Retinoic Acid-Mediated Control of Energy Metabolism Is Essential for Lung Branching Morphogenesis.

Lung branching morphogenesis relies on intricate epithelial-mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal-distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA's pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders.

Intraluminal chloride regulates lung branching morphogenesis: involvement of PIEZO1/PIEZO2.

BACKGROUND: Clinical and experimental evidence shows lung fluid volume as a modulator of fetal lung growth with important value in treating fetal lung hypoplasia. Thus, understanding the mechanisms underlying these morphological dynamics has been the topic of multiple investigations with, however, limited results, partially due to the difficulty of capturing or recapitulating these movements in the lab. In this sense, this study aims to establish an ex vivo model allowing the study of lung fluid function in branching morphogenesis and identify the subsequent molecular/ cellular mechanisms. METHODS: Ex vivo lung explant culture was selected as a model to study branching morphogenesis, and intraluminal injections were performed to change the composition of lung fluid. Distinct chloride (Cl-) concentrations (5.8, 29, 143, and 715 mM) or Cl- channels inhibitors [antracene-9-carboxylic acid (A9C), cystic fibrosis transmembrane conductance regulator inhibitor172 (CFTRinh), and calcium-dependent Cl- channel inhibitorA01 (CaCCinh)] were injected into lung lumen at two timepoints, day0 (D0) and D2. At D4, morphological and molecular analyses were performed in terms of branching morphogenesis, spatial distribution (immunofluorescence), and protein quantification (western blot) of mechanoreceptors (PIEZO1 and PIEZO2), neuroendocrine (bombesin, ghrelin, and PGP9.5) and smooth muscle [alpha-smooth muscle actin (α-SMA) and myosin light chain 2 (MLC2)] markers. RESULTS: For the first time, we described effective intraluminal injections at D0 and D2 and demonstrated intraluminal movements at D4 in ex vivo lung explant cultures. Through immunofluorescence assay in in vivo and ex vivo branching morphogenesis, we show that PGP9.5 colocalizes with PIEZO1 and PIEZO2 receptors. Fetal lung growth is increased at higher [Cl-], 715 mM Cl-, through the overexpression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. In contrast, intraluminal injection of CFTRinh or CaCCinh decreases fetal lung growth and the expression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. Finally, the inhibition of PIEZO1/PIEZO2 by GsMTx4 decreases branching morphogenesis and ghrelin, bombesin, MLC2, and α-SMA expression in an intraluminal injection-independent manner. CONCLUSIONS: Our results identify PIEZO1/PIEZO2 expressed in neuroendocrine cells as a regulator of fetal lung growth induced by lung fluid.

Comparative study of e-cigarette aerosol and cigarette smoke effect on ex vivo embryonic chick lung explants.

Electronic cigarette usage has significantly expanded among young people and pregnant women in the last decade. Although there are already some data regarding the short- and long-term consequences of e-cigarettes on human health, their effect on embryo and lung development still needs to be fully disclosed. In this sense, this study describes, for the first time, the impact of electronic cigarette aerosol on early lung development. For this purpose, ex vivo chick (Gallus gallus) embryonic lungs were cultured in vitro for 48 h in e-cigarette aerosol exposed-medium or unexposed medium. Chick lung explants were also cultured in a cigarette smoke-exposed medium for comparison purposes. Lung explants were morphologically analyzed to assess the impact on lung growth. Additionally, TNF-α levels were determined in the supernatant as a marker of pro-inflammatory response. The results suggest that electronic cigarette aerosol impairs lung growth and promotes lung inflammation. However, its impact on early lung growth seems less detrimental than conventional cigarette smoke. This work provides significant data regarding the impact of e-cig aerosol, adding to the efforts to fully understand its effect on embryo development. The validation of these effects may eventually lead to new tobacco control recommendations for pregnant women.

Molecular insights of Hippo signaling in the chick developing lung.

