA intercalator DAPI showed the distribution of cardiac microtissue cells and parasites DNA in cardiac microtissues. Cardiac microtissue presented a mean of 68 % of cardiomyocytes (Supplementary Fig. S1) and nuclear staining (Fig. 3
A). In infected microtissue, the parasites showed nuclei and kinetoplast stained (Fig. 3B and D) and T. cruzi infection was not only in the microtissue surface as showed in the confocal reconstruction image (Supplementary Fig. S2). In infected and treated microtissues it was possible to visualize fragments of T. cruzi DNA (Fig. 3C and E), while the nuclei of cardiac cells were intact, indicating selective parasite cell death. This data was confirmed by the cell count of slices from not treated and treated cardiac microtissue, which revealed no significant difference in the number of cells per field [(106 ± 44 and 107 ± 61, respectively) Supplementary Fig. S3].
To confirm the trypanocidal effect of POS in this 3D culture system we evaluated the T. cruzi load by quantitative real-time PCR. It was found that 5 nM POS treatment reduced significantly (51.6 %, p < 0.05) the parasite load when compared to non-treated TCFCM (Fig. 3F), showing that even in a complex multilayer 3D structure, the compound was able to reach the parasites maintaining the trypanocidal activity observed in monolayer.
3.3
Posaconazole treatment reduces ECM proteins expression in T. cruzi fibrotic cardiac microtissues
Since we previously found fibrosis and hypertrophy in TCFCM at 144 h post-infection [21], we evaluated the alteration of ECM proteins associated with the reduction of the parasite load after 96 h of POS treatment that was initiated 144 h post-infection. Immunofluorescence revealed that non-treated TCFCM presented increased laminin (Fig. 4
B) and fibronectin (Fig. 4E) staining compared to POS treated ones.
After 96 h of treatment with 5 nM of POS, we observed a marked reduction of staining for both proteins in TCFCM (Fig. 4C and F). We also performed western blotting and the results confirmed our observations in immunofluorescence, showing a reduction in the expression levels of laminin (53.8 %, p < 0.05) (Fig. 5
A) and fibronectin (45 %, p < 0.05) (Fig. 5B) after treatment with POS. These results show that POS treatment was able to revert fibrosis induced by T. cruzi in cardiac microtissues.
Trying to understand the effect of POS treatment on mechanisms of ECM synthesis in TCFCM, we decided to investigate the expression of α-smooth muscle actin (α-SMA). This protein is expressed in activated myofibroblasts in the development of fibrosis [28]. By western blotting, we observed a 3.3-fold increase (p < 0.05) in α-SMA in non-treated TCFCM (Fig. 5C), suggesting that one of the mechanisms involved in the induction of ECM proteins expression in TCFCM is the differentiation of fibroblasts to myofibroblasts. We also found that the treatment with POS did not change the levels of this protein (Fig. 5C), showing that neither the reduction on parasite load nor POS per se are sufficient to reduce myofibroblast activation.
3.4
Posaconazole treatment alters levels of ECM regulators (TGF-β, MMP-9 and TIMP-4) in T. cruzi fibrotic cardiac microtissues
We also investigated molecules directly involved in the degradation of ECM proteins (metalloprotease MMP-9 and the inhibitor of metalloproteases TIMP-4). We did not observe statistically significant changes in MMP-9 levels in T. cruzi infected microtissues (Fig. 6
A). In contrast, the infection induced a 6-fold increase (p < 0.001) in the expression of TIMP-4 (Fig. 6B), suggesting that T. cruzi inhibit the mechanisms involved on the degradation of ECM, while treatment with POS reduced 2.4-fold (p < 0.05) the levels of TIMP-4 in T. cruzi-infected cultures (Fig. 6B).
Finally, we investigated TGF-β levels, since its involvement in cardiac fibrosis during Chagas cardiomyopathy is well established [16]. We performed ELISA of cultures supernatant to evaluate the effect of POS treatment on TGF-β secretion (Fig. 6C). We found that T. cruzi infection increased the levels of TGF-β secretion (20 %, p < 0.05) in the supernatant of TCFCM when compared to non-infected cardiac microtissue. Treatment of TCFCM with POS increased by 50 % the TGF-β secretion levels (p < 0.001) when compared to non-treated TCFCM and 70 % (p < 0.001) when compared to non-infected ones. These results show that POS changes TGF-β secretion levels in infected cardiac microtissues, suggesting an immunomodulatory effect of this compound on cardiac cells.
4
Discussion
CD is mostly silent, the symptoms during the acute phase are generic, making difficult the diagnosis and consequently the specific treatment with BZ or NF, which can reach 80 % of cure if started during the early stages of the disease. However, these drugs can present adverse effects and have poor efficacy in the chronic stages of infection [3,29].
CCC results from parasite persistence and chronic inflammation [30], which cause microvascular abnormalities, myocytolysis, necrosis, fibrosis and compensatory hypertrophy. These clinical alterations contribute to cardiac dysfunction and consequently to heart failure, observed in advanced stages of CD [11].
For that reason, the treatment with trypanocidal agents, despite contributing to parasite load reduction, is in many cases not sufficient to reverse the cardiac alterations. In this context, is necessary to find treatment strategies that not only kill the parasite but also reduce cardiac injury. Since fibrosis is one of the more nefarious cardiac alterations observed in CCC, it should be considered as a target in the treatment of chronic CD.
Ergosterol biosynthesis inhibitors have been tested as new therapeutic agents for Chagas disease [31]. Here we showed that the treatment with POS has potent dose and time-dependent trypanocidal effect in T. cruzi-infected cardiac cells in monolayer, corroborating previous data [8,32,33]. POS reduced T. cruzi infection also in experimental models of acute and chronic Chagas disease in mice [7,19].
In humans, the CHAGASAZOL [10] and STOP CHAGAS [34] clinical trials showed that POS had trypanocidal and trypanostatic activity in chronic patients with Chagas disease, although less than that of BZ, most probably because the plasma exposure levels in the patients resulting from the dose of the drug used in these studies (400 mg b.i.d.) were just 10–20 % of optimal dose attained in mice (20 mg/kg.d) [3].
In an experimental model of acute CD in mice, the treatment with POS started 4 days after T. cruzi infection reduced the parasitemia and prevented fibronectin deposit in the heart of infected mice [19]. Since fibrosis contributes to electrophysiological changes and compensatory ventricular dilation leading up to clinical manifestations of CCC, such as heart failure [34], evaluation of POS treatment on progression of cardiac alterations in CD is necessary.
In this perspective, in this study we decided to evaluate the effect of POS in vitro in a model of cardiac fibrosis induced by T. cruzi, here called T. cruzi infected fibrotic cardiac microtissue (TCFCM). TCFCM mimics aspects observed in CCC including fibrosis and hypertrophy, even in the absence of inflammatory cells [21]. Cardiac microtissues are formed without any artificial scaffold in an agarose substrate, where the isolated cardiac cells agglomerate, retaining the ECM proteins secreted by them and forming a spheroidal 3D structure. It mimics cell-cell and cell-ECM interactions, similar to tissues in vivo, an advantage over 2D culture in monolayer [22,35].
Moreover, similar to what is observed during chagasic cardiomyopathy [16], the fibrosis induced by T. cruzi in cardiac microtissues, also results in TGF-β increased levels, which are reduced by inhibitors of TGF-β signaling pathway [16,23]. Here, we evaluated in TCFCM the effect of POS treatment on parasite load and ECM proteins levels (laminin and fibronectin), including its regulators (MMP-9, TIMP-4 and TGF-β).
After the series of experiments in monolayers, we selected the concentration of 5 nM POS to perform the experiments in TCFCM. Starting the treatment at 144 h post-infection, this concentration maintained for 96 h, POS treatment reduced parasite load as evaluated by qPCR. We found that besides reducing the parasite load, POS also reduced the expression levels of fibronectin and laminin in TCFCM, two proteins strongly involved in cardiac fibrosis during the CCC [15,36]. However, if this effect was a consequence of the reduction in parasite load or a direct effect of POS over fibrosis mechanisms, is difficult to conclude.
Our results showing an increase of TIMP-4 expression in TCFCM suggests that the fibrosis observed in this system is influenced by an unbalance in the regulation of the ECM. MMPs are zinc-dependent proteases crucial for physiological tissue remodeling and TIMPs inhibit the activity of MMPs [37]. Any change in the balance of these proteins can modify the myocardial structure. TIMP-4 is a low-molecular-weight protein of 23 kDa that is highly expressed in the heart [37], and modifications in its levels may influence the myocardial matrix remodeling. TIMP-4 is a key endogenous modulator of MMP-9 and a reduction in the levels of this protein has been described in heart failure [38]. Here, the treatment of TCFCM with POS reduced the levels of TIMP-4 that had been before increased by T. cruzi infection.
We also observed an increase in TGF-β secretion induced by T. cruzi infection. This finding corroborates previous observations in cardiomyocytes monolayers, in animal experimental models of CD and in humans, which associate this molecule to the host cell-parasite invasion and cardiac fibrosis during CD [16]. Moreover, this cytokine presents an anti-inflammatory effect in CD [39] and it was previously showed that POS regulates the trypanocidal immune response by controlling unspecific splenocyte proliferation in the early acute phase of CD [19].
TGF-β is a molecule that remains attached to the ECM after its production in a latent form [40]. Since we observed a higher level of secreted TGF-β in infected and treated supernatant of TCFCM, an explanation could be lower retention of TGF-β in ECM linkage sites, that could result from the reduction in fibronectin and laminin levels induced by the drug. Fibronectin is a critical protein for the incorporation of latent TGF beta-binding protein-1 [41]. More studies involving the understanding of mechanisms of fibrosis and inflammation in the context of treatment with POS and TGF-β response during T. cruzi infection are necessary.
We conclude that POS, apart from its potent intrinsic anti-T. cruzi activity may contribute to the prevention or reversal of CCC by reducing fibrosis of the infected cardiac tissues, due to an intrinsic reorganization of the myocardial tissue resulting from a decreased parasite load.
Funding
This study was supported by CNPq and POM/Fiocruz.
Data availability statement
All datasets generated for this study are included in the manuscript.
CRediT authorship contribution statement
Lindice Mitie Nisimura: Conceptualization, Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Patrícia Mello Ferrão: Investigation, Formal analysis. Alanderson da Rocha Nogueira: Investigation. Mariana Caldas Waghabi: Supervision, Formal analysis. Marcelo Meuser-Batista: Investigation, Formal analysis. Otacílio C. Moreira: Formal analysis, Investigation. Julio A. Urbina: Writing - review & editing, Funding acquisition. Luciana Ribeiro Garzoni: Supervision, Investigation, Formal analysis, Writing - original draft, Writing - review & editing, Funding acquisition.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors would like to thank the Program for Technological Development in Tools for Health (PDTIS-FIOCRUZ) for the use of its facilities.
Appendix A
Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.molbiopara.2020.111283.
Appendix A
Supplementary data
The following are Supplementary data to this article:
References
[1]
E.
Cunha-Neto
C.
Chevillard
Chagas disease cardiomyopathy: immunopathology and genetics
Mediators Inflamm.
2014
2014
683230-683230
E. Cunha-Neto, C. Chevillard, Chagas disease cardiomyopathy: immunopathology and genetics, Mediators of inflammation 2014 (2014) 683230-683230.
[2]
W.H.O. (WHO)
Chagas’ Disease (American Trypanosomiasis) Factsheet
2017
http://www.who.int/mediacentre/factsheets/fs340/en/index.html
W.H.O. (WHO), Chagas’ disease (American trypanosomiasis) Factsheet 2017. http://www.who.int/mediacentre/factsheets/fs340/en/index.html
[3]
J.A.
Urbina
Recent clinical trials for the etiological treatment of chronic chagas disease: advances, challenges and perspectives
J. Eukaryot. Microbiol.
62
1
2015
149
156
J.A. Urbina, Recent clinical trials for the etiological treatment of chronic chagas disease: advances, challenges and perspectives, J Eukaryot Microbiol 62(1) (2015) 149-56.
[4]
C.A.
Morillo
J.A.
Marin-Neto
A.
Avezum
S.
Sosa-Estani
A.
Rassi
Jr.
F.
Rosas
E.
Villena
R.
Quiroz
R.
Bonilla
C.
Britto
F.
Guhl
E.
Velazquez
L.
Bonilla
B.
Meeks
P.
Rao-Melacini
J.
Pogue
A.
Mattos
J.
Lazdins
A.
Rassi
S.J.
Connolly
S.
Yusuf
Randomized trial of benznidazole for chronic chagas’ cardiomyopathy
N. Engl. J. Med.
373
14
2015
1295
1306
C.A. Morillo, J.A. Marin-Neto, A. Avezum, S. Sosa-Estani, A. Rassi, Jr., F. Rosas, E. Villena, R. Quiroz, R. Bonilla, C. Britto, F. Guhl, E. Velazquez, L. Bonilla, B. Meeks, P. Rao-Melacini, J. Pogue, A. Mattos, J. Lazdins, A. Rassi, S.J. Connolly, S. Yusuf, Randomized Trial of Benznidazole for Chronic Chagas' Cardiomyopathy, N Engl J Med 373(14) (2015) 1295-306.
[5]
D.S.
Kwon
E.
