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Objective: We aimed to assess the expression and localization of renin-angiotensin system (RAS) receptors in lung tissue and the plasma concentration of related peptides in IPF patients. Materials and Methods: This case–control study involved 19 patients from southern Brazil undergoing lung resection or transplantation. Plasma levels of Angiotensin I, II, A, 1-7, Alamandine were measured via liquid chromatography–tandem mass spectrometry. Lung tissue expression and localization of angiotensin type 1 (AT1), Mas, and Mas-related G-protein-coupled receptor D (MrgD) receptors were evaluated using Western blot and immunohistochemistry. Clinical data and the 6-min walk test were analyzed to correlate receptor expression with lung function and oxygen dependence. Results: IPF patients showed reduced forced vital capacity (FVC) at 49 ± 13% and forced expiratory volume (FEV1) at 51 ± 14%, with a 60% increase in oxygen dependence. Plasma peptide concentrations were similar between the groups, except for Angiotensin I, which was significantly higher in the control group. In IPF lungs, AT1 and Mas receptors were expressed 2.31 and 2.13 times more, respectively, while MrgD expression was lower. Mas receptors were mostly found in bronchiole areas, whereas MrgD was predominant in the lung parenchyma. Conclusions: This study indicates that the RAS operates independently within tissue, in addition to its systemic functions, highlighting distinct differences between tissue and plasma RAS activities. The distinct roles of MrgD and Mas receptors in lung structure and function could be pivotal for new therapies, potentially leading to more effective IPF treatments.

The syncytiotrophoblast is a multinucleated layer that plays a critical role in regulating functions of the human placenta during pregnancy. Maintaining the syncytiotrophoblast layer relies on ongoing fusion of mononuclear cytotrophoblasts throughout pregnancy, and errors in this fusion process are associated with complications such as preeclampsia. While biochemical factors are known to drive fusion, the role of disease-specific extracellular biophysical cues remains undefined. Since substrate mechanics play a crucial role in several diseases, and preeclampsia is associated with placental stiffening, we hypothesize that trophoblast fusion is mechanically regulated by substrate stiffness. We developed stiffness-tunable polyacrylamide substrate formulations that match the linear elasticity of placental tissue in normal and disease conditions, and evaluated trophoblast morphology, fusion, and function on these surfaces. Our results demonstrate that morphology, fusion, and hormone release is mechanically-regulated via myosin-II; optimal on substrates that match healthy placental tissue stiffness; and dysregulated on disease-like and supraphysiologically-stiff substrates. We further demonstrate that stiff regions in heterogeneous substrates provide dominant physical cues that inhibit fusion, suggesting that even focal tissue stiffening limits widespread trophoblast fusion and tissue function. These results confirm that mechanical microenvironmental cues influence fusion in the placenta, provide critical information needed to engineer better in vitro models for placental disease, and may ultimately be used to develop novel mechanically-mediated therapeutic strategies to resolve fusion-related disorders during pregnancy.

Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor pathways. Hypoxia/reoxygenation (H/R) induces oxidative damage and apoptosis. These consequences activate autophagy, which degrades damaged cellular content, as well as activates Nrf2 the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor, and thereby the expression of protective genes. Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and Nrf2 pathways. The current study shows melatonin to differentially modulate autophagy and Nrf2 pathways in tumor and normal placental cells exposed to H/R. BeWo, a human placental choriocarcinoma cell line, and primary villous cytotrophoblasts isolated from normal term placenta, were maintained in normoxia (8% O2) for 24 h or exposed to hypoxia (0.5% of O2 for 4 h) followed by 20 h of normoxia, creating a situation of H/R, in the presence or absence of 1 mM melatonin. Melatonin induced a 7-fold increase in the activation of 5' adenosine monophosphate-activated protein kinase (AMPK)α, an upstream modulator of autophagy, rising to a 16-fold increase in BeWo cells co-exposed to H/R and melatonin, compared to controls. H/R induced autophagosome formation via the increased expression of Beclin-1 (by 94%) and ATG7 (by 97%) in BeWo cells. Moreover, H/R also induced autophagic activity, indicated by the by the 630% increase in P62, and increased Nrf2 by 314% in BeWo cells. In H/R conditions, melatonin reduced autophagic activity by 74% and Nrf2 expression activation by 300%, leading to BeWo cell apoptosis. In contrast, In human primary villous cytotrophoblasts, H/R induced autophagy and Nrf2, which melatonin further potentiated, thereby affording protection against H/R. This study demonstrates that melatonin differentially modulates autophagy and the Nrf2 pathway in normal vs. tumor trophoblast cells, being cytoprotective in normal cells whilst increasing apoptosis in tumoral trophoblast cells.

