FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1.

The mechanisms of blood vessel maturation into distinct parts of the blood vasculature such as arteries, veins, and capillaries have been the subject of intense investigation over recent years. In contrast, our knowledge of lymphatic vessel maturation is still fragmentary. In this study, we provide a molecular and morphological characterization of the major steps in the maturation of the primary lymphatic capillary plexus into collecting lymphatic vessels during development and show that forkhead transcription factor Foxc2 controls this process. We further identify transcription factor NFATc1 as a novel regulator of lymphatic development and describe a previously unsuspected link between NFATc1 and Foxc2 in the regulation of lymphatic maturation. We also provide a genome-wide map of FOXC2-binding sites in lymphatic endothelial cells, identify a novel consensus FOXC2 sequence, and show that NFATc1 physically interacts with FOXC2-binding enhancers. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Glucocorticoids induce retinal toxicity through mechanisms mainly associated with paraptosis.

PURPOSE: Corticosteroids have recorded beneficial clinical effects and are widely used in medicine. In ophthalmology, besides their treatment benefits, side effects, including ocular toxicity have been observed especially when intraocular delivery is used. The mechanism of these toxic events remains, however, poorly understood. In our present study, we investigated the mechanisms and potential pathways of corticosteroid-induced retinal cell death. METHODS: Rats were sacrificed 24 h and 8 days after an intravitreous injection of 1 microl (40 microg) of Kenacort Retard. The eyes were processed for ultra structure analysis and detection of activated caspase-3, cytochrome-C, apoptosis-inducing factor (AIF), LEI-L-Dnase II, terminal transferase dUTP nick end labeling (TUNEL), and microtubule-associated protein 1-light chain 3 (MAP-LC3). In vitro, rat retinal pigment epithelial cells (RPE), retinal Müller glial cells (RMG) and human ARPE-19 cells were treated with triamcinolone acetonide (TA) or other glucocorticoids. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) assay and cell counts. Nuclei staining, TUNEL assay, annexin-V binding, activated caspase-3 and lactate dehydrogenase (LDH) production characterized cell death. Localization of cytochrome-C, AIF, LEI-and L-Dnase II, and staining with MAP-LC3 or monodansylcadaverine were also carried out. Finally, ARPE-19 cells transfected with AIP-1/Alix were exposed to TA. RESULTS: In vitro incubation of retinal cell in the presence of corticosteroids induced a specific and dose-dependent reduction of cell viability. These toxic events were not associated with the anti-inflammatory activity of these compounds but depended on the hydro solubility of their formulation. Before cell death, extensive cytoplasmic vacuolization was observed in the retinal pigment epithelial (RPE) cells in vivo and in vitro. The cells however, did not show known caspase-dependent or caspase-independent apoptotic reactions. These intracellular vacuoles were negative for MAP-LC3 but some stained positive for monodansylcadaverine. Furthermore, over expression of AIP-1/Alix inhibited RPE cell death. CONCLUSIONS: These observations suggest that corticosteroid-induced retinal cell death may be carried out mainly through a paraptosis pathway.

Pharmacokinetic and pharmacodynamic effects of YM087, a combined V1/V2 vasopressin receptor antagonist in normal subjects.

OBJECTIVE: The pharmacokinetic and pharmacodynamic properties of YM087, (4'-[(2-methyl-1,4,5,6- tetrahydroimidazo[4,5-d][1]benzazepin-6-yl)-carbonyl]-2-p henylbenzanilide monohydrochloride), a new orally active, dual V1/V2 receptor antagonist were characterised in healthy normotensive subjects. METHODS: Six subjects were randomly allocated to receive, at 1-week intervals, a single oral dose of 60 mg YM087 and a single i.v. dose of 50 mg YM087 in an open-label, crossover study. RESULTS: YM087 had an oral bioavailability of 44% and a short half-life. Upon oral and i.v. administration of YM087, a significant sevenfold increase in urine flow rate and a fall in urinary osmolality (from 600 mosmol/l to less than 100-mosmol/l) were observed with a peak effect 2 h after drug intake suggesting effective vasopressin V2 receptor blockade. Simultaneously, significant increases in plasma osmolality (from 283 +/- 1.3 mosmol/l to 288 +/- 1.0 mosmol/l after i.v. and from 283 +/- 2.1 mosmol/l to 289 +/- 1.7-mosmol/l after oral administration) and vasopressin levels (from 1.5 +/- 0.3 pg/ml to 3.7 +/- 0.6 pg/ml after i.v. and from 0.9 +/- 0.1 pg/ml to 3.9 +/- 0.7 pg/ml after oral administration) were found. When administered i.v., YM087 inhibited the vasopressin-induced skin vasoconstriction, suggesting a blockade of V1 receptors. However, the YM087-induced antagonism of V1 receptors was less pronounced than V2 receptor blockade. CONCLUSION: These data show that YM087 is an effective dual V1/V2 receptor antagonist in man.

Activation of GPER-1 estradiol receptor downregulates production of testosterone in isolated rat Leydig cells and adult human testis.

PURPOSE: Estradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis. MATERIALS AND METHODS: Isolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. RESULTS: GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20-30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs. CONCLUSIONS: Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.

Effects of triamcinolone acetonide on vessels of the posterior segment of the eye.

PURPOSE: This study investigates the effects of triamcinolone acetonide (TA) on retinal endothelial cells in vitro and explores the potential vascular toxic effect of TA injected into the vitreous cavity of rats in vivo. METHODS: Subconfluent endothelial cells were treated with either 0.1 mg/ml or 1 mg/ml TA in 1% ethanol. Control cells were either untreated or exposed to 1% ethanol. Cell viability was evaluated at 24 h, 72 h, and five days using the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) and lactate dehydrogenase (LDH) assays. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) test. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL assay), annexin-binding, and caspase 3 activation. Caspase-independent cell deaths were investigated by immunohistochemistry using antibodies against apoptosis inducing factor (AIF), cytochrome C, microtubule-associated protein (MAP)-light chain 3 (MAP-LC3), and Leukocyte Elastase Inhibitor/Leukocyte Elastase Inhibitor-derived DNase II (LEI/L-DNase II). In vivo, semithin and ultrathin structure analysis and vascular casts were performed to examine TA-induced changes of the choroidal vasculature. In addition, outer segments phagocytosis assay on primary retinal pigment epithelium (RPE) cells was performed to assess cyclooxygenase (COX-2) and vascular endothelial growth factor (VEGF) mRNAs upregulation with or without TA. RESULTS: The inhibitory effect of TA on cell proliferation could not explain the significant reduction in cell viability. Indeed, TA induced a time-dependent reduction of bovine retinal endothelial cells viability. Annexin-binding positive cells were observed. Cytochrome C was not released from mitochondria. L-DNase II was found translocated to the nucleus, meaning that LEI was changed into L-DNase II. AIF was found nuclearized in some cells. LC3 labeling showed the absence of autophagic vesicles. No autophagy or caspase dependent apoptosis was identified. At 1 mg/ml TA induced necrosis while exposure to lower concentrations for 3 to 5 days induced caspase independent apoptosis involving AIF and LEI/L-DNase II. In vivo, semithin and ultrathin structure analysis and vascular casts revealed that TA mostly affected the choroidal vasculature with a reduction of choroidal thickness and increased the avascular areas of the choriocapillaries. Experiments performed on primary RPE cells showed that TA downregulates the basal expression of COX-2 and VEGF and inhibits the outer segments (OS)-dependent COX-2 induction but not the OS-dependent VEGF induction. CONCLUSIONS: This study demonstrates for the first time that glucocorticoids exert direct toxic effect on endothelial cells through caspase-independent cell death mechanisms. The choroidal changes observed after TA intravitreous injection may have important implications regarding the safety profile of TA use in human eyes.

Anti-cancer activity of 5-O-alkyl 1,4-imino-1,4-dideoxyribitols.

New derivatives of 1,4-dideoxy-1,4-imino-D-ribitol have been prepared and evaluated for their cytotoxicity on solid and haematological malignancies. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-D-ribitol (13, IC(50) &#8764;2 &#956;M) and its C(18)-analogues (IC(50) <10 &#956;M) are cytotoxic toward SKBR3 (breast cancer) cells. 13 also inhibits (IC(50) &#8764;8 &#956;M) growth of JURKAT cells.

Prox1 interacts with Atoh1 and Gfi1, and regulates cellular differentiation in the inner ear sensory epithelia.