Mylonakis
Posaconazole: a new broad-spectrum antifungal agent
Expert Opin. Pharmacother.
8
8
2007
1167
1178
D.S. Kwon, E. Mylonakis, Posaconazole: a new broad-spectrum antifungal agent, Expert Opin Pharmacother 8(8) (2007) 1167-78.
[6]
G.I.
Lepesheva
F.
Villalta
M.R.
Waterman
Targeting Trypanosoma cruzi sterol 14alpha-demethylase (CYP51)
Adv. Parasitol.
75
2011
65
87
G.I. Lepesheva, F. Villalta, M.R. Waterman, Targeting Trypanosoma cruzi sterol 14alpha-demethylase (CYP51), Adv Parasitol 75 (2011) 65-87.
[7]
J.
Molina
O.
Martins-Filho
Z.
Brener
A.J.
Romanha
D.
Loebenberg
J.A.
Urbina
Activities of the triazole derivative SCH 56592 (posaconazole) against drug-resistant strains of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi in immunocompetent and immunosuppressed murine hosts
Antimicrob. Agents Chemother.
44
1
2000
150
155
J. Molina, O. Martins-Filho, Z. Brener, A.J. Romanha, D. Loebenberg, J.A. Urbina, Activities of the triazole derivative SCH 56592 (posaconazole) against drug-resistant strains of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi in immunocompetent and immunosuppressed murine hosts, Antimicrob Agents Chemother 44(1) (2000) 150-5.
[8]
J.A.
Urbina
G.
Payares
L.M.
Contreras
A.
Liendo
C.
Sanoja
J.
Molina
M.
Piras
R.
Piras
N.
Perez
P.
Wincker
D.
Loebenberg
Antiproliferative effects and mechanism of action of SCH 56592 against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies
Antimicrob. Agents Chemother.
42
7
1998
1771
1777
J.A. Urbina, G. Payares, L.M. Contreras, A. Liendo, C. Sanoja, J. Molina, M. Piras, R. Piras, N. Perez, P. Wincker, D. Loebenberg, Antiproliferative effects and mechanism of action of SCH 56592 against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies, Antimicrob Agents Chemother 42(7) (1998) 1771-7.
[9]
M.J.
Pinazo
G.
Espinosa
M.
Gallego
P.L.
Lopez-Chejade
J.A.
Urbina
J.
Gascon
Successful treatment with posaconazole of a patient with chronic Chagas disease and systemic lupus erythematosus
Am. J. Trop. Med. Hyg.
82
4
2010
583
587
M.J. Pinazo, G. Espinosa, M. Gallego, P.L. Lopez-Chejade, J.A. Urbina, J. Gascon, Successful treatment with posaconazole of a patient with chronic Chagas disease and systemic lupus erythematosus, Am J Trop Med Hyg 82(4) (2010) 583-7.
[10]
I.
Molina
J.
Gomez i Prat
F.
Salvador
B.
Trevino
E.
Sulleiro
N.
Serre
D.
Pou
S.
Roure
J.
Cabezos
L.
Valerio
A.
Blanco-Grau
A.
Sanchez-Montalva
X.
Vidal
A.
Pahissa
Randomized trial of posaconazole and benznidazole for chronic Chagas’ disease
N. Engl. J. Med.
370
20
2014
1899
1908
I. Molina, J. Gomez i Prat, F. Salvador, B. Trevino, E. Sulleiro, N. Serre, D. Pou, S. Roure, J. Cabezos, L. Valerio, A. Blanco-Grau, A. Sanchez-Montalva, X. Vidal, A. Pahissa, Randomized trial of posaconazole and benznidazole for chronic Chagas' disease, N Engl J Med 370(20) (2014) 1899-908.
[11]
H.B.
Tanowitz
L.V.
Kirchhoff
D.
Simon
S.A.
Morris
L.M.
Weiss
M.
Wittner
Chagas’ disease
Clin. Microbiol. Rev.
5
4
1992
400
419
H.B. Tanowitz, L.V. Kirchhoff, D. Simon, S.A. Morris, L.M. Weiss, M. Wittner, Chagas' disease, Clin Microbiol Rev 5(4) (1992) 400-19.
[12]
F.L.
de Oliveira
T.C.
Araujo-Jorge
E.M.
de Souza
G.M.
de Oliveira
W.M.
Degrave
J.J.
Feige
S.
Bailly
M.C.
Waghabi
Oral administration of GW788388, an inhibitor of transforming growth factor beta signaling, prevents heart fibrosis in Chagas disease
PLoS Negl. Trop. Dis.
6
6
2012
e1696
F.L. de Oliveira, T.C. Araujo-Jorge, E.M. de Souza, G.M. de Oliveira, W.M. Degrave, J.J. Feige, S. Bailly, M.C. Waghabi, Oral administration of GW788388, an inhibitor of transforming growth factor beta signaling, prevents heart fibrosis in Chagas disease, PLoS neglected tropical diseases 6(6) (2012) e1696.
[13]
G.
Vilar-Pereira
I.
Resende Pereira
L.A.
de Souza Ruivo
O.
Cruz Moreira
A.A.
da Silva
C.
Britto
J.
Lannes-Vieira
Combination chemotherapy with suboptimal doses of benznidazole and pentoxifylline sustains partial reversion of experimental chagas’ heart disease
Antimicrob. Agents Chemother.
60
7
2016
4297
4309
G. Vilar-Pereira, I. Resende Pereira, L.A. de Souza Ruivo, O. Cruz Moreira, A.A. da Silva, C. Britto, J. Lannes-Vieira, Combination Chemotherapy with Suboptimal Doses of Benznidazole and Pentoxifylline Sustains Partial Reversion of Experimental Chagas' Heart Disease, Antimicrob Agents Chemother 60(7) (2016) 4297-309.
[14]
I.R.
Pereira
G.
Vilar-Pereira
O.C.
Moreira
I.P.
Ramos
D.
Gibaldi
C.
Britto
M.O.
Moraes
J.
Lannes-Vieira
Pentoxifylline reverses chronic experimental Chagasic cardiomyopathy in association with repositioning of abnormal CD8+ T-cell response
PLoS Negl. Trop. Dis.
9
3
2015
e0003659
I.R. Pereira, G. Vilar-Pereira, O.C. Moreira, I.P. Ramos, D. Gibaldi, C. Britto, M.O. Moraes, J. Lannes-Vieira, Pentoxifylline reverses chronic experimental Chagasic cardiomyopathy in association with repositioning of abnormal CD8+ T-cell response, PLoS Negl Trop Dis 9(3) (2015) e0003659.
[15]
Z.A.
Andrade
S.G.
Andrade
R.
Correa
M.
Sadigursky
V.J.
Ferrans
Myocardial changes in acute Trypanosoma cruzi infection. Ultrastructural evidence of immune damage and the role of microangiopathy
Am. J. Pathol.
144
6
1994
1403
1411
Z.A. Andrade, S.G. Andrade, R. Correa, M. Sadigursky, V.J. Ferrans, Myocardial changes in acute Trypanosoma cruzi infection. Ultrastructural evidence of immune damage and the role of microangiopathy, Am J Pathol 144(6) (1994) 1403-11.
[16]
T.C.
Araujo-Jorge
M.C.
Waghabi
S.
Bailly
J.J.
Feige
The TGF-beta pathway as an emerging target for Chagas disease therapy
Clin. Pharmacol. Ther.
92
5
2012
613
621
T.C. Araujo-Jorge, M.C. Waghabi, S. Bailly, J.J. Feige, The TGF-beta pathway as an emerging target for Chagas disease therapy, Clin Pharmacol Ther 92(5) (2012) 613-21.
[17]
Y.Y.
Li
C.F.
McTiernan
A.M.
Feldman
Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling
Cardiovasc. Res.
46
2
2000
214
224
Y.Y. Li, C.F. McTiernan, A.M. Feldman, Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling, Cardiovasc Res 46(2) (2000) 214-24.
[18]
R.C.
Fares
A.
Gomes Jde
L.R.
Garzoni
M.C.
Waghabi
R.M.
Saraiva
N.I.
Medeiros
R.
Oliveira-Prado
L.H.
Sangenis
C.
Chambela Mda
F.F.
de Araujo
A.
Teixeira-Carvalho
M.P.
Damasio
V.A.
Valente
K.S.
Ferreira
G.R.
Sousa
M.O.
Rocha
R.
Correa-Oliveira
Matrix metalloproteinases 2 and 9 are differentially expressed in patients with indeterminate and cardiac clinical forms of Chagas disease
Infect. Immun.
81
10
2013
3600
3608
R.C. Fares, A. Gomes Jde, L.R. Garzoni, M.C. Waghabi, R.M. Saraiva, N.I. Medeiros, R. Oliveira-Prado, L.H. Sangenis, C. Chambela Mda, F.F. de Araujo, A. Teixeira-Carvalho, M.P. Damasio, V.A. Valente, K.S. Ferreira, G.R. Sousa, M.O. Rocha, R. Correa-Oliveira, Matrix metalloproteinases 2 and 9 are differentially expressed in patients with indeterminate and cardiac clinical forms of Chagas disease, Infection and immunity 81(10) (2013) 3600-8.
[19]
B.P.
Olivieri
J.T.
Molina
S.L.
de Castro
M.C.
Pereira
C.M.
Calvet
J.A.
Urbina
T.C.
Araujo-Jorge
A comparative study of posaconazole and benznidazole in the prevention of heart damage and promotion of trypanocidal immune response in a murine model of Chagas disease
Int. J. Antimicrob. Agents
36
1
2010
79
83
B.P. Olivieri, J.T. Molina, S.L. de Castro, M.C. Pereira, C.M. Calvet, J.A. Urbina, T.C. Araujo-Jorge, A comparative study of posaconazole and benznidazole in the prevention of heart damage and promotion of trypanocidal immune response in a murine model of Chagas disease, International journal of antimicrobial agents 36(1) (2010) 79-83.
[20]
A.
Amelian
K.
Wasilewska
D.
Megias
K.
Winnicka
Application of standard cell cultures and 3D in vitro tissue models as an effective tool in drug design and development
Pharmacol. Rep.
69
5
2017
861
870
A. Amelian, K. Wasilewska, D. Megias, K. Winnicka, Application of standard cell cultures and 3D in vitro tissue models as an effective tool in drug design and development, Pharmacol Rep 69(5) (2017) 861-870.
[21]
L.R.
Garzoni
D.
Adesse
M.J.
Soares
M.I.
Rossi
R.
Borojevic
N.
de Meirelles Mde
Fibrosis and hypertrophy induced by Trypanosoma cruzi in a three-dimensional cardiomyocyte-culture system
J. Infect. Dis.
197
6
2008
906
915
L.R. Garzoni, D. Adesse, M.J. Soares, M.I. Rossi, R. Borojevic, N. de Meirelles Mde, Fibrosis and hypertrophy induced by Trypanosoma cruzi in a three-dimensional cardiomyocyte-culture system, The Journal of infectious diseases 197(6) (2008) 906-15.
[22]
L.R.
Garzoni
M.I.
Rossi
A.P.
de Barros
V.
Guarani
M.
Keramidas
L.B.
Balottin
D.
Adesse
C.M.
Takiya
P.P.
Manso
I.B.
Otazu
N.
Meirelles Mde
R.
Borojevic
Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells
Exp. Cell Res.
315
19
2009
3406
3418
L.R. Garzoni, M.I. Rossi, A.P. de Barros, V. Guarani, M. Keramidas, L.B. Balottin, D. Adesse, C.M. Takiya, P.P. Manso, I.B. Otazu, N. Meirelles Mde, R. Borojevic, Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells, Experimental cell research 315(19) (2009) 3406-18.
[23]
P.M.
Ferrao
L.M.
Nisimura
Od.C.
Moreira
M.G.
Land
M.C.
Pereira
Ld.
Mendonca-Lima
T.C.
Araujo-Jorge
M.C.
Waghabi
L.R.
Garzoni
Inhibition of TGF-beta pathway reverts extracellular matrix remodeling in T. cruzi-infected cardiac spheroids
Exp. Cell Res.
362
2
2018
260
267
P.M. Ferrao, L.M. Nisimura, O.d.C. Moreira, M.G. Land, M.C. Pereira, L.d. Mendonca-Lima, T.C. Araujo-Jorge, M.C. Waghabi, L.R. Garzoni, Inhibition of TGF-beta pathway reverts extracellular matrix remodeling in T. cruzi-infected cardiac spheroids, Exp Cell Res 362(2) (2018) 260-267.
[24]
L.
Hudson
F.
Grover
W.E.
Gutteridge
R.A.
Klein
W.
Peters
R.A.
Neal
M.A.
Miles
M.T.
Scott
R.
Nourish
B.P.
Ager
Suggested guidelines for work with live Trypanosoma cruzi
Trans. R. Soc. Trop. Med. Hyg.
77
3
1983
416
419
L. Hudson, F. Grover, W. E. Gutteridge, R. A. Klein, W. Peters, R. A. Neal, M. A. Miles, M. T. Scott, R. Nourish, and B. P. Ager, Suggested guidelines for work with live Trypanosoma cruzi, Trans R Soc Trop Med Hyg 77(3) (1983) 416-9.
[25]
M.N.
Meirelles
T.C.
de Araujo-Jorge
C.F.
