The apelinergic system in the developing lung: expression and signaling

Apelin and its receptor APJ constitute a signaling pathway best recognized as an important regulator of cardiovascular homeostasis. This multifunctional peptidergic system is currently being described to be involved in embryonic events which extend into vascular, ocular and heart development. Additionally, it is highly expressed in pulmonary tissue. Therefore, the aim of this study was to investigate the role of apelinergic system during fetal lung development. Immunohistochemistry and Western blot analysis were used to characterize apelin and APJ expression levels and cellular localization in normal fetal rat lungs, at five different gestational ages as well as in the adult. Fetal rat lung explants were cultured in vitro with increasing doses of apelin. Treated lung explants were morphometrically analyzed and assessed for MAPK signaling modifications. Both components of the apelinergic system are constitutively expressed in the developing lung, with APJ exhibiting monomeric, dimeric and oligomeric forms in the pulmonary tissue. Pulmonary epithelium also displayed constitutive nuclear localization of the receptor. Fetal apelin expression is higher than adult expression. Apelin supplementation inhibitory effect on branching morphogenesis was associated with a dose dependent decrease in p38 and JNK phosphorylation. The results presented provide the first evidence of the presence of an apelinergic system operating in the developing lung. Our findings also suggest that apelin inhibits fetal lung growth by suppressing p38 and JNK signaling pathways.This project was funded by Fundação para a Ciência e a Tecnologia (PTDC/SAU-OBD/108051/2008), PP was supported by Fundação para a Ciência e a Tecnologia (reference SFRH/BD/33410/2008). RSM was supported by Fundação para a Ciência e a Tecnologia (reference SFRH/BPD/15408/2005)

STATs in lung development: distinct early and late expression, growth modulation and signaling dysregulation in congenital diaphragmatic hernia

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.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.Competitiveness Factors Operational Programme (COMPETE), Northern Portugal Regional Operational Programme (NORTE 2020) - NORTE-01-0145-FEDER-000013, Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER).info:eu-repo/semantics/publishedVersio

Leukemia Inhibitory Factor in Rat Fetal Lung Development: Expression and Functional Studies

Background: Leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) are members of the family of the glycoprotein 130 (gp130)-type cytokines. These cytokines share gp130 as a common signal transducer, which explains why they show some functional redundancy. Recently, it was demonstrated that IL-6 promotes fetal lung branching. Additionally, LIF has been implicated in developmental processes of some branching organs. Thus, in this study LIF expression pattern and its effects on fetal rat lung morphogenesis were assessed. Methodology/Principal Findings: LIF and its subunit receptor LIFRa expression levels were evaluated by immunohistochemistry and western blot in fetal rat lungs of different gestational ages, ranging from 13.5 to 21.5 days post-conception. Throughout all gestational ages studied, LIF was constitutively expressed in pulmonary epithelium, whereas LIFRa was first mainly expressed in the mesenchyme, but after pseudoglandular stage it was also observed in epithelial cells. These results point to a LIF epithelium-mesenchyme cross-talk, which is known to be important for lung branching process. Regarding functional studies, fetal lung explants were cultured with increasing doses of LIF or LIF neutralizing antibodies during 4 days. MAPK, AKT, and STAT3 phosphorylation in the treated lung explants was analyzed. LIF supplementation significantly inhibited lung growth in spite of an increase in p44/42 phosphorylation. On the other hand, LIF inhibition significantly stimulated lung growth via p38 and Akt pathways

Improved lymphocyte response to H2O2 after regular intake of Sage tea (Salvia officinalis) involves induction of HSP70