Inner ear hair cells and supporting cells arise from common precursors and, in mammals, do not show phenotypic conversion. Here, we studied the role of the homeodomain transcription factor Prox1 in the inner ear sensory epithelia. Adenoviral-mediated Prox1 transduction into hair cells in explant cultures led to strong repression of Atoh1 and Gfi1, two transcription factors critical for hair cell differentiation and survival. Luciferase assays showed that Prox1 can repress transcriptional activity of Gfi1 independently of Atoh1. Prox1 transduction into cochlear outer hair cells resulted in degeneration of these cells, consistent with the known phenotype of Gfi1-deficient mice. These results together with the widespread expression of endogenous Prox1 within the population of inner ear supporting cells point to the role for Prox1 in antagonizing the hair cell phenotype in these non-sensory cells. Further, in vivo analyses of hair cells from Gfi1-deficient mice suggest that the cyclin-dependent kinase inhibitor p57(Kip2) mediates the differentiation- and survival-promoting functions of Gfi1. These data reveal novel gene interactions and show that these interactions regulate cellular differentiation within the inner ear sensory epithelia. The data point to the tight regulation of phenotypic characteristics of hair cells and supporting cells.

International variation in management of screen-detected ductal carcinoma in situ of the breast.

BACKGROUND: Ductal carcinoma in situ (DCIS) incidence has grown with the implementation of screening and its detection varies across International Cancer Screening Network (ICSN) countries. The aim of this survey is to describe the management of screen-detected DCIS in ICSN countries and to evaluate the potential for treatment related morbidity. METHODS: We sought screen-detected DCIS data from the ICSN countries identified during 2004-2008. We adopted standardised data collection forms and analysis and explored DCIS diagnosis and treatment processes ranging from pre-operative diagnosis to type of surgery and radiotherapy. RESULTS: Twelve countries contributed data from a total of 15 screening programmes, all from Europe except the United States of America and Japan. Among women aged 50-69years, 7,176,050 screening tests and 5324 screen-detected DCIS were reported. From 21% to 93% of DCIS had a pre-operative diagnosis (PO); 67-90% of DCIS received breast conservation surgery (BCS), and in 41-100% of the cases this was followed by radiotherapy; 6.4-59% received sentinel lymph node biopsy (SLNB) only and 0.8-49% axillary dissection (ALND) with 0.6% (range by programmes 0-8.1%) being node positive. Among BCS patients 35% received SLNB only and 4.8% received ALND. Starting in 2006, PO and SLNB use increased while ALND remained stable. SLNB and ALND were associated with larger size and higher grade DCIS lesions. CONCLUSIONS: Variation in DCIS management among screened women is wide and includes lymph node surgery beyond what is currently recommended. This indicates the presence of varying levels of overtreatment and the potential for its reduction.

Phosphorylation regulates FOXC2-mediated transcription in lymphatic endothelial cells.

One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.

Characterisation of two soluble metalloexopeptidases in the protozoan parasite Leishmania major.

Two soluble exopeptidases were identified in promastigotes of Leishmania major, using an iodinated model tetrapeptide (LIAY) as substrate. Similar activities were also detected in L. major amastigotes and in different species of Leishmania promastigotes. A carboxy- and an aminopeptidase activity were resolved and isolated by anion exchange and gel permeation chromatographies. A single polypeptide of 62 kDa co-purified with the aminopeptidase activity. Optimum pH was neutral for the carboxypeptidase and neutral to alkaline for the aminopeptidase. Both activities were able to hydrolyse a dipeptide substrate (YL), and were inhibited by 20 microM bestatin and 200 microM 1,10-phenanthroline, but not by leupeptin, iodoacetamide and a range of other inhibitors. These results strongly suggest that both enzymes are metalloexopeptidases and thus represent a novel class of soluble peptidases in Leishmania.

Genome-wide association study to identify common variants associated with brachial circumference: a meta-analysis of 14 cohorts.

Brachial circumference (BC), also known as upper arm or mid arm circumference, can be used as an indicator of muscle mass and fat tissue, which are distributed differently in men and women. Analysis of anthropometric measures of peripheral fat distribution such as BC could help in understanding the complex pathophysiology behind overweight and obesity. The purpose of this study is to identify genetic variants associated with BC through a large-scale genome-wide association scan (GWAS) meta-analysis. We used fixed-effects meta-analysis to synthesise summary results across 14 GWAS discovery and 4 replication cohorts comprising overall 22,376 individuals (12,031 women and 10,345 men) of European ancestry. Individual analyses were carried out for men, women, and combined across sexes using linear regression and an additive genetic model: adjusted for age and adjusted for age and BMI. We prioritised signals for follow-up in two-stages. We did not detect any signals reaching genome-wide significance. The FTO rs9939609 SNP showed nominal evidence for association (p&lt;0.05) in the age-adjusted strata for men and across both sexes. In this first GWAS meta-analysis for BC to date, we have not identified any genome-wide significant signals and do not observe robust association of previously established obesity loci with BC. Large-scale collaborations will be necessary to achieve higher power to detect loci underlying BC.

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

Genome-wide association and longitudinal analyses reveal genetic loci linking pubertal height growth, pubertal timing and childhood adiposity.

The pubertal height growth spurt is a distinctive feature of childhood growth reflecting both the central onset of puberty and local growth factors. Although little is known about the underlying genetics, growth variability during puberty correlates with adult risks for hormone-dependent cancer and adverse cardiometabolic health. The only gene so far associated with pubertal height growth, LIN28B, pleiotropically influences childhood growth, puberty and cancer progression, pointing to shared underlying mechanisms. To discover genetic loci influencing pubertal height and growth and to place them in context of overall growth and maturation, we performed genome-wide association meta-analyses in 18 737 European samples utilizing longitudinally collected height measurements. We found significant associations (P &lt; 1.67 × 10(-8)) at 10 loci, including LIN28B. Five loci associated with pubertal timing, all impacting multiple aspects of growth. In particular, a novel variant correlated with expression of MAPK3, and associated both with increased prepubertal growth and earlier menarche. Another variant near ADCY3-POMC associated with increased body mass index, reduced pubertal growth and earlier puberty. Whereas epidemiological correlations suggest that early puberty marks a pathway from rapid prepubertal growth to reduced final height and adult obesity, our study shows that individual loci associating with pubertal growth have variable longitudinal growth patterns that may differ from epidemiological observations. Overall, this study uncovers part of the complex genetic architecture linking pubertal height growth, the timing of puberty and childhood obesity and provides new information to pinpoint processes linking these traits.

Medication use in pregnancy: a cross-sectional, multinational web-based study.

OBJECTIVES: Intercountry comparability between studies on medication use in pregnancy is difficult due to dissimilarities in study design and methodology. This study aimed to examine patterns and factors associated with medications use in pregnancy from a multinational perspective, with emphasis on type of medication utilised and indication for use. DESIGN: Cross-sectional, web-based study performed within the period from 1 October 2011 to 29 February 2012. Uniform collection of drug utilisation data was performed via an anonymous online questionnaire. SETTING: Multinational study in Europe (Western, Northern and Eastern), North and South America and Australia. PARTICIPANTS: Pregnant women and new mothers with children less than 1 year of age. PRIMARY AND SECONDARY OUTCOME MEASURES: Prevalence of and factors associated with medication use for acute/short-term illnesses, chronic/long-term disorders and over-the-counter (OTC) medication use. RESULTS: The study population included 9459 women, of which 81.2% reported use of at least one medication (prescribed or OTC) during pregnancy. Overall, OTC medication use occurred in 66.9% of the pregnancies, whereas 68.4% and 17% of women reported use of at least one medication for treatment of acute/short-term illnesses and chronic/long-term disorders, respectively. The extent of self-reported medicated illnesses and types of medication used by indication varied across regions, especially in relation to urinary tract infections, depression or OTC nasal sprays. Women with higher age or lower educational level, housewives or women with an unplanned pregnancy were those most often reporting use of medication for chronic/long-term disorders. Immigrant women in Western (adjusted OR (aOR): 0.55, 95% CI 0.34 to 0.87) and Northern Europe (aOR: 0.50, 95% CI 0.31 to 0.83) were less likely to report use of medication for chronic/long-term disorders during pregnancy than non-immigrants. CONCLUSIONS: In this study, the majority of women in Europe, North America, South America and Australia used at least one medication during pregnancy. There was a substantial inter-region variability in the types of medication used.