Miranda
W.
de Souza
H.S.
Barbosa
Interaction of Trypanosoma cruzi with heart muscle cells: ultrastructural and cytochemical analysis of endocytic vacuole formation and effect upon myogenesis in vitro
Eur. J. Cell Biol.
41
2
1986
198
206
M.N. Meirelles, T.C. de Araujo-Jorge, C.F. Miranda, W. de Souza, H.S. Barbosa, Interaction of Trypanosoma cruzi with heart muscle cells: ultrastructural and cytochemical analysis of endocytic vacuole formation and effect upon myogenesis in vitro, Eur J Cell Biol 41(2) (1986) 198-206.
[26]
O.C.
Moreira
J.D.
Ramirez
E.
Velazquez
M.F.
Melo
C.
Lima-Ferreira
F.
Guhl
S.
Sosa-Estani
J.A.
Marin-Neto
C.A.
Morillo
C.
Britto
Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma cruzi parasitemia in patients with chronic Chagas disease cardiomyopathy: a substudy from the BENEFIT trial
Acta Trop.
125
1
2013
23
31
O.C. Moreira, J.D. Ramirez, E. Velazquez, M.F. Melo, C. Lima-Ferreira, F. Guhl, S. Sosa-Estani, J.A. Marin-Neto, C.A. Morillo, C. Britto, Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma cruzi parasitemia in patients with chronic Chagas disease cardiomyopathy: a substudy from the BENEFIT trial, Acta Trop 125(1) (2013) 23-31.
[27]
J.C.
Ramirez
C.I.
Cura
O.
da Cruz Moreira
E.
Lages-Silva
N.
Juiz
E.
Velazquez
J.D.
Ramirez
A.
Alberti
P.
Pavia
M.D.
Flores-Chavez
A.
Munoz-Calderon
D.
Perez-Morales
J.
Santalla
P.
Marcos da Matta Guedes
J.
Peneau
P.
Marcet
C.
Padilla
D.
Cruz-Robles
E.
Valencia
G.E.
Crisante
G.
Greif
I.
Zulantay
J.A.
Costales
M.
Alvarez-Martinez
N.E.
Martinez
R.
Villarroel
S.
Villarroel
Z.
Sanchez
M.
Bisio
R.
Parrado
L.
Maria da Cunha Galvao
A.C.
Jacome da Camara
B.
Espinoza
B.
Alarcon de Noya
C.
Puerta
A.
Riarte
P.
Diosque
S.
Sosa-Estani
F.
Guhl
I.
Ribeiro
C.
Aznar
C.
Britto
Z.E.
Yadon
A.G.
Schijman
Analytical validation of quantitative real-time PCR methods for quantification of trypanosoma cruzi DNA in blood samples from chagas disease patients
J. Mol. Diagn.
17
5
2015
605
615
J.C. Ramirez, C.I. Cura, O. da Cruz Moreira, E. Lages-Silva, N. Juiz, E. Velazquez, J.D. Ramirez, A. Alberti, P. Pavia, M.D. Flores-Chavez, A. Munoz-Calderon, D. Perez-Morales, J. Santalla, P. Marcos da Matta Guedes, J. Peneau, P. Marcet, C. Padilla, D. Cruz-Robles, E. Valencia, G.E. Crisante, G. Greif, I. Zulantay, J.A. Costales, M. Alvarez-Martinez, N.E. Martinez, R. Villarroel, S. Villarroel, Z. Sanchez, M. Bisio, R. Parrado, L. Maria da Cunha Galvao, A.C. Jacome da Camara, B. Espinoza, B. Alarcon de Noya, C. Puerta, A. Riarte, P. Diosque, S. Sosa-Estani, F. Guhl, I. Ribeiro, C. Aznar, C. Britto, Z.E. Yadon, A.G. Schijman, Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients, J Mol Diagn 17(5) (2015) 605-15.
[28]
T.A.
Wynn
T.R.
Ramalingam
Mechanisms of fibrosis: therapeutic translation for fibrotic disease
Nat. Med.
18
7
2012
1028
1040
T.A. Wynn, T.R. Ramalingam, Mechanisms of fibrosis: therapeutic translation for fibrotic disease, Nat Med 18(7) (2012) 1028-40.
[29]
J.R.
Cancado
Long term evaluation of etiological treatment of chagas disease with benznidazole
Rev. Inst. Med. Trop. Sao Paulo
44
1
2002
29
37
J.R. Cancado, Long term evaluation of etiological treatment of chagas disease with benznidazole, Rev Inst Med Trop Sao Paulo 44(1) (2002) 29-37.
[30]
L.
Zhang
R.L.
Tarleton
Parasite persistence correlates with disease severity and localization in chronic Chagas’ disease
J. Infect. Dis.
180
2
1999
480
486
L. Zhang, R.L. Tarleton, Parasite persistence correlates with disease severity and localization in chronic Chagas' disease, The Journal of infectious diseases 180(2) (1999) 480-6.
[31]
J.A.
Urbina
Ergosterol biosynthesis and drug development for Chagas disease
Mem. Inst. Oswaldo Cruz
104
Suppl 1
2009
311
318
J.A. Urbina, Ergosterol biosynthesis and drug development for Chagas disease, Memorias do Instituto Oswaldo Cruz 104 Suppl 1 (2009) 311-8.
[32]
G.
Benaim
J.M.
Sanders
Y.
Garcia-Marchan
C.
Colina
R.
Lira
A.R.
Caldera
G.
Payares
C.
Sanoja
J.M.
Burgos
A.
Leon-Rossell
J.L.
Concepcion
A.G.
Schijman
M.
Levin
E.
Oldfield
J.A.
Urbina
Amiodarone has intrinsic anti-Trypanosoma cruzi activity and acts synergistically with posaconazole
J. Med. Chem.
49
3
2006
892
899
G. Benaim, J.M. Sanders, Y. Garcia-Marchan, C. Colina, R. Lira, A.R. Caldera, G. Payares, C. Sanoja, J.M. Burgos, A. Leon-Rossell, J.L. Concepcion, A.G. Schijman, M. Levin, E. Oldfield, J.A. Urbina, Amiodarone has intrinsic anti-Trypanosoma cruzi activity and acts synergistically with posaconazole, Journal of medicinal chemistry 49(3) (2006) 892-9.
[33]
P.
Veiga-Santos
E.S.
Barrias
J.F.
Santos
T.L.
de Barros Moreira
T.M.
de Carvalho
J.A.