L’apport en oxygène dans le placenta est finement régulé et directement lié avec le bon déroulement de la grossesse et du développement foetal. Dans les pathologies de grossesses associées avec un remodelage artériel incomplet, telles que la prééclampsie, l’apport d’oxygène est plus faible et intermittent entraînant une hypoxie/réoxygénation (H/R). Cette H/R est associée avec une augmentation du stress oxydatif et de l’apoptose des trophoblastes villeux. Il n’existe aucun traitement thérapeutique ou préventif pour la prééclampsie excepté l’accouchement. La mélatonine est produite de novo par les cellules trophoblastiques humaines. Cette indolamine exerce diverses actions protectrices telles que antioxydante, anti-inflammatoire et régulation de l’autophagie. Au niveau placentaire, la mélatonine protège les trophoblastes des dommages induits par l’H/R, entre autres, par une action antioxydante directe et indirecte, via ses récepteurs MT1 et MT2. Par contre, sa capacité de diminuer l’inflammation et de réguler l’autophagie induite par une H/R trophoblastique n’a jamais été étudiée. Les hypothèses de recherche sont : la mélatonine (1) via la régulation de l’autophagie et de l’inflammation protège le trophoblaste villeux contre les dommages induits par l’H/R ; et (2) module différemment l’autophagie pour maintenir la survie/l’homéostasie des cellules trophoblastiques normales et induire la mort des cellules tumorales. L’objectif général est de comprendre comment la mélatonine module l’autophagie des cellules placentaires (primaires et tumorales) en situation de stress. Les objectifs spécifiques sont 1) Déterminer si et par quelle voie de signalisation la mélatonine régule l’inflammation et l’autophagie dans le syncytiotrophoblaste primaire humain cultivé en normoxie et H/R ; 2) Déterminer si la mélatonine régule différemment l’autophagie dans les cellules trophoblastiques normales (primocultures) et tumorales (cellules BeWo). Les expériences ont été réalisées avec la lignée cellulaire BeWo (lignée cellulaire de choriocarcinome placentaire humain) et avec des cultures primaires de trophoblastes villeux de placentas normaux à terme. Toutes les expériences ont été conduites sous conditions de normoxie (8 % O2) mimant l’espace intervilleux. Pour mimer l’H/R, la normoxie a été suivie par 4 h d’hypoxie (0,5 % O2) puis 18 h de normoxie. Les cellules ont été traitées ou non avec 1 mM de mélatonine à toutes les 24 h. L’expression protéique des biomarqueurs de l’autophagie, de l’inflammation et de l’apoptose a été déterminée par immunobuvardage de type western, par immunofluorescence, par immunohistochimie et à l’aide de la trousse MILLIPLEX MAP ; la mélatonine a été mesurée par ELISA. L’exposition in vitro du syncytiotrophoblaste à l’H/R réduit la production endogène de mélatonine et induit une réponse pro-inflammatoire par l’augmentation des facteurs TNF, IL-6 et NFκB. Au contraire, le traitement avec 1 mM mélatonine augmente les taux d'IL-10 et rétablit les taux de TNF, d'IL-6 et NFκB aux taux observés en normoxie. L’H/R augmente également l'autophagie dans le syncytiotrophoblaste. La mélatonine et l'inhibition spécifique du NFKB induisent également l'autophagie et augmente la survie cellulaire en condition d’H/R. Dans les cellules BeWo, l’H/R avec et sans mélatonine réduit la viabilité cellulaire et induit l'activation des capteurs d'énergie cellulaire comme le AMPK et le PP2A-C, qui sont des modulateurs en amont de l’autophagie. Dans les BeWo, l'H/R a induit la formation de l'autophagosome, l'activité autophagique et l’activité du facteur de transcription Nrf2. La mélatonine, dans les conditions d'H/R, a réduit l'activation de l’autophagie et du Nrf2, ce qui a entrainé l'activation de l'apoptose. Dans les primocultures de cytotrophoblastes villeux, l’H/R a aussi induit l'autophagie et Nrf2. Par contre, la mélatonine a augmenté l’activité de l’autophagie et du Nrf2 protégeant les cellules primaires contre les effets de l’H/R. Puisque la mélatonine est impliquée dans les mécanismes de survie cellulaire des trophoblastes primaires, ses actions pourraient avoir un effet bénéfique sur la santé de la grossesse et sur le développement du foetus.

Hypoxia/reoxygenation (H/R) induces oxidative damage and apoptosis. These consequences activate autophagy, which degrades damaged cellular content, as well as inducing the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor, and thereby the expression of protective genes. Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and Nrf2 pathways. The current study shows melatonin to differentially modulate autophagy and Nrf2 pathways in tumor and normal placental cells exposed to H/R. BeWo, a human placental choriocarcinoma cell line, and primary villous cytotrophoblasts isolated from normal term placenta, were maintained in normoxia (8% O2) for 24 h or exposed to hypoxia (0.5% of O2 for 4 h) followed by 20 h of normoxia, creating a H/R, in the presence or absence of 1 mM melatonin. Melatonin induced a 7-fold increase in the activation of 5' adenosine monophosphate-activated protein kinase (AMPK) , an upstream modulator of autophagy, rising to a 16-fold increase in cells co-exposed to H/R and melatonin, compared to controls. H/R induced autophagosome formation via the increased expression of Beclin-1 (by 94 %) and ATG7 (by 97%). H/R also induced autophagic activity, indicated by the by the 630% increase in P62, and increased Nrf2 by 314%. In H/R conditions, melatonin reduced autophagy by 74% and Nrf2 expression by 66%, leading to BeWo cell apoptosis. In contrast, in human primary villous cytotrophoblasts, H/R induced autophagy and Nrf2, which melatonin further potentiated, thereby affording protection against H/R. This study demonstrates that melatonin differentially modulates autophagy and the Nrf2 pathway in normal vs. tumor trophoblast cells, being cytoprotective in normal cells whilst increasing apoptosis in tumoral trophoblast cells.