Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis

Funding Information: This work was supported by European Regional Development Fund (FEDER) through the Operational Program for Competitiveness Factors (COMPETE) [HealthyAging2020 CENTRO-01-0145-FEDER-000012-N2323, POCI-01-0145-FEDER-016385, POCI-01-0145-FEDER-007440 to CNC.IBILI, POCI-01-0145-FEDER-007274 to i3S/INEB and NORTE-01-0145-FEDER-000012 to T.L.L.]; national funds through the Portuguese Foundation for Science and Technology (FCT) [PTDC/SAU-ORG/119296/2010, PTDC/ NEU-OSD/0312/2012, PESTC/ SAU/UI3282/2013-2014, MITP-TB/ECE/0013/ 2013, FCT-UID/NEU/04539/2013], PD/BD/52294/2013 to T.M.R.R., SFRH/ BD/85556/2012 (co-financed by QREN) to V.C.S]; Lisboa Portugal Regional Operational Programme (LISBOA 2020) and Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement; and by INFARMED Autoridade Nacional do Medicamento e Produtos de Saúde, I.P. [FIS-FIS-2015-01_CCV_20150630-157]. Publisher Copyright: © 2017 The Author.Aims Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). Methods and results Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. Conclusions This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply.publishersversionpublishe

Interdigital cell death in the embryonic limb is associated with depletion of Reelin in the extracellular matrix

Interdigital cell death is a physiological regression process responsible for sculpturing the digits in the embryonic vertebrate limb. Changes in the intensity of this degenerative process account for the different patterns of interdigital webbing among vertebrate species. Here, we show that Reelin is present in the extracellular matrix of the interdigital mesoderm of chick and mouse embryos during the developmental stages of digit formation. Reelin is a large extracellular glycoprotein which has important functions in the developing nervous system, including neuronal survival; however, the significance of Reelin in other systems has received very little attention. We show that reelin expression becomes intensely downregulated in both the chick and mouse interdigits preceding the establishment of the areas of interdigital cell death. Furthermore, fibroblast growth factors, which are cell survival signals for the interdigital mesoderm, intensely upregulated reelin expression, while BMPs, which are proapototic signals, downregulate its expression in the interdigit. Gene silencing experiments of reelin gene or its intracellular effector Dab-1 confirmed the implication of Reelin signaling as a survival factor for the limb undifferentiated mesoderm. We found that Reelin activates canonical survival pathways in the limb mesoderm involving protein kinase B and focal adhesion kinase. Our findings support that Reelin plays a role in interdigital cell death, and suggests that anoikis (apoptosis secondary to loss of cell adhesion) may be involved in this process

Nanopore native RNA sequencing of a human poly(A) transcriptome

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes

Measuring children’s involvement as an indicator of curriculum effectiveness : a curriculum evaluation of a selected child study centre in Singapore

This paper presents one aspect of a research project evaluating a curriculum model of a selected child study centre in Singapore. An issue of worldwide interest and concern is the ‘quality of learning’ debate as it relates to early childhood centres. In Singapore, the government is focusing on expansion in child care settings and increases in the amount of funded training. One of the issues surrounding prior-to-school education raises the question of how one measures the quality of teaching and learning, to describe the value of using, funding and promoting early education. The research reported in this study used a quasi experimental research paradigm to assess one aspect of the quality of a curriculum programme in a child study centre in Singapore. Children aged between 18 months and 6 years (N = 81) participated in the research. Using the observation scale of Laevers’ Child Involvement Scale, the active involvement of children in learning experiences was measured. The findings are presented and discussed

Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell

Construction and analysis of a modular model of caspase activation in apoptosis

<p>Abstract</p> <p>Background</p> <p>A key physiological mechanism employed by multicellular organisms is apoptosis, or programmed cell death. Apoptosis is triggered by the activation of caspases in response to both extracellular (extrinsic) and intracellular (intrinsic) signals. The extrinsic and intrinsic pathways are characterized by the formation of the death-inducing signaling complex (DISC) and the apoptosome, respectively; both the DISC and the apoptosome are oligomers with complex formation dynamics. Additionally, the extrinsic and intrinsic pathways are coupled through the mitochondrial apoptosis-induced channel via the Bcl-2 family of proteins.</p> <p>Results</p> <p>A model of caspase activation is constructed and analyzed. The apoptosis signaling network is simplified through modularization methodologies and equilibrium abstractions for three functional modules. The mathematical model is composed of a system of ordinary differential equations which is numerically solved. Multiple linear regression analysis investigates the role of each module and reduced models are constructed to identify key contributions of the extrinsic and intrinsic pathways in triggering apoptosis for different cell lines.</p> <p>Conclusion</p> <p>Through linear regression techniques, we identified the feedbacks, dissociation of complexes, and negative regulators as the key components in apoptosis. The analysis and reduced models for our model formulation reveal that the chosen cell lines predominately exhibit strong extrinsic caspase, typical of type I cell, behavior. Furthermore, under the simplified model framework, the selected cells lines exhibit different modes by which caspase activation may occur. Finally the proposed modularized model of apoptosis may generalize behavior for additional cells and tissues, specifically identifying and predicting components responsible for the transition from type I to type II cell behavior.</p

Knock-Down of Cathepsin D Affects the Retinal Pigment Epithelium, Impairs Swim-Bladder Ontogenesis and Causes Premature Death in Zebrafish

The lysosomal aspartic protease Cathepsin D (CD) is ubiquitously expressed in eukaryotic organisms. CD activity is essential to accomplish the acid-dependent extensive or partial proteolysis of protein substrates within endosomal and lysosomal compartments therein delivered via endocytosis, phagocytosis or autophagocytosis. CD may also act at physiological pH on small-size substrates in the cytosol and in the extracellular milieu. Mouse and fruit fly CD knock-out models have highlighted the multi-pathophysiological roles of CD in tissue homeostasis and organ development. Here we report the first phenotypic description of the lack of CD expression during zebrafish (Danio rerio) development obtained by morpholino-mediated knock-down of CD mRNA. Since the un-fertilized eggs were shown to be supplied with maternal CD mRNA, only a morpholino targeting a sequence containing the starting ATG codon was effective. The main phenotypic alterations produced by CD knock-down in zebrafish were: 1. abnormal development of the eye and of retinal pigment epithelium; 2. absence of the swim-bladder; 3. skin hyper-pigmentation; 4. reduced growth and premature death. Rescue experiments confirmed the involvement of CD in the developmental processes leading to these phenotypic alterations. Our findings add to the list of CD functions in organ development and patho-physiology in vertebrates

The methylation status of the embryonic limb skeletal progenitors determines their cell fate in chicken

Digits shape is sculpted by interdigital programmed cell death during limb development. Here, we show that DNA breakage in the periphery of 5-methylcytosine nuclei foci of interdigital precursors precedes cell death. These cells showed higher genome instability than the digit-forming precursors when exposed to X-ray irradiation or local bone morphogenetic protein (BMP) treatments. Regional but not global DNA methylation differences were found between both progenitors. DNA-Methyl-Transferases (DNMTs) including DNMT1, DNMT3B and, to a lesser extent, DNMT3A, exhibited well-defined expression patterns in regions destined to degenerate, as the interdigital tissue and the prospective joint regions. Dnmt3b functional experiments revealed an inverse regulation of cell death and cartilage differentiation, by transcriptional regulation of key genes including Sox9, Scleraxis, p21 and Bak1, via differential methylation of CpG islands across their promoters. Our findings point to a regulation of cell death versus chondrogenesis of limb skeletal precursors based on epigenetic mechanisms.We thank Prof. Miguel Lafarga for helpful comments and advice. We thank Dr Jose E Gomez-Arozamena for helping us with the irradiation experiments. We are grateful to Montse Fernandez Calderon, Susana Dawalibi, and Sonia Perez Mantecon, for excellent technical assistance. This work was supported by a Grant (BFU2017–84046-P) from the Spanish Science and Innovation Ministry to JAM. C.S.F is recipient of a FPI grant (BES-2015–074267)

Distinct Merkel Cell Polyomavirus Molecular Features in Tumour and Non Tumour Specimens from Patients with Merkel Cell Carcinoma

Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083). Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease