No evidence for a functional role of bi-directional Notch signaling during angiogenesis.

The Delta-Notch pathway is a signal exchanger between adjacent cells to regulate numerous differentiation steps during embryonic development. Blood vessel formation by sprouting angiogenesis requires high expression of the Notch ligand DLL4 in the leading tip cell, while Notch receptors in the trailing stalk cells are activated by DLL4 to achieve strong Notch signaling activity. Upon ligand binding, Notch receptors are cleaved by ADAM proteases and gamma-secretase. This releases the intracellular Notch domain that acts as a transcription factor. There is evidence that also Notch ligands (DLL1, DLL4, JAG1, JAG2) are processed upon receptor binding to influence transcription in the ligand-expressing cell. Thus, the existence of bi-directional Delta-Notch signaling has been proposed. We report here that the Notch ligands DLL1 and JAG1 are processed in endothelial cells in a gamma-secretase-dependent manner and that the intracellular ligand domains accumulate in the cell nucleus. Overexpression of JAG1 intracellular domain (ICD) as well as DLL1-ICD, DLL4-ICD and NOTCH1-ICD inhibited endothelial proliferation. Whereas NOTCH1-ICD strongly repressed endothelial migration and sprouting angiogenesis, JAG1-ICD, DLL1-ICD and DLL4-ICD had no significant effects. Consistently, global gene expression patterns were only marginally affected by the processed Notch ligands. In addition to its effects as a transcription factor, NOTCH1-ICD promotes cell adhesion to the extracellular matrix in a transcription-independent manner. However, JAG1-ICD, DLL1-ICD and DLL4-ICD did not influence endothelial cell adhesion. In summary, reverse signaling of Notch ligands appears to be dispensable for angiogenesis in cellular systems

Localisation of Notch ligand intracellular domains.

<p>(A) Scheme of the Notch ligand (DSL) intracellular domain (ICD) expression cassette in adenoviral vectors. Western blotting revealed expression of the fusion proteins in total lysates. (B) JAG1-ICD was localized predominantly in the nucleus, whereas DLL1-ICD and DLL4-ICD proteins were also localized in the cytoplasm. Scale bar, 100 µm. (C) Ligand-ICDs fused with Citrine were detected only in nuclear fractions (N) but not in the cytoplasmic fractions (C). HUVEC expressing full-length JAG1 or DLL1 fused to a c-terminal myc or V5-tag were lysed to obtain nuclear and cytoplasmic fractions. The processed intracellular fragments were detected only in the nuclear extracts by Western blotting.</p

JAG1 is processed by gamma-secretase.

<p>HUVEC were transduced with adenoviruses expressing GFP, full length JAG1. The intracellular JAG1 domain fused to Citrine, or DLL1 tagged with V5 at the carboxyterminus. (A) JAG1 was processed and the cleavage products have the predicted size of the transmembrane-intracellular domain fragment (TM-ICD) and the free intracellular domain (ICD). The membrane was blotted with JAG1 antibodies recognizing the carboxyterminal intracellular domain. (B) Treatment with VEGF or FGF2 did not significantly increase ligand cleavage compared to solvent as control. Inhibition of gamma-secretase activity with 25 µM DAPT prevented the formation of the intracellular domain and led to accumulation of the uncleaved TM-ICD fragment. (C) The predicted size of the DLL1 intracellular domain, tagged with V5, was detected in HUVEC lysates by Western blotting.</p

Intracellular domains of DLL4, DLL1 and JAG1 are highly conserved.

<p>(A) ClustalW alignment of the intracellular domains of human DLL4, DLL1, and JAG1 with their respective homologues from mouse, cow, chicken, xenopus, zebrafish, and fugu revealed a high grade of conservation between all vertebrate species. The carboxyterminal PDZ motif (highlighted in yellow) is 100% conserved in all cases. (B) A dendrogram from a ClustalW alignment of the <u>full-length</u> proteins from vertebrates and drosophila proves that the proteins as a whole are also highly conserved between species. Colored boxes show the closer relationship of DLL1 and DLL4 compared to JAG1.</p

Notch ligand intracellular domains inhibit cellular proliferation but not migration and adhesion.