Urbina
W.
de Souza
Effects of amiodarone and posaconazole on the growth and ultrastructure of Trypanosoma cruzi
Int. J. Antimicrob. Agents
40
1
2012
61
71
P. Veiga-Santos, E.S. Barrias, J.F. Santos, T.L. de Barros Moreira, T.M. de Carvalho, J.A. Urbina, W. de Souza, Effects of amiodarone and posaconazole on the growth and ultrastructure of Trypanosoma cruzi, International journal of antimicrobial agents 40(1) (2012) 61-71.
[34]
A.
Rassi
Jr.
A.
Rassi
W.C.
Little
Chagas’ heart disease
Clin. Cardiol.
23
12
2000
883
889
A. Rassi, Jr., A. Rassi, W.C. Little, Chagas' heart disease, Clin Cardiol 23(12) (2000) 883-9.
[35]
J.M.
Kelm
E.
Ehler
L.K.
Nielsen
S.
Schlatter
J.C.
Perriard
M.
Fussenegger
Design of artificial myocardial microtissues
Tissue Eng.
10
1–2
2004
201
214
J.M. Kelm, E. Ehler, L.K. Nielsen, S. Schlatter, J.C. Perriard, M. Fussenegger, Design of artificial myocardial microtissues, Tissue engineering 10(1-2) (2004) 201-14.
[36]
M.L.
Higuchi
S.
Fukasawa
T.
De Brito
L.C.
Parzianello
G.
Bellotti
J.A.
Ramires
Different microcirculatory and interstitial matrix patterns in idiopathic dilated cardiomyopathy and Chagas’ disease: a three dimensional confocal microscopy study
Heart
82
3
1999
279
285
M.L. Higuchi, S. Fukasawa, T. De Brito, L.C. Parzianello, G. Bellotti, J.A. Ramires, Different microcirculatory and interstitial matrix patterns in idiopathic dilated cardiomyopathy and Chagas' disease: a three dimensional confocal microscopy study, Heart 82(3) (1999) 279-85.
[37]
I.
Koskivirta
Z.
Kassiri
O.
Rahkonen
R.
Kiviranta
G.Y.
Oudit
T.D.
McKee
V.
Kyto
A.
Saraste
E.
Jokinen
P.P.
Liu
E.
Vuorio
R.
Khokha
Mice with tissue inhibitor of metalloproteinases 4 (Timp4) deletion succumb to induced myocardial infarction but not to cardiac pressure overload
J. Biol. Chem.
285
32
2010
24487
24493
I. Koskivirta, Z. Kassiri, O. Rahkonen, R. Kiviranta, G.Y. Oudit, T.D. McKee, V. Kyto, A. Saraste, E. Jokinen, P.P. Liu, E. Vuorio, R. Khokha, Mice with tissue inhibitor of metalloproteinases 4 (Timp4) deletion succumb to induced myocardial infarction but not to cardiac pressure overload, J Biol Chem 285(32) (2010) 24487-93.
[38]
F.G.
Spinale
Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function
Physiol. Rev.
87
4
2007
1285
1342
F.G. Spinale, Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function, Physiological reviews 87(4) (2007) 1285-342.
[39]
J.S.
Silva
D.R.
Twardzik
S.G.
Reed
Regulation of Trypanosoma cruzi infections in vitro and in vivo by transforming growth factor beta (TGF-beta)
J. Exp. Med.
174
3
1991
539
545
J.S. Silva, D.R. Twardzik, S.G. Reed, Regulation of Trypanosoma cruzi infections in vitro and in vivo by transforming growth factor beta (TGF-beta), J Exp Med 174(3) (1991) 539-45.
[40]
I.B.
Robertson
M.
Horiguchi
L.
Zilberberg
B.
Dabovic
K.
Hadjiolova
D.B.
Rifkin
Latent TGF-β-binding proteins
Matrix Biol.
47
2015
44
53
I.B. Robertson, M. Horiguchi, L. Zilberberg, B. Dabovic, K. Hadjiolova, D.B. Rifkin, Latent TGF-β-binding proteins, Matrix biology : journal of the International Society for Matrix Biology 47 (2015) 44-53.
[41]
S.L.
Dallas
P.
Sivakumar
C.J.
Jones
Q.
Chen
D.M.
Peters
D.F.
Mosher
M.J.
Humphries
C.M.
Kielty
Fibronectin regulates latent transforming growth factor-beta (TGF beta) by controlling matrix assembly of latent TGF beta-binding protein-1
J. Biol. Chem.
280
19
2005
18871
18880
S.L. Dallas, P. Sivakumar, C.J. Jones, Q. Chen, D.M. Peters, D.F. Mosher, M.J. Humphries, C.M. Kielty, Fibronectin regulates latent transforming growth factor-beta (TGF beta) by controlling matrix assembly of latent TGF beta-binding protein-1, The Journal of biological chemistry 280(19) (2005) 18871-80.
MOLBIO
111283
111283
S0166-6851(20)30047-5
10.1016/j.molbiopara.2020.111283
Effect of Posaconazole in an in vitro model of cardiac fibrosis induced by Trypanosoma cruzi
Lindice Mitie
Nisimura
Conceptualization
Investigation
Formal analysis
Writing - original draft
Writing - review & editing
a
Patrícia Mello
Ferrão
Investigation
Formal analysis
a
b
Alanderson da Rocha
Nogueira
Investigation
c
Mariana Caldas
Waghabi
Supervision
Formal analysis
b
Marcelo
Meuser-Batista
Investigation
Formal analysis
a
Otacílio C.
Moreira
Formal analysis
Investigation
d
Julio A.
Urbina
Writing - review & editing
Funding acquisition
e
Luciana Ribeiro
Garzoni
Supervision
Investigation
Formal analysis
Writing - original draft
Writing - review & editing
Funding acquisition
a
*
largarz@ioc.fiocruz.br
a
Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Laboratório de Inovações em Terapias
Ensino e Bioprodutos
Instituto Oswaldo Cruz
Fundação Oswaldo Cruz
Rio de Janeiro
RJ
Brazil
Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
b
Laboratório de Genômica Funcional e Bioinformática, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Laboratório de Genômica Funcional e Bioinformática
Instituto Oswaldo Cruz
Fundação Oswaldo Cruz
Rio de Janeiro
RJ
Brazil
Laboratório de Genômica Funcional e Bioinformática, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
c
Laboratório de Ultra-estrutura Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Laboratório de Ultra-estrutura Celular
Instituto Oswaldo Cruz
Fundação Oswaldo Cruz
Rio de Janeiro
RJ
Brazil
Laboratório de Ultra-estrutura Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
d
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Laboratório de Biologia Molecular e Doenças Endêmicas
Instituto Oswaldo Cruz
Fundação Oswaldo Cruz
Rio de Janeiro
RJ
Brazil
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
e
Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela
Instituto Venezolano de Investigaciones Cientificas
Caracas
Venezuela
Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela
⁎
Corresponding author at: Av. Brasil, 4365 - CEP. 21045-900, Rio de Janeiro, RJ, Brazil.
