Looking for mechanisms regulating lung growth in CDH: rat and human studies

Tese de Doutoramento em Ciências da SaúdeLung development is a complex process that involves a coordinated orchestration of several signalling pathways and mechanisms in order to growth normally. Pulmonary hypoplasia emerges as a result of foetal abnormal lung development and consequently it leads to high rates of morbidity and mortality. The understanding of normal and abnormal foetal lung growth has became clinical relevant because it can offer new perspectives in the treatment of lung diseases. Regardless the recent advances in the knowledge of lung development, there are still a lot of mechanisms that need to be elucidated and investigated. The main aim of this PhD dissertation was to investigate new physiological regulators of foetal lung growth in order to find a prenatal therapy to revert foetal lung hypoplasia in Congenital Diaphragmatic Hernia (CDH) context. Moreover, two already well-known and studied signalling pathways (retinoic acid and neuroendocrine factors) were further investigated and an important link between them was discovered/established. Using molecular/histological techniques and gain-loss of function studies with nitrofen rat model and human samples we tried to achieve our goals. In this PhD thesis, it was demonstrated that ephrins B1, -B2 and eph B4 receptor were expressed during all foetal lung developmental stages. Moreover ephrin B1 and eph B4 receptor mainly presented mesenchymal expressions whereas ephrin B2 presented an epithelial expression. Ephrin B1 and –B2 administration contributed to increase foetal lung branching, but the observed increase has not significant biological meaning. Another interesting finding was the importance of microRNAs in human CDH. MicroRNA 10a and microRNA 200b are up-regulated in CDH human lungs when compared to normal lungs and they are also changed in the tracheal fluid of CDH babies that respond or did not respond to FETO. In FETO responders, after removing the plug, tracheal fluid presented higher expression of miR-10a and miR-200 family when compared to non-responders. Moreover, in responders the expression of those microRNAs increased significantly after removing the plug when compared to the expression observed at the moment of plug insertion. In line with the second aim mentioned above, we decided to look into two already known and apparently unrelated signalling pathways and discover a link between them because both of them are altered in CDH (retinoic acid was down-regulated and neuroendocrine factors were up-regulated). We found that neuroendocrine factors act as regulators of lung growth, sensitizing the lungs to the action of retinoic acid through RAR α and RAR γ up-regulation. To conclude, in this thesis, we showed that ephrins seem to be strong candidates to act as morphogens or being involved in vascular regulation instead of being a promisor mechanism to regulate lung growth. Additionally, we discovered that microRNAs are an important tool as a prognostic biomarker for CDH new-borns outcome. Moreover, we established a novel physiological link showing that neuroendocrine factors and retinoic acid signalling pathways interact with each other during foetal lung growth regulation. With this work, new insights into normal and abnormal foetal lung development were brought, discovering novel mechanisms to further explore as potential therapeutic targets into CDH field.O desenvolvimento pulmonar é um processo complexo que envolve a combinação e funcionamento coordenados de várias vias e mecanismos de sinalização, a fim do crescimento pulmonar normal. A hipoplasia pulmonar surge como resultado do desenvolvimento anormal do pulmão fetal estando associada a elevadas taxas de morbilidade e mortalidade. A compreensão do crescimento do pulmão fetal normal e anormal, tornou-se clinicamente relevante pelo facto de poder oferecer novas perspetivas no tratamento de doenças pulmonares. Independentemente dos recentes avanços no conhecimento do desenvolvimento pulmonar, ainda há muito que é preciso fazer, ser elucidado e investigado. O objetivo principal desta dissertação de doutoramento foi investigar novos reguladores fisiológicos de crescimento do pulmão fetal, a fim de encontrar uma terapia pré-natal para reverter a hipoplasia pulmonar fetal no contexto da Hérnia Diafragmática Congénita (HDC). Por outro lado, foi descoberto e estabelecido um elo de ligação entre duas vias de sinalização (via do ácido retinóico e fatores neuroendócrinos) já bem conhecidas e estudadas como sendo importantes ao longo do desenvolvimento pulmonar fetal. Foram utilizadas técnicas moleculares/histológicas e estudos de ganho-perda de função com o modelo do nitrofeno em rato e com amostras humanas. Nesta tese de doutoramento, foi demonstrado que as efrinas B1,-B2 e o recetor eph B4 são expressos durante todas as fases de desenvolvimento pulmonar fetal. Além disso, a efrina B1 e o recetor eph B4 expressaram-se essencialmente no mesenquima enquanto que a efrina B2 apresentou ter uma expressão epitelial. A administração de efrina B1 e –B2 em culturas de explantes pulmonares, contribuiu para aumentar a ramificação pulmonar fetal, embora o aumento observado não tenha significado biológico relevante. Outro achado interessante ao longo deste trabalho de Doutoramento, foi a importância dos microRNAs na fisiopatologia da HDC em humanos. O microRNA 10a e o microRNA 200b estão aumentados em pulmões humanos com HDC quando comparados com pulmões normais e a sua expressão está também alterada no fluido traqueal em crianças que sobrevivem ou morrem à oclusão fetal da traqueia (FETO). Nos pacientes que sobrevivem depois do FETO, após se remover o balão da traqueia, o fluido traqueal apresentou uma maior expressão de miR-10a e da família do miR-200 quando comparados com os não sobreviventes. Por outro lado, nos sobreviventes, a expressão desses microRNAs aumentou significativamente após a remoção do balão quando comparada com a expressão observada no momento da inserção do balão na traqueia. Em linha com o segundo objetivo acima mencionado, decidimos revesitar duas vias de sinalização já conhecidas e aparentemente não relacionadas, e descobrir uma ligação entre elas, pelo facto de ambas estarem alteradas na HDC (défice de ácido retinóico e aumento dos fatores neuroendócrinos nomeadamente da bombesina e da grelina). Descobrimos que os fatores neuroendócrinos atuam como reguladores do crescimento pulmonar, sensibilizando os pulmões para a ação do ácido retinóico através do aumento da expressão dos seus recetores (RAR α e RAR γ). Para concluir, nesta tese, mostramos que as efrinas parecem ser fortes candidatos para atuar como morfogéneos ou como fatores importantes na regulação vascular ao invés de serem um mecanismo importante de regulação do crescimento pulmonar. Por outro lado, descobrimos que os microRNAs são uma ferramenta importante que poderá vir a ser utilizada como um biomarcador de prognóstico em crianças com HDC. Adicionalmente, estabelecemos uma nova ligação fisiológica mostrando que os fatores neuroendócrinos e a via de sinalização do ácido retinóico interagem entre elas durante a regulação do crescimento fetal pulmonar. Com este trabalho, hipotetizámos novas abordagens sobre o desenvolvimento/regulação do pulmão fetal normal e anormal, descobrindo-se novos mecanismos para continuar a explorar como potenciais alvos terapêuticos para o tratamento da HDC

The role of Ephrins-B1 and - B2 during fetal rat lung development

Background/Aims: The knowledge of the molecular network that governs fetal lung branching is an essential step towards the discovery of novel therapeutic targets against pulmonary pathologies. Lung consists of two highly branched systems: airways and vasculature. Ephrins and its receptors, Eph, have been implicated in cardiovascular development, angiogenesis and vascular remodeling. This study aims to clarify the role of these factors during lung morphogenesis. Methods: Ephrins-B1, -B2 and receptor EphB4 expression pattern was assessed in fetal rat lungs between 15.5 and 21.5 days post-conception, by immunohistochemistry. Fetal rat lungs were harvested at 13.5 dpc, cultured during 4 days and treated with increasing doses of ephrins-B1 and -B2 and the activity of key signaling pathways was assessed. Results: Ephrin-B1 presents mesenchymal expression, whereas ephrin-B2 and its receptor EphB4 were expressed by the epithelium. Both ephrins stimulated pulmonary branching. Moreover, while ephrin-B1 did not affect the pathways studied, ephrin-B2 supplementation decreased activity of JNK, ERK and STAT. This study characterizes the expression pattern of ephrins-B1, -B2 and EphB4 receptor throughout rat lung development. Conclusion: Our data highlight a possible role of ephrins as molecular stimulators of lung morphogenesis. Moreover, it supports the idea that classical vascular factors might play a role as airway growth promoters.We would like to thank Luis Martins and Miguel Carneiro for histological technical support and help on animal euthanasia. This project was funded by Fundacao para a Ciencia e a Tecnologia (PTDC/SAU-OBD/108051/2008). PPT was supported by POPH/ FSE by Fundacao para a Ciencia e a Tecnologia (reference SFRH/BD/73660/2010) and FOP was supported by Fundacao para a Ciencia e a Tecnologia (reference UMINHO/BII/172/2009)

Neuroendocrine factors regulate retinoic acid receptors in normal and hypoplastic lung development

Congenital diaphragmatic hernia (CDH) is characterised by a spectrum of lung hypoplasia and consequent pulmonary hypertension, leading to high morbidity and mortality rates. Moreover, CDH has been associated with an increase in the levels of pulmonary neuroendocrine factors, such as bombesin and ghrelin, and a decrease in the action of retinoic acid (RA). The present study aimed to elucidate the interaction between neuroendocrine factors and RA. In vitro analyses were performed on Sprague-Dawley rat embryos. Normal lung explants were treated with bombesin, ghrelin, a bombesin antagonist, a ghrelin antagonist, dimethylsulfoxide (DMSO), RA dissolved in DMSO, bombesin plus RA and ghrelin plus RA. Hypoplastic lung explants (nitrofen model) were cultured with bombesin, ghrelin, bombesin antagonist or ghrelin antagonist. The lung explants were analysed morphometrically, and retinoic acid receptor (RAR) α, β and γ expression levels were assessed via Western blotting. Immunohistochemistry analysis of RAR was performed in normal and hypoplastic lungs 17.5 days post-conception (dpc). Compared with the controls, hypoplastic lungs exhibited significantly higher RARα/γ expression levels. Furthermore considering hypoplastic lungs, bombesin and ghrelin antagonists decreased RARα/γ expression. Normal lung explants (13.5 dpc) treated with RA, bombesin plus RA, ghrelin plus RA, bombesin or ghrelin exhibited increased lung growth. Moreover, bombesin and ghrelin increased RARα/γ expression levels, whereas the bombesin and ghrelin antagonists decreased RARα/γ expression. This study demonstrates for the first time that neuroendocrine factors function as lung growth regulators, sensitising the lung to the action of RA through up-regulation of RARα and RARγ.P.P.-T. was supported by the Fundação para a Ciência e a Tecnologia (ref. SFRH/BD/73660/2010). R.S.M. was supported by the ON.2 SR&TD Integrated Program (N-01-01-01-24-01-07) (ref. UMINHO/BPD/31/2013). The funding bodies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Unique tracheal fluid microRNA signature predicts response to FETO in patients with congenital diaphragmatic hernia

"Epub ahead of print 2015 Jan 5"OBJECTIVE AND BACKGROUND: Our objective was to determine the fetal in vivo microRNA signature in hypoplastic lungs of human fetuses with severe isolated congenital diaphragmatic hernia (CDH) and changes in tracheal and amniotic fluid of fetuses undergoing fetoscopic endoluminal tracheal occlusion (FETO) to reverse severe lung hypoplasia due to CDH. METHODS:: We profiled microRNA expression in prenatal human lungs by microarray analysis. We then validated this signature with real-time quantitative polymerase chain reaction in tracheal and amniotic fluid of CDH patients undergoing FETO. We further explored the role of miR-200b using semiquantitative in situ hybridization and immunohistochemistry for TGF-ß2 in postnatal lung sections. We investigated miR-200b effects on TGF-ß signaling using a SMAD-luciferase reporter assay and Western blotting for phospho-SMAD2/3 and ZEB-2 in cultures of human bronchial epithelial cells. RESULTS:: CDH lungs display an increased expression of 2 microRNAs: miR-200b and miR-10a as compared to control lungs. Fetuses undergoing FETO display increased miR-200 expression in their tracheal fluid at the time of balloon removal. Future survivors of FETO display significantly higher miR-200 expression than those with a limited response. miR-200b was expressed in bronchial epithelial cells and vascular endothelial cells. TGF-ß2 expression was lower in CDH lungs. miR-200b inhibited TGF-ß-induced SMAD signaling in cultures of human bronchial epithelial cells. CONCLUSIONS:: Human fetal hypoplastic CDH lungs have a specific miR-200/miR-10a signature. Survival after FETO is associated with increased miR-200 family expression. miR-200b overexpression in CDH lungs results in decreased TGF-ß/SMAD signaling