<p>(A,B) Adenoviral expression of Delta and Jagged intracellular domains (ICD) in HUVEC inhibited BrdU incorporation indicating decreased cell proliferation. NOTCH1-ICD had stronger repression activity that was not altered by co-expression of DLL4-ICD or JAG1-ICD. n = 3 independent experiments. (C) Chemotactic migration of HUVEC through a transwell filter was not altered by ligand-ICDs but strongly impaired by NOTCH1-ICD expression. n = 3 independent experiments. (D) Adhesion of HUVEC to extracellular matrix proteins was enhanced by NOTCH1-ICD but not by ligand-ICDs. n = 3 independent experiments. Data are presented as mean +SD. *, p<0.05.</p

Transcripts regulated by Notch ligand intracellular domains.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053074#pone-0053074-t001" target="_blank"><b>Table 1</b></a><b>.</b> mRNA transcripts significantly changed 36 h after adenoviral infection of HUVEC with DLL1-ICD, DLL4-ICD or JAG1-ICD. Adenoviral GFP expression served as control.</p

Loss of Mpdz impairs ependymal cell integrity leading to perinatal‐onset hydrocephalus in mice

Abstract Hydrocephalus is a common congenital anomaly. LCAM1 and MPDZ (MUPP1) are the only known human gene loci associated with non‐syndromic hydrocephalus. To investigate functions of the tight junction‐associated protein Mpdz, we generated mouse models. Global Mpdz gene deletion or conditional inactivation in Nestin‐positive cells led to formation of supratentorial hydrocephalus in the early postnatal period. Blood vessels, epithelial cells of the choroid plexus, and cilia on ependymal cells, which line the ventricular system, remained morphologically intact in Mpdz‐deficient brains. However, flow of cerebrospinal fluid through the cerebral aqueduct was blocked from postnatal day 3 onward. Silencing of Mpdz expression in cultured epithelial cells impaired barrier integrity, and loss of Mpdz in astrocytes increased RhoA activity. In Mpdz‐deficient mice, ependymal cells had morphologically normal tight junctions, but expression of the interacting planar cell polarity protein Pals1 was diminished and barrier integrity got progressively lost. Ependymal denudation was accompanied by reactive astrogliosis leading to aqueductal stenosis. This work provides a relevant hydrocephalus mouse model and demonstrates that Mpdz is essential to maintain integrity of the ependyma

New Onset of DiabetEs in aSsociation with pancreatic ductal adenocarcinoma (NODES Trial): protocol of a prospective, multicentre observational trial

Introduction Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with an overall 5-year survival of approximately 8%. The success in reducing the mortality rate of PDAC is related to the discovery of new therapeutic agents, and to a significant extent to the development of early detection and prevention programmes. Patients with new-onset diabetes mellitus (DM) represent a high-risk group for PDAC as they have an eightfold higher risk of PDAC than the general population. The proposed screening programme may allow the detection of PDAC in the early, operable stage. Diagnosing more patients in the curable stage might decrease the morbidity and mortality rates of PDAC and additionally reduce the burden of the healthcare.Methods and analysis This is a prospective, multicentre observational cohort study. Patients ≥60 years old diagnosed with new-onset (≤6 months) diabetes will be included. Exclusion criteria are (1) Continuous alcohol abuse; (2) Chronic pancreatitis; (3) Previous pancreas operation/pancreatectomy; (4) Pregnancy; (5) Present malignant disease and (6) Type 1 DM. Follow-up visits are scheduled every 6 months for up to 36 months. Data collection is based on questionnaires. Clinical symptoms, body weight and fasting blood will be collected at each, carbohydrate antigen 19–9 and blood to biobank at every second visit. The blood samples will be processed to plasma and analysed with mass spectrometry (MS)-based metabolomics. The metabolomic data will be used for biomarker validation for early detection of PDAC in the high-risk group patients with new-onset diabetes. Patients with worrisome features will undergo MRI or endoscopic ultrasound investigation, and surgical referral depending on the radiological findings. One of the secondary end points is the incidence of PDAC in patients with newly diagnosed DM.Ethics and dissemination The study has been approved by the Scientific and Research Ethics Committee of the Hungarian Medical Research Council (41085-6/2019). We plan to disseminate the results to several members of the healthcare system includining medical doctors, dietitians, nurses, patients and so on. We plan to publish the results in a peer-reviewed high-quality journal for professionals. In addition, we also plan to publish it for lay readers in order to maximalise the dissemination and benefits of this trial.Trial registration number ClinicalTrials.gov NCT0416460