Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in ...this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood.
Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival.
Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease.
Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires ...multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.
MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial ...microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5′ phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH.
Regulation of microRNA (miRNA) expression and function in the context of activity‐dependent synaptic plasticity in the adult brain is little understood. Here, we examined miRNA expression during ...long‐term potentiation (LTP) in the dentate gyrus of adult anesthetized rats. Microarray expression profiling identified a subpopulation of regulated mature miRNAs 2 h after the induction of LTP by high‐frequency stimulation (HFS) of the medial perforant pathway. Real‐time polymerase chain reaction analysis confirmed modest upregulation of miR‐132 and miR‐212, and downregulation of miR‐219, while no changes occurred at 10 min post‐HFS. Surprisingly, pharmacological blockade of N‐methyl‐d‐aspartate receptor (NMDAR)‐dependent LTP enhanced expression of these mature miRNAs. This HFS‐evoked expression was abolished by local infusion of the group 1 metabotropic glutamate receptor (mGluR) antagonist, (RS)‐1‐aminoindan‐1,5‐dicarboxylic acid (AIDA). AIDA had no effect on LTP induction or maintenance, but blocked activity‐dependent depotentiation of LTP. Turning to the analysis of miRNA precursors, we show that HFS elicits 50‐fold elevations of primary (pri) and precursor (pre) miR‐132/212 that is transcription dependent and mGluR dependent, but insensitive to NMDAR blockade. Primary miR‐219 expression was unchanged during LTP. In situ hybridization showed upregulation of the pri‐miR‐132/212 cluster restricted to dentate granule cell somata. Thus, HFS induces transcription miR‐132/212 that is mGluR dependent and functionally correlated with depotentiation rather than LTP. In contrast, NMDAR activation selectively downregulates mature miR‐132, ‐212 and ‐219 levels, indicating accelerated decay of these mature miRNAs. This study demonstrates differential regulation of primary and mature miRNA expression by mGluR and NMDAR signaling following LTP induction, the function of which remains to be defined.
The characterization of post-transcriptional gene regulation by small regulatory (20–30 nt) RNAs, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite ...for characterizing small RNAs is their identification and quantification across different developmental stages, and in normal and disease tissues, as well as model cell lines. Here we present a step-by-step protocol for generating barcoded small RNA cDNA libraries compatible with Illumina HiSeq sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections.
Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to ...understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut; and the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine
Submitted 29 January 2006
; accepted in ...final form 8 May 2006
Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 ± 66 to 2,604 ± 286 µeq·h 1 ·g 1 ; the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH 2 terminus with seven cysteine residues, and has three N -glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 ± 2 to 127 ± 34 µS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.
Squalus acanthias ; molecular cloning; Xenopus oocytes
Address for reprint requests and other correspondence: J. N. Forrest, Jr., Dept. of Internal Medicine, Yale Univ. School of Medicine, New Haven, Connecticut 06510 (e-mail: john.forrest{at}yale.edu )
MicroRNAs (miRNAs) are small regulatory RNAs with many biological functions and disease associations. We showed that
in situ
hybridization (ISH) using conventional formaldehyde fixation results in ...significant miRNA loss from mouse tissue sections, which can be prevented by fixation with 1–ethyl–3–(3–dimethylaminopropyl) carbodiimide (EDC) that irreversibly immobilizes the miRNA at its 5' phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid (LNA) probes by recording nucleic acid melting temperature during ISH.
Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of ...the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 ± 66 to 2,604 ± 286 µeq*h^sup -1^*g^sup -1^; the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-Ri. It maintains a long, extracellular NH^sub 2^ terminus with seven cysteine residues, and has three N- glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 ± 2 to 127 ± 34 µS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R. PUBLICATION ABSTRACT
Summary
The purpose of this registry study was to provide an overview of trends and results of liver transplantation (LT) in Europe from 1968 to 2016. These data on LT were collected prospectively ...from 169 centers from 32 countries, in the European Liver Transplant Registry (ELTR) beginning in 1968. This overview provides epidemiological data, as well as information on evolution of techniques, and outcomes in LT in Europe over more than five decades; something that cannot be obtained from only a single center experience.
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