Emanuel Carvalho-Dias

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 immunohistochemistry 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.

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 LIFRα 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 LIFRα 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. Conclusions/Significance The present study describes that LIF and its subunit receptor LIFRα are constitutively expressed during fetal lung development and that they have an inhibitory physiological role on fetal lung branching.

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.

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.

Video-Based Surgical Learning: Improving Trainee Education and Preparation for Surgery

BACKGROUND Since the end of the XIX century, teaching of surgery has remained practically unaltered until now. With the dawn of video-assisted laparoscopy, surgery has faced new technical and learning challenges. Due to technological advances, from Internet access to portable electronic devices, the use of online resources is part of the educational armamentarium. In this respect, videos have already proven to be effective and useful, however the best way to benefit from these tools is still not clearly defined. AIMS To assess the importance of video-based learning, using an electronic questionnaire applied to residents and specialists of different surgical fields. METHODS Importance of video-based learning was assessed in a sample of 141 subjects, using a questionnaire distributed by a GoogleDoc online form. RESULTS We found that 98.6% of the respondents have already used videos to prepare for surgery. When comparing video sources by formation status, residents were found to use Youtube significantly more often than specialists (p < 0.001). Additionally, residents placed more value on didactic illustrations and procedure narration than specialists (p < 0.001). On the other hand, specialists prized surgeons technical skill and the presence of tips and tricks much more than residents (p < 0.001). CONCLUSION Video-based learning is currently a hallmark of surgical preparation among residents and specialists working in Portugal. Based on these findings we believe that the creation of quality and scientifically accurate videos, and subsequent compilation in available video-libraries appears to be the future landscape for video-based learning.

MP45-09 EFFECTS OF CASTRATION AND TESTOSTERONE REPLACEMENT OVER SEROTONIN (PROSTATIC AND PLASMATIC): A MICE IN VIVO STUDY.

Here, we hypothesized that modulating the plasmatic levels of 5-HT through a tryptophan-rich diet we could inhibit prostatic growth. METHODS: C57BL6 adult male mice were divided in 2 groups, Group 1: fed with a normal diet and Group 2: fed with, for a 3-month period. After the sacrifice of the mice, the prostate was dissected and weighted. By ELISA, prostatic 5-HT concentration was determined and by Western Blot, AR expression was evaluated in both groups. RESULTS: We observed that mice fed with a tryptophan-rich diet for 3 months present a significant reduction in the prostatic weight comparatively to mice fed with normal diet (fig1). Intra-prostatic 5-HT concentration was significantly increased in mice fed with a tryptophanrich diet (fig2). Furthermore, we demonstrated that the expression of AR in prostatic tissue from mice who underwent tryptophan supplementation was inferior to the AR expression in the normal diet group (fig3). CONCLUSIONS: Modulating diet through tryptophan enrichment, with the consequent increase in prostatic serotonin, decreases prostate size and down-regulates AR. These results suggests that a tryptophan rich diet could be potential used to prevent or treat BPH.

MP17-14 DEPLETION OF PERIPHERAL SEROTONIN SYNTHESIS INDUCES BENIGN PROSTATIC GROWTH IN MICE: MORE EVIDENCE FOR THE NEW “NEUROENDOCRINE THEORY” IN BPH ETIOLOGY

up-regulated collagen 1 and aSMA protein expression in response to IL-4 or IL-13 treatment. These effects were ablated through co-treatment with IL-4 and IL-13 receptors antibodies. Low concentrations of IL-4 and IL-13 significantly up-regulated N1 and primary prostate cell proliferation. Prostate fibroblasts treated with IL-4 or IL-13 significantly and almost equivalently up-regulated IL-13 protein expression, suggesting the establishment of an autocrine IL-13 expression loop. Treatment with IL-4 alone up-regulated IL-4, but not IL-13, protein expression. Neither IL-4 nor IL-13 affected TGFbeta expression levels. CONCLUSIONS: The results of these studies show that IL-4 and IL-13 promote myofibroblast phenoconversion and collagen accumulation, and thus support a role for IL-4/IL-13 promotion of prostatic fibrosis associated with LUTD. A major new discovery reported here is that Th2 cytokines, particularly IL-13, can establish autocrine expression loops in prostate fibroblasts that may promote continual myofibroblast phenoconversion in the lower urinary tract. These findings support the investigation of small molecules or antibodies that target Th2 IL-4/IL13 activities for the prevention or treatment of lower urinary tract fibrosis.

V5-10 EXTRAPERITEONAL SIMPLE PROSTATECTOMY: A SURGERY FOR BEGINNERS?

RESULTS: After initial inspection of the bladder demonstrated asymptomatic ureteroceles, an incision was made at the bladder neck and carried to the depth of the surgical capsule distally to the verumontanum. The lateral dissection expresses purulence almost immediately and a large abscess pocket is encountered during the anterior dissection. The remainder of the enucleation is performed without complication. The patient was spontaneously voiding by POD 2 and discharged with IV antibiotics for 6 weeks to treat his osteomyelitis. CONCLUSIONS: Holmium laser enucleation of a prostatic abscess can be performed safely and effectively to both maximally reduce the abscess cavity and risk of excessive thermal injury when treating an apically located abscess.

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.