Av. Brasil
4365 - CEP. 21045-900
Rio de Janeiro
RJ
Brazil
Graphical abstract
Highlights
•
Fibrosis in heart tissue and cardiac hypertrophy are features in Chagas disease.
•
Trypanosoma cruzi fibrotic cardiac microtissue is a three-dimensional culture.
•
Posaconazole has potent and selective anti-T. cruzi activity.
•
Posaconazole induced reorganization of the myocardial tissue.
Abstract
Posaconazole (POS) is an inhibitor of ergosterol biosynthesis in clinical use for treating invasive fungal infections. POS has potent and selective anti-Trypanosoma cruzi activity and has been evaluated as a possible treatment for Chagas disease. Microtissues are a 3D culture system that has been shown to reproduce better tissue architecture and functionality than cell cultures in monolayer (2D). It has been used to evaluate chemotropic response as in vitro disease models. We previously developed an in vitro model that reproduces aspects of cardiac fibrosis observed in Chagas cardiomyopathy, using microtissues formed by primary cardiac cells infected by the T. cruzi, here called T. cruzi fibrotic cardiac microtissue (TCFCM). We also showed that the treatment of TCFCM with a TGF-β pathway inhibitor reduces fibrosis. Here, we aimed to evaluate the effect of POS in TCFCM, observing parasite load and molecules involved in fibrosis. To choose the concentration of POS to be used in TCFCM we first performed experiments in a monolayer of primary cardiac cell cultures and, based on the results, TCFCM was treated with 5 nM of POS for 96 h, starting at 144 h post-infection. Our previous studies showed that at this time the TCFCM had established fibrosis, resulting from T. cruzi infection. Treatment with POS of TCFCM reduced 50 % of parasite load as observed by real-time PCR and reduced markedly the fibrosis as observed by western blot and immunofluorescence, associated with a strong reduction in the expression of fibronectin and laminin (45 % and 54 %, respectively). POS treatment also changed the expression of proteins involved in the regulation of extracellular matrix proteins (TGF-β and TIMP-4, increased by 50 % and decreased by 58 %, respectively) in TCFCM. In conclusion, POS presented a potent trypanocidal effect both in 2D and in TCFCM, and the reduction of the parasite load was associated with a reduction of fibrosis in the absence of external immunological effectors.
Keywords
Three-dimensional culture
Cardiac microtissues
Chagas disease
Posaconazole
Cardiac fibrosis
Extracellular matrix protein
KBJ00000000013850
2020-10-30T18:42:58
S300.3
S300
S0166-6851(20)30047-5
10.1016/j.molbiopara.2020.111283
MOLBIO
0166-6851
111283
111283
FLA
NON-CRC
UNLIMITED
NONE
2020-06-18T15:13:32Z
01666851/v238sC/S0166685120300475/main.xml
126562
MAIN
JA 5.6.0 ARTICLE
FULL-TEXT
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IMAGE-DOWNSAMPLED
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IMAGE-DOWNSAMPLED
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IMAGE-DOWNSAMPLED
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IMAGE-DOWNSAMPLED
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46460
IMAGE-DOWNSAMPLED
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49546
IMAGE-DOWNSAMPLED
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27161
IMAGE-DOWNSAMPLED
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10879
IMAGE-DOWNSAMPLED
01666851/v238sC/S0166685120300475/main.assets/ga1.sml
13410
IMAGE-THUMBNAIL
01666851/v238sC/S0166685120300475/main.assets/gr1.sml
15565
IMAGE-THUMBNAIL
01666851/v238sC/S0166685120300475/main.assets/gr2.sml
10401
IMAGE-THUMBNAIL
01666851/v238sC/S0166685120300475/main.assets/gr3.sml
15189
IMAGE-THUMBNAIL
01666851/v238sC/S0166685120300475/main.assets/gr4.sml
26657
IMAGE-THUMBNAIL
01666851/v238sC/S0166685120300475/main.assets/gr5.sml
5807
IMAGE-THUMBNAIL
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5367
IMAGE-THUMBNAIL
01666851/v238sC/S0166685120300475/main.assets/mmc2.sml
12239
IMAGE-THUMBNAIL
01666851/v238sC/S0166685120300475/main.assets/mmc3.sml
21741
IMAGE-THUMBNAIL
01666851/v238sC/S0166685120300475/main.assets/mmc4.sml
4723
IMAGE-THUMBNAIL
01666851/v238sC/S0166685120300475/main.assets/mmc1.docx
31527
APPLICATION
01666851/v238sC/S0166685120300475/main.pdf
9292225
MAIN
1.7 7.0
DISTILLED OPTIMIZED BOOKMARKED
01666851/v238sC/S0166685120300475/main.raw
56421
S0166-6851(20)X0004-1
MOLBIO
0166-6851
238
C
202007
S0166-6851(20)30047-5
10.1016/j.molbiopara.2020.111283
111283
main.pdf
PDF
1.7
local
1604323634688
collectiebehoudsniveau 1
2020-11-02T12:00:14.702+01:00
local
1604323635200
0
SHA-512
01f21adadae0c2aedae6b81706d2d29aec584e6738e06398783787af333050d227d31a179bce21d72e7b4a82ca2262322930b5779fc8e6d6b6ed0e72973cf06f
java.security.MessageDigest
9292225
Adobe Acrobat Document
1.7
DIAS
62
DIAS tentative identification
main.pdf
local
1604323635201
0
SHA-512
edbc62c434ad131944036f3e7fea9e5bac66139005033657f800e6831212e7b091997b9153a96faa7f867eeb6cca68e813b672f6254952e21c3865883bd99d22
java.security.MessageDigest
56421
not checked
main.raw
local
1604323635202
0
SHA-512
2d3433ef6c73451532f1478256803c0a353cd95d50377b34cec24f2b428473a1a48655f2827982fcb5bdcf584835e4eeeec7df9c341e673591aa83a3a496cfa0