Sage (Salvia officinalis) has recently been shown to have plasma glucose lowering potential. This suggests an antidiabetic potential for this plant alongside with other well-known properties that include antioxidant, anti-inflammatory and anti-mutagenic activities. Diabetes, a disease caused by loss of control of glucose homeostasis, also involves imbalances in the internal metabolic environment that can lead to irreversible oxidative damage in some cell populations. In the present study, we investigated the protective effect of the regular intake of Sage tea on the antioxidant response of peripheral blood lymphocytes (PBLs) challenged with H2O2. Lymphocytes were isolated from six healthy human volunteers at several times during a pilot trial of four weeks of sage tea treatment (twice a day). We also collected blood samples in the pre- and post-treatment periods referred to as baseline and wash-out, respectively. Damage to DNA was evaluated by Comet Assay. Significant reduction of damage induced by 200 μM H2O2 after two weeks of treatment were observed, indicative of improved cell defences. To further explore the mechanisms of cellular protection conferred by tea drinking to lymphocytes, we assessed effects on Hsp70 expression levels in PBLs by use of Western Blotting analysis. Besides being associated with improved stress resistance, the increased expression of Hsp is regarded as one of the most powerful means of cytoprotection against protein misfolding and aggregation. Our results show Hsp70 levels were significantly elevated in treatment compared with the baseline, which suggests that an induction of Hsp70 may be, at least in part, responsible for the improved cellular response to H2O2 induced damage.Fundação para a Ciência e a Tecnologia (FCT) - grant SFR/BD/12527/200, POCTI/AGR/62040/2004

Sage drinking improves plasma lipid profile, erythrocyte antioxidant defences and increases lymphocyte HSP70

Salvia officinalis (common sage) is a medicinal plant to which antioxidant, anti-inflammatory and antimutagenic properties have been attributed. Recent results from our laboratory showed cellular and in vivo antioxidant effects of sage as well as metformin-like effects at the rat liver level, suggesting an antidiabetic potential for sage. In order to test these effects in humans, we performed a pilot trial with six healthy female volunteers. The trial was carried out in three phases, which includes two weeks of baseline, four weeks of sage treatment (drinking of a sage infusion twice a day) and two weeks of wash-out. Sage treatment positively affected the erythrocyte antioxidant status as shown by increased SOD and CAT activities. Cholesterol and LDL levels significantly decreased and HDL levels significantly increased after treatment, indicating benefits also in lipid metabolism. However, no changes in glucose clearance were observed in the oral glucose tolerance tests at the end of treatment period. In addition, a reduction of in vitro lymphocyte DNA damage induced by H2O2 was observed during the treatment period, which was maintained through the wash-out period. During the S. officinalis drinking period, lymphocyte Hsp70 protein expression was significantly increased (about 2.25 times) and decreased to baseline following the wash-out period. Overall these results confirm the health improving potential of sage infusion drinking

Effects of the regular intake of sage tea (Salvia Officinalis) in humans : a pilot trial

Cells are constantly subjected to oxidative stress and oxidative damage is frequently involved in the development of several pathologies, such as cancer, hypertension and diabetes. Salvia officinalis (common sage) extracts and constituents are known for their antioxidant, antiinflammatory and anti-mutagenic proprieties. Recent experimental studies have shown that a water extract of Salvia officinalis reduces liver glucose production and fasting plasma glucose levels in normal rats, suggesting an antidiabetic potential. Therefore, we believe that common sage may be beneficial to diabetic patients where it may improve antioxidant defences and have a role in glycaemia control. The present work was undertaken to assess the effects of a sage water extract (tea) in human volunteers. To evaluate the effects of water extract of Salvia officinalis, a medicinal plant, on blood parameters and on erythrocyte antioxidant enzymes activities, we developed a pilot trial with six healthy volunteers. This trial was carried out in three phases which include two weeks of baseline, four weeks of sage tea treatment (sage tea was taken twice a day) and two weeks of wash out. During the study, blood samples were collected and analysed for haemoglobin as well as ALT and AST activities, fasting and post-prandial glucose, LDL, HDL, and cholesterol. Erythrocyte antioxidant enzymes activities (SOD and CAT) were measured from haemolysed samples. Effects on weight, blood pressure, heart rate at rest, perceived negative events (and concomitant medication) were recorded at the end of every week. The results indicate that sage tea had no toxic effects to the liver enzyme activities (ALT, AST) and increased SOD and CAT activities, which indicate that two weeks of sage tea treatment positively affect the antioxidant status. Cholesterol and LDL levels significantly decreased and HDL levels significantly increased after four weeks of treatment, which in turn suggests a beneficial effect also in lipid metabolism. We also found that two weeks of sage treatment increased blood haemoglobin levels and did not change significantly plasma glucose following oral glucose tolerance tests. Despite the fact that there were no effects on blood glucose, improvements on lipid and antioxidant balance suggest health improving effects to sage tea drinking.Fundação para a Ciência e a Tecnologia (FCT)FCT Bolsa SFRH/BD/6942/2001, POCTI/AGR/62040/200

The role of glycoprotein 130 family of cytokines in fetal rat lung development

The glycoprotein 130 (gp130) dependent family of cytokines comprises interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), cardiotrophin-like cytokine (CLC), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and oncostatin M (OSM). These cytokines share the membrane gp130 as a common signal transducer. Recently, it was demonstrated that IL-6 promotes, whereas LIF inhibits fetal lung branching. Thus, in this study, the effects on fetal lung morphogenesis of the other classical members of the gp130-type cytokines (IL-11, CLC, CNTF, CT-1 and OSM) were investigated. We also provide the first description of these cytokines and their common gp130 receptor protein expression patterns during rat lung development. Fetal rat lung explants were cultured in vitro with increasing concentrations of IL-11, CLC, CNTF, CT-1 and OSM. Treated lung explants were morphometrically analyzed and assessed for MAPK, PI3K/AKT and STAT3 signaling modifications. IL-11, which similarly to IL-6 acts through a gp130 homodimer receptor, significantly stimulated lung growth via p38 phosphorylation. On the other hand, CLC, CNTF, CT-1 and OSM, whose receptors are gp130 heterodimers, inhibited lung growth acting in different signal-transducing pathways. Thus, the present study demonstrated that although cytokines of the gp130 family share a common signal transducer, there are specific biological activities for each cytokine on lung development. Indeed, cytokine signaling through gp130 homodimers stimulate, whereas cytokine signaling through gp130 heterodimers inhibit lung branching.This project was funded by Fundacao para a Ciencia e a Tecnologia (PTDC/SAU-OBD/108051/2008) and by Seccao de Neonatologia da Sociedade Portuguesa de Pediatria (Grant ZERU 2008). PP was supported by Fundacao para a Ciencia e a Tecnologia (reference SFRH/BD/33410/2008). RSM was supported by Fundacao para a Ciencia e a Tecnologia (reference SFRH/BPD/15408/2005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Local Fetal Lung Renin-Angiotensin System as a Target to Treat Congenital Diaphragmatic Hernia

Antenatal stimulation of lung growth is a reasonable approach to treat congenital diaphragmatic hernia (CDH), a disease characterized by pulmonary hypoplasia and hypertension. Several evidences from the literature demonstrated a possible involvement of renin-angiotensin system (RAS) during fetal lung development. Thus, the expression pattern of renin, angiotensin-converting enzyme, angiotensinogen, type 1 (AT1) and type 2 (AT2) receptors of angiotensin II (ANGII) was assessed by immunohisto-chemistry throughout gestation, whereas the function of RAS in the fetal lung was evaluated using fetal rat lung explants. These were morphometrically analyzed and intracellular pathway alterations assessed by Western blot. In nitrofen-induced CDH model, pregnant rats were treated with saline or PD-123319. In pups, lung growth, protein/DNA ratio, radial saccular count, epithelial differentiation and lung maturation, vascular morphometry, right ventricular hypertrophy and overload molecular markers, gasometry and survival time were evaluated. Results demonstrated that all RAS components were constitutively expressed in the lung during gestation and that ANGII had a stimulatory effect on lung branching, mediated by AT1 receptor, through p44/42 and Akt phosphorylation. This stimulatory effect on lung growth was mimicked by AT2-antagonist (PD-123319) treatment. In vivo antenatal PD-123319 treatment increased lung growth, ameliorated indirect parameters of pulmonary hypertension, improved lung function and survival time in nonventilated CDH pups, without maternal or fetal deleterious effects. Therefore, this study demonstrated a local and physiologically active RAS during lung morphogenesis. Moreover, selective inhibition of AT2 receptor is presented as a putative antenatal therapy for CDH