Hippo signaling pathway and its effector YAP have been recognized as an essential growth regulator during embryonic development. Hippo has been studied in different contexts; nevertheless, its role during chick lung branching morphogenesis remains unknown. Therefore, this work aims to determine Hippo role during early pulmonary organogenesis in the avian animal model. The current study describes the spatial distribution of Hippo signaling members in the embryonic chick lung by in situ hybridization. Overall, their expression is comparable to their mammalian counterparts. Moreover, the expression levels of phosphorylated-YAP (pYAP) and total YAP revealed that Hippo signaling is active in the embryonic chick lung. Furthermore, the presence of pYAP in the cytoplasm demonstrated that the Hippo machinery distribution is maintained in this tissue. In vitro studies were performed to assess the role of the Hippo signaling pathway in lung branching. Lung explants treated with a YAP/TEAD complex inhibitor (verteporfin) displayed a significant reduction in lung size and branching and decreased expression of ctgf (Hippo target gene) compared to the control. This approach also revealed that Hippo seems to modulate the expression of key molecular players involved in lung branching morphogenesis (sox2, sox9, axin2, and gli1). Conversely, when treated with dobutamine, an upstream regulator that promotes YAP phosphorylation, explant morphology was not severely affected. Overall, our data indicate that Hippo machinery is present and active in the early stages of avian pulmonary branching and that YAP is likely involved in the regulation of lung growth.

Developmental Pathways Underlying Lung Development and Congenital Lung Disorders.

Lung organogenesis is a highly coordinated process governed by a network of conserved signaling pathways that ultimately control patterning, growth, and differentiation. This rigorously regulated developmental process culminates with the formation of a fully functional organ. Conversely, failure to correctly regulate this intricate series of events results in severe abnormalities that may compromise postnatal survival or affect/disrupt lung function through early life and adulthood. Conditions like congenital pulmonary airway malformation, bronchopulmonary sequestration, bronchogenic cysts, and congenital diaphragmatic hernia display unique forms of lung abnormalities. The etiology of these disorders is not yet completely understood; however, specific developmental pathways have already been reported as deregulated. In this sense, this review focuses on the molecular mechanisms that contribute to normal/abnormal lung growth and development and their impact on postnatal survival.

Lung branching morphogenesis is accompanied by temporal metabolic changes towards a glycolytic preference.

BACKGROUND: Lung branching morphogenesis is characterized by epithelial-mesenchymal interactions that ultimately define the airway conducting system. Throughout this process, energy and structural macromolecules are necessary to sustain the high proliferative rates. The extensive knowledge of the molecular mechanisms underlying pulmonary development contrasts with the lack of data regarding the embryonic lung metabolic requirements. Here, we studied the metabolic profile associated with the early stages of chicken pulmonary branching. METHODS: In this study, we used an ex vivo lung explant culture system and analyzed the consumption/production of extracellular metabolic intermediates associated with glucose catabolism (alanine, lactate, and acetate) by 1H-NMR spectroscopy in the culture medium. Then, we characterized the transcript levels of metabolite membrane transporters (glut1, glut3, glut8, mct1, mct3, mct4, and mct8) and glycolytic enzymes (hk1, hk2, pfk1, ldha, ldhb, pdha, and pdhb) by qPCR. ldha and ldhb mRNA spatial localization was determined by in situ hybridization. Proliferation was analyzed by directly assessing DNA synthesis using an EdU-based assay. Additionally, we performed western blot to analyze LDHA and LDHT protein levels. Finally, we used a Clark-Type Electrode to assess the lung explant's respiratory capacity. RESULTS: Glucose consumption decreases, whereas alanine, lactate, and acetate production progressively increase as branching morphogenesis proceeds. mRNA analysis revealed variations in the expression levels of key enzymes and transporters from the glycolytic pathway. ldha and ldhb displayed a compartment-specific expression pattern that resembles proximal-distal markers. In addition, high proliferation levels were detected at active branching sites. LDH protein expression levels suggest that LDHB may account for the progressive rise in lactate. Concurrently, there is a stable oxygen consumption rate throughout branching morphogenesis. CONCLUSIONS: This report describes the temporal metabolic changes that accompany the early stages of chicken lung branching morphogenesis. Overall, the embryonic chicken lung seems to shift to a glycolytic lactate-based metabolism as pulmonary branching occurs. Moreover, this metabolic rewiring might play a crucial role during lung development.

Effects of testosterone replacement on serotonin levels in the prostate and plasma in a murine model of hypogonadism.

Benign prostate hyperplasia is a dysfunctional disease with an elevated prevalence. Despite the accepted impact of aging and testosterone (TES) in its pathophysiology, its aetiology remains unknown. Recent studies described that serotonin (5-HT) inhibits benign prostate growth through the modulation of the androgen receptor, in the presence of TES. Accordingly, this work aimed to determine the impact of castration and TES replacement in plasmatic and prostatic 5-HT regulation. C57BL/6 mice were submitted to surgical castration and divided into three groups, continually exposed to either vehicle or different TES doses for 14 days. Plasmatic 5-HT concentration was measured before and after castration, and after TES reintroduction. Finally, total prostatic weight and intra-prostatic 5-HT were determined in the different groups. Our results demonstrate that mice prostate exhibits high 5-HT tissue levels and that intra-prostatic total 5-HT was independent of castration or TES reintroduction, in all studied groups. Also, 5-HT plasmatic concentration significantly increased after castration and then normalized after TES administration. Our findings revealed that mice prostate has a high 5-HT content and that total prostatic 5-HT levels do not depend on androgens' action. On the other hand, castration induced a significant increase in plasmatic 5-HT concentration, raising the hypothesis that androgens might be regulating the production of extra-prostatic 5-HT.

Retinoic Acid: A Key Regulator of Lung Development.

Retinoic acid (RA) is a key molecular player in embryogenesis and adult tissue homeostasis. In embryo development, RA plays a crucial role in the formation of different organ systems, namely, the respiratory system. During lung development, there is a spatiotemporal regulation of RA levels that assures the formation of a fully functional organ. RA signaling influences lung specification, branching morphogenesis, and alveolarization by regulating the expression of particular target genes. Moreover, cooperation with other developmental pathways is essential to shape lung organogenesis. This review focuses on the events regulated by retinoic acid during lung developmental phases and pulmonary vascular development; also, it aims to provide a snapshot of RA interplay with other well-known regulators of lung development.

Retinoic Acid as a Modulator of Proximal-Distal Patterning and Branching Morphogenesis of the Avian Lung.

Retinoic acid modulates numerous cellular events, namely, proliferation, differentiation, apoptosis, and patterning, hence influencing both embryo development and adult homeostasis. In vitro explant culture is a valuable technique for studying the impact of growth factors and signaling molecules, such as retinoic acid, in organ development since tissue architecture is maintained. This technique allows controlled supplementation of culture medium and straightforward analysis of its effect on morphogenesis. This chapter describes the detailed protocol for culturing embryonic chick lung explants and testing the impact of retinoic acid in branching and patterning, based on morphometric and molecular analysis.

STATs in Lung Development: Distinct Early and Late Expression, Growth Modulation and Signaling Dysregulation in Congenital Diaphragmatic Hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a life-threatening developmental anomaly, intrinsically combining severe pulmonary hypoplasia and hypertension. During development, signal transducers and activators of transcription (STAT) are utilized to elicit cell growth, differentiation, and survival. METHODS: We used the nitrofen-induced CDH rat model. At selected gestational time points, lungs were divided into two experimental groups, i.e., control or CDH. We performed immunohistochemistry and western blotting analysis to investigate the developmental expression profile of the complete family of STATs (STAT1-6), plus specific STATs activation (p-STAT3, p-STAT6) and regulation by SOCS (SOCS3) in normal lungs against those of diseased lungs. The normal fetal lung explants were treated with piceatannol (STAT3 inhibitor) in vitro followed by morphometrical analysis. RESULTS: Molecular profiling of STATs during the lung development revealed distinct early and late expression signatures. Experimental CDH altered the STATs expression, activation, and regulation in the fetal lungs. In particular, STAT3 and STAT6 were persistently over-expressed and early over-activated. Piceatannol treatment dose-dependently stimulated the fetal lung growth. CONCLUSION: These findings suggest that STATs play an important role during normal fetal lung development and CDH pathogenesis. Moreover, functionally targeting STAT signaling modulates fetal lung growth, which highlights that STAT3 and STAT6 signaling might be promising therapeutic targets in reducing or preventing pulmonary hypoplasia in CDH.

Serotonin regulates prostate growth through androgen receptor modulation.