Lower NPAS3 expression during the later stages of abnormal lung development in rat congenital diaphragmatic hernia

Purpose Congenital diaphragmatic hernia (CDH) is characterized by a developmental defect in the diaphragm, pulmonary hypoplasia and pulmonary hypertension. NPAS3 is a PAS domain transcription factor regulating Drosophila tracheogenesis. NPAS3 null mice develop pulmonary hypoplasia in utero and die after birth due to respiratory failure. We aimed to evaluate NPAS3 expres- sion during normal and abnormal lung development due to CDH. Methods CDH was induced by administering 100 mg/ml nitrofen to time-pregnant dams on embryonic day (E) 9 of gestation. Lungs were isolated on E15, E18 and E21 and NPAS3 localization was determined by immunohisto- chemistry and quantified using Western blotting. Results We found that only E21 hypoplastic CDH lungs have reduced expression of NPAS3 in the terminal sac- cules. Western blotting confirmed the down-regulation of NPAS3 protein in the nitrofen-induced hypoplastic lungs. Conclusions We demonstrate for the first time that ni- trofen-induced hypoplastic CDH lungs have reduced NPAS3 expression in the terminal saccules during the later stages of abnormal lung development. Our findings suggest that NPAS3 is associated with pulmonary hypoplasia in CDH.Supported by the Children’s Hospital Research Institute of Manitoba; RK is the recipient of a Career Enhancement Award from the Canadian Child Health Clinician Scientist Program and a New Investigator Salary Award from the Canadian Institutes of Health Research, Manitoba Lung Association and the Children’s Hospital Research Institute

Retinoic acid regulates avian lung branching through a molecular network

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rar alpha, and rar beta) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgf beta 2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rar beta, meis2, hoxb5, tgf beta 2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgf beta 2, and id2 spatial distribution; to increase rar beta, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal-distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.The authors would like to thank Ana Lima for slide sectioning and Rita Lopes for contributing to the initiation of this project. This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the Project POCI-01-0145-FEDER-007038; and by the Project NORTE-01-0145- FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

ATLANTIC-PRIMATES: a dataset of communities and occurrences of primates in the Atlantic Forests of South America

Primates play an important role in ecosystem functioning and offer critical insights into human evolution, biology, behavior, and emerging infectious diseases. There are 26 primate species in the Atlantic Forests of South America, 19 of them endemic. We compiled a dataset of 5,472 georeferenced locations of 26 native and 1 introduced primate species, as hybrids in the genera Callithrix and Alouatta. The dataset includes 700 primate communities, 8,121 single species occurrences and 714 estimates of primate population sizes, covering most natural forest types of the tropical and subtropical Atlantic Forest of Brazil, Paraguay and Argentina and some other biomes. On average, primate communities of the Atlantic Forest harbor 2 ± 1 species (range = 1–6). However, about 40% of primate communities contain only one species. Alouatta guariba (N = 2,188 records) and Sapajus nigritus (N = 1,127) were the species with the most records. Callicebus barbarabrownae (N = 35), Leontopithecus caissara (N = 38), and Sapajus libidinosus (N = 41) were the species with the least records. Recorded primate densities varied from 0.004 individuals/km 2 (Alouatta guariba at Fragmento do Bugre, Paraná, Brazil) to 400 individuals/km 2 (Alouatta caraya in Santiago, Rio Grande do Sul, Brazil). Our dataset reflects disparity between the numerous primate census conducted in the Atlantic Forest, in contrast to the scarcity of estimates of population sizes and densities. With these data, researchers can develop different macroecological and regional level studies, focusing on communities, populations, species co-occurrence and distribution patterns. Moreover, the data can also be used to assess the consequences of fragmentation, defaunation, and disease outbreaks on different ecological processes, such as trophic cascades, species invasion or extinction, and community dynamics. There are no copyright restrictions. Please cite this Data Paper when the data are used in publications. We also request that researchers and teachers inform us of how they are using the data. © 2018 by the The Authors. Ecology © 2018 The Ecological Society of Americ

ATLANTIC EPIPHYTES: a data set of vascular and non-vascular epiphyte plants and lichens from the Atlantic Forest

Epiphytes are hyper-diverse and one of the frequently undervalued life forms in plant surveys and biodiversity inventories. Epiphytes of the Atlantic Forest, one of the most endangered ecosystems in the world, have high endemism and radiated recently in the Pliocene. We aimed to (1) compile an extensive Atlantic Forest data set on vascular, non-vascular plants (including hemiepiphytes), and lichen epiphyte species occurrence and abundance; (2) describe the epiphyte distribution in the Atlantic Forest, in order to indicate future sampling efforts. Our work presents the first epiphyte data set with information on abundance and occurrence of epiphyte phorophyte species. All data compiled here come from three main sources provided by the authors: published sources (comprising peer-reviewed articles, books, and theses), unpublished data, and herbarium data. We compiled a data set composed of 2,095 species, from 89,270 holo/hemiepiphyte records, in the Atlantic Forest of Brazil, Argentina, Paraguay, and Uruguay, recorded from 1824 to early 2018. Most of the records were from qualitative data (occurrence only, 88%), well distributed throughout the Atlantic Forest. For quantitative records, the most common sampling method was individual trees (71%), followed by plot sampling (19%), and transect sampling (10%). Angiosperms (81%) were the most frequently registered group, and Bromeliaceae and Orchidaceae were the families with the greatest number of records (27,272 and 21,945, respectively). Ferns and Lycophytes presented fewer records than Angiosperms, and Polypodiaceae were the most recorded family, and more concentrated in the Southern and Southeastern regions. Data on non-vascular plants and lichens were scarce, with a few disjunct records concentrated in the Northeastern region of the Atlantic Forest. For all non-vascular plant records, Lejeuneaceae, a family of liverworts, was the most recorded family. We hope that our effort to organize scattered epiphyte data help advance the knowledge of epiphyte ecology, as well as our understanding of macroecological and biogeographical patterns in the Atlantic Forest. No copyright restrictions are associated with the data set. Please cite this Ecology Data Paper if the data are used in publication and teaching events. © 2019 The Authors. Ecology © 2019 The Ecological Society of Americ

EGCG prevents the loss of pontine noradrenergic neurons induced by diabetes: a role in diabetic neuropathic pain

[Extract] Diabetes is a major health problem with an alarming increasing prevalence, and is the most frequent cause of neuropathy worldwide. Neuropathy affects 50-60% of diabetic patients, being a major life-quality impairment for a quarter of these patients(undefined

Lichen Xanthones as Models for New Antifungal Agents

Due to the emergence of multidrug-resistant pathogenic microorganisms, the search for new antimicrobial compounds plays an important role in current medicinal chemistry research. Inspired by lichen antimicrobial xanthones, a series of novel chlorinated xanthones was prepared using five chlorination methods (Methods A–E) to obtain different patterns of substitution in the xanthone scaffold. All the synthesized compounds were evaluated for their antimicrobial activity. Among them, 3-chloro-4,6-dimethoxy-1-methyl-9H-xanthen-9-one 15 showed promising antibacterial activity against E. faecalis (ATCC 29212 and 29213) and S. aureus ATCC 29213. 2,7-Dichloro-3,4,6-trimethoxy-1-methyl-9H-xanthen-9-one 18 revealed a potent fungistatic and fungicidal activity against dermatophytes clinical strains (T. rubrum, M. canis, and E. floccosum (MIC = 4–8 µg/mL)). Moreover, when evaluated for its synergistic effect for T. rubrum, compound 18 exhibited synergy with fluconazole (ΣFIC = 0.289). These results disclosed new hit xanthones for both antibacterial and antifungal activity