java.security.MessageDigest
126562
not checked
main.xml
local
1604323635203
0
SHA-512
d6fc38d2cdaaf799451e268c3b174ef67b2d7f31b320a7f5c16a8cd086dc9cae575625e9477c2efbcaf34fe15226949dcdb178d508b4d3f4917464decfc920f3
java.security.MessageDigest
21114
not checked
ga1.jpg
local
1604323635204
0
SHA-512
0d26c9e8fe158c18cc5740368e501ec1a0d31205c41276493eb72180dd2db20d793ebb3f88db39a148a41a9d808a4f010a1b38becd1f3ee4165905f8c306704f
java.security.MessageDigest
13410
not checked
ga1.sml
local
1604323635205
0
SHA-512
31c7c4c51671296cf0ba3f0954318ec62ba53d041f3d9a12a49686be9856fbe1f37cd0b6d17c09a416f1cba5ad1e81eb010beea7a02b293abdfa383a4bdb8405
java.security.MessageDigest
182821
not checked
gr1.jpg
local
1604323635206
0
SHA-512
97e6c136a1928edd0361a42bc46794b85f3b094d03b8eb24da6b7d9565c8c83c42ec83bc5a5e216ee8df2250753ced3e2677640881b83b7e47d7364c6dfab9ad
java.security.MessageDigest
15565
not checked
gr1.sml
local
1604323635207
0
SHA-512
0aa917a4aaba994d6704af7e71a30f7a5e4eecb40360a938e39f124240f03e9112b2ddb1e68d837dc5c45c61602796556323234717136d33fb0d7fb6cd344197
java.security.MessageDigest
19332
not checked
gr2.jpg
local
1604323635208
0
SHA-512
fbc4310baceb2aa3c08e332cb41eb78e27ae550e08332d69e242e88bfca6c167d187b7fa804c73d61e86e41a88f779d13a61d29f249ae0bc4ffa3b177d0224e2
java.security.MessageDigest
10401
not checked
gr2.sml
local
1604323635209
0
SHA-512
da1bb9109888aa562079c3fd112f2eefd543fafbc41435e1c661e147d5852d63f9e08332312c3254c123415b213eb4db3a819f738f068a3006d8d43799548590
java.security.MessageDigest
98717
not checked
gr3.jpg
local
1604323635210
0
SHA-512
bfd8d85d884cfc8d25615f33280a1cbf2458afeacb73fb2faf0a4bc78dfd6b824d9e1722155ab88e8e8eea4af5cab0ccd152196fafe8f9a19413800e2b689c46
java.security.MessageDigest
15189
not checked
gr3.sml
local
1604323635211
0
SHA-512
21ee73010a65c1f0631103dfe49d2931e3600ca72a2d04b766b189dd57f74f968942f768bf7192785562a78c71f974824e49e8f1b866772784c8e0f445886e8f
java.security.MessageDigest
108587
not checked
gr4.jpg
local
1604323635212
0
SHA-512
26fb8f79beb693f5454a48d1c5a8e5cb4e0497728dedcff7cc54ca661868efcf09b80a604b5a23dad808b2121545c836d58171509293d28f6d15a8e23fc87ee0
java.security.MessageDigest
26657
not checked
gr4.sml
local
1604323635213
0
SHA-512
ec074f86e6ad4ce7cfb15a59a1aae6386bc959e16ed1e185e06c9bcdd78ecee90c857e7d4cfe5de14bc6d6f4897a0a8c3288411d7b6b2c5c0f70398a1c55cdd8
java.security.MessageDigest
52104
not checked
gr5.jpg
local
1604323635214
0
SHA-512
47158c6a4a8095c0bffdc7e324ea7889c9ccdb7473a9a1c5d3f86c13a963b8e1c825a7a1f1404b4d39e20e1bc1b6b058d86d1a540f94d3ecb5189f26d7b2893a
java.security.MessageDigest
5807
not checked
gr5.sml
local
1604323635215
0
SHA-512
7709f96ac167b72d3bc0393803fec44b850ef048b94a0c9b2bbdeb7e0d45d3acdf57e7d2bd6ada697b619f4ea1c366c86260275a482c26084cd1a50d5d096c0d
java.security.MessageDigest
46460
not checked
gr6.jpg
local
1604323635216
0
SHA-512
f09e77f13450fce7f810d962b9ff5718fdf8c0dbdc190583ae311f219b2dac281fcb139ebbdd5ae107877035dabb06087ecf1db82b85c685c3488396dcc0a553
java.security.MessageDigest
5367
not checked
gr6.sml
local
1604323635217
0
SHA-512
150815cb7e77f526108ee92f97600134ff1e55c36de9fb500d7b0920f843009fc9f27251153ef239ca7ea8e11dcdd76de0524b7fa2f0b221474ac60692f7cab7
java.security.MessageDigest
31527
not checked
mmc1.docx
local
1604323635218
0
SHA-512
cc3116c101183d122453f18f79fb6dfc342a3c7473ccdee3e0a60312c760bd1f904075174cbd08453cb455c5ee4de76fb55a6acd2f5ec2b4f966d1539834e301
java.security.MessageDigest
49546
not checked
mmc2.jpg
local
1604323635219
0
SHA-512
f1eea9b1d966d55b8c7beaa3cb1b150c8efbd50a72082facac59df7c6239553e56349cb18af48cf6cb4a2fcd7b0dc4646b8eb6cc3690d25b947efc5039361ff3
java.security.MessageDigest
12239
not checked
mmc2.sml
local
1604323635220
0
SHA-512
bdac47f9705efbfff65d5f9cff092d1eecc700016bbf38d20b330344fc5576dc48cd2a69531f655b33c5717feefd6ae19c1d050b3837189f4f39e7e96053b125
java.security.MessageDigest
27161
not checked
mmc3.jpg
local
1604323635221
0
SHA-512
92b30eb8c49adaf92a23bb78e5da736f14513c6763324327b0b0b9122a35451d32f928b1f17587e6dc6f27ef34f0df61b5bbc8a6445c43c84bef90e0af51bf72
java.security.MessageDigest
21741
not checked
mmc3.sml
local
1604323635222
0
SHA-512
b61feab86a682a1d13c430d86a93fa51ef3d5a4525aa541b59be48448912dec1a0b98a910a932c5e63e5f42ca8157c82e3213c19e5c00118b44bec8024eadbe4
java.security.MessageDigest
10879
not checked
mmc4.jpg
local
1604323635223
0
SHA-512
659d69ac0ac2653a83be070df6be834ceced3757c63dd49ee230b208b708be9b13e10c6bbacb084bc37cc0aada29ec36ed1a11ca07547c679556e513b5ffc133
java.security.MessageDigest
4723
not checked
mmc4.sml
local
1604323635224
0
SHA-512
b32b33b0322c09b52100c69cdbc573a24320ded2870ca2f61028551d5bbdd55e8d5a2e488a76435b782bf85cf919d2a456ab370aad1e137d0cfdbf9741b6a643
java.security.MessageDigest
25368
not checked
metadata.xml
free
00001
Published by Elsevier B.V.
KB-agent-id
1
supplier
KB-owner-id
00001
KB-agent-id
1
Elsevier
organization
Published by Elsevier B.V.
ingestion
2020-11-02T12:00:14.702+01:00
Connector
software
Digitaal Magazijn release 1.5
ejournals_esp_1
streamprofile
ingestion2020-11-02T14:29:45.604+01:00Generic IngestsoftwareDigitaal Magazijn release 1.5