Aging and testosterone almost inexorably cause benign prostatic hyperplasia (BPH) in Human males. However, etiology of BPH is largely unknown. Serotonin (5-HT) is produced by neuroendocrine prostatic cells and presents in high concentration in normal prostatic transition zone, but its function in prostate physiology is unknown. Previous evidence demonstrated that neuroendocrine cells and 5-HT are decreased in BPH compared to normal prostate. Here, we show that 5-HT is a strong negative regulator of prostate growth. In vitro, 5-HT inhibits rat prostate branching through down-regulation of androgen receptor (AR). This 5-HT's inhibitory mechanism is also present in human cells of normal prostate and BPH, namely in cell lines expressing AR when treated with testosterone. In both models, 5-HT's inhibitory mechanism was replicated by specific agonists of 5-Htr1a and 5-Htr1b. Since peripheral 5-HT production is specifically regulated by tryptophan hydroxylase 1(Tph1), we showed that Tph1 knockout mice present higher prostate mass and up-regulation of AR when compared to wild-type, whereas 5-HT treatment restored the prostate weight and AR levels. As 5-HT is decreased in BPH, we present here evidence that links 5-HT depletion to BPH etiology through modulation of AR. Serotoninergic prostate pathway should be explored as a new therapeutic target for BPH.

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, rarα, and rarß) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgfß2, 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, rarß, meis2, hoxb5, tgfß2, 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, tgfß2, and id2 spatial distribution; to increase rarß, 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.

Canonical Sonic Hedgehog Signaling in Early Lung Development.

The canonical hedgehog (HH) signaling pathway is of major importance during embryonic development. HH is a key regulatory morphogen of numerous cellular processes, namely, cell growth and survival, differentiation, migration, and tissue polarity. Overall, it is able to trigger tissue-specific responses that, ultimately, contribute to the formation of a fully functional organism. Of all three HH proteins, Sonic Hedgehog (SHH) plays an essential role during lung development. In fact, abnormal levels of this secreted protein lead to severe foregut defects and lung hypoplasia. Canonical SHH signal transduction relies on the presence of transmembrane receptors, such as Patched1 and Smoothened, accessory proteins, as Hedgehog-interacting protein 1, and intracellular effector proteins, like GLI transcription factors. Altogether, this complex signaling machinery contributes to conveying SHH response. Pulmonary morphogenesis is deeply dependent on SHH and on its molecular interactions with other signaling pathways. In this review, the role of SHH in early stages of lung development, specifically in lung specification, primary bud formation, and branching morphogenesis is thoroughly reviewed.

Expression analysis of Shh signaling members in early stages of chick lung development.

Lung organogenesis is guided by epithelial-mesenchymal interactions that coordinate cellular events responsible for the formation of the respiratory system. Several signaling pathways have been implicated in this process; among them, sonic hedgehog (Shh) signaling has emerged as a crucial regulator of branching morphogenesis in the mammalian lung. Canonical Shh signaling requires the presence of patched (Ptch) and smoothened (Smo) transmembrane receptors in order to induce the activation of glioblastoma (Gli) zinc finger transcription factors that are the true effectors of the pathway. Signal transduction is finely regulated by Ptch1, Gli, and Hhip (hedgehog-interacting protein). The present work characterizes, for the first time, the expression pattern of shh, ptch1, smo, gli1, and hhip in early stages of the embryonic chick lung. In situ hybridization studies revealed that these genes are expressed in the same cellular compartments as their mammalian counterparts, although their proximo-distal distribution is slightly changed. Moreover, the molecular interactions between fibroblast growth factor (FGF) and Shh signaling pathway were assessed, in vitro, by grafting beads soaked in SU5402 (an FGF receptor inhibitor). In the chick lung, Shh signaling seems to have some features that are species specific since shh is not a downstream target of FGF signaling. Nonetheless and despite the observed differences, these findings suggest a role for Shh signaling in the epithelial-mesenchymal interactions that control chick lung morphogenesis.

Characterization of miRNA processing machinery in the embryonic chick lung.

Lung development is a very complex process that relies on the interaction of several signaling pathways that are controlled by precise regulatory mechanisms. Recently, microRNAs (miRNAs), small non-coding regulatory RNAs, have emerged as new players involved in gene expression regulation controlling several biological processes, such as cellular differentiation, apoptosis and organogenesis, in both developmental and disease processes. Failure to correctly express some specific miRNAs or a component of their biosynthetic machinery during embryonic development is disastrous, resulting in severe abnormalities. Several miRNAs have already been identified as modulators of lung development. Regarding the spatial distribution of the processing machinery of miRNAs, only two of its members (dicer1 and argonaute) have been characterized. The present work characterizes the expression pattern of drosha, dgcr8, exportin-5 and dicer1 in early stages of the embryonic chick lung by whole mount in situ hybridization and cross-section analysis. Overall, these genes are co-expressed in dorsal and distal mesenchyme and also in growing epithelial regions. The expression pattern of miRNA processing machinery supports the previously recognized regulatory role of this mechanism in epithelial and mesenchymal morphogenesis.

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

KEY POINTS: Retinoic acid (RA) and ghrelin levels are altered in human hypoplastic lungs when compared to healthy lungs. Although considerable data have been obtained about RA, ghrelin and bombesin in the congenital diaphragmatic hernia (CDH) rat model, neuroendocrine factors have never been associated with the RA signalling pathway in this animal model. In this study, the interaction between neuroendocrine factors and RA was explored in the CDH rat model. The authors found that normal fetal lung explants treated with RA, bombesin and ghrelin showed an increase in lung growth. Hypoplastic lungs presented higher expression levels of the RA receptors α and γ. Moreover bombesin and ghrelin supplementation, in vitro, to normal lungs increased RA receptor α/γ expression whereas administration of bombesin and ghrelin antagonists to normal and hypoplastic lungs decreased it. These data reveal for the first time that there is a link between neuroendocrine factors and RA, and that neuroendocrine factors sensitise the lung to the RA action through RA receptor modulation. ABSTRACT: 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, ß 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.

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

UNLABELLED: 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.

Canonical Wnt signaling activity in early stages of chick lung development.

Wnt signaling pathway is an essential player during vertebrate embryonic development which has been associated with several developmental processes such as gastrulation, body axis formation and morphogenesis of numerous organs, namely the lung. Wnt proteins act through specific transmembrane receptors, which activate intracellular pathways that regulate cellular processes such as cell proliferation, differentiation and death. Morphogenesis of the fetal lung depends on epithelial-mesenchymal interactions that are governed by several growth and transcription factors that regulate cell proliferation, fate, migration and differentiation. This process is controlled by different signaling pathways such as FGF, Shh and Wnt among others. Wnt signaling is recognized as a key molecular player in mammalian pulmonary development but little is known about its function in avian lung development. The present work characterizes, for the first time, the expression pattern of several Wnt signaling members, such as wnt-1, wnt-2b, wnt-3a, wnt-5a, wnt-7b, wnt-8b, wnt-9a, lrp5, lrp6, sfrp1, dkk1, ß-catenin and axin2 at early stages of chick lung development. In general, their expression is similar to their mammalian counterparts. By assessing protein expression levels of active/total ß-catenin and phospho-LRP6/LRP6 it is revealed that canonical Wnt signaling is active in this embryonic tissue. In vitro inhibition studies were performed in order to evaluate the function of Wnt signaling pathway in lung branching. Lung explants treated with canonical Wnt signaling inhibitors (FH535 and PK115-584) presented an impairment of secondary branch formation after 48 h of culture along with a decrease in axin2 expression levels. Branching analysis confirmed this inhibition. Wnt-FGF crosstalk assessment revealed that this interaction is preserved in the chick lung. This study demonstrates that Wnt signaling is crucial for precise chick lung branching and further supports the avian lung as a good model for branching studies since it recapitulates early mammalian pulmonary development.

Diabetes causes bone marrow autonomic neuropathy and impairs stem cell mobilization via dysregulated p66Shc and Sirt1.

Diabetes compromises the bone marrow (BM) microenvironment and reduces the number of circulating CD34(+) cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in patients with diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34(+) cells. Experimental diabetes (streptozotocin-induced and ob/ob mice) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin(-)cKit(+)Sca1(+) (LKS) cell and endothelial progenitor cell (EPC; CD34(+)Flk1(+)) mobilization, and vascular recovery after ischemia. DAN increased the expression of the 66-kDa protein from the src homology and collagen homology domain (p66Shc) and reduced the expression of sirtuin 1 (Sirt1) in mice and humans. p66Shc knockout (KO) in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, whereas hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1, diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L. CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the life-span regulators p66Shc and Sirt1.

The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression.