Die Bedeutung von microRNAs für die Funktion von Endothelzellen

Dicer and Drosha are the major enzymes involved in microRNA processing. Using siRNA targeting Dicer and Drosha, thereby downregulating a substantial number of microRNAs in EC, we demonstrate a crucial role of both enzymes in angiogenic processes. Interestingly, Dicer inhibition exerts more profound effects on processes like migration and viability of EC in comparison to Drosha inhibition. Moreover, Dicer effects in vivo angiogenesis, a process which is unaffected by Drosha. This discrepancy might be partially due to the involvement of Dicer in other cellular processes like heterochromatin formation and to the fact that Dicer and Drosha target mainly different subsets of microRNAs. In addition, we identified miR-92a as a novel endogenous repressor of the angiogenic program in EC, which impairs their angiogenic functions in vitro and in vivo. Consistent with these data, blocking miR-92a by systemic infusion of antagomirs enhances neovascularization and functional recovery after ischemia in vivo. At first sight, the anti-angiogenic function of miR-92a in EC appears to contradict the previously identified anti-apoptotic and pro-angiogenic activities of the miR-17~92 cluster in tumor cells. However, this apparent discrepancy might be well rationalized by a predominant function of miR-18a and miR-19a in tumor cells, which are responsible for the tumorigenic and non-cell autonomous pro-angiogenic functions of the miR-17~92 cluster. Instead, miR-92a expression is specifically upregulated in ischemic tissues and appears to cell-autonomously repress the angiogenic potential of EC. Among the various targets and verified regulated genes identified by microarray, we confirmed the downregulation of Integrin a5 in vitro and in vivo. The relevance of this miR-92a target is evidenced by severe vascular defects in the absence of Integrin a5. In addition, endothelial miR-92a interferes with the expression pattern of genes controlling key EC functions at various levels, some of which, e.g. eNOS, might be secondarily affected by directly targeted genes. Obviously, our data do not formally exclude effects of antagomir-92a on perivascular and other cell types, but surely include effects on EC. Regardless of this, the capacity of miR-92a to target various downstream effectors might be an advantage of miRNA-based therapeutic strategies and may overcome the limited therapeutic capacity of single growth factor or single gene therapies in ischemic diseases, since the highly organized process of vessel growth, maturation and functional maintenance is well known to require the fine-tuned regulation of a set of genes.Angiogenese, die Bildung von Kapillaren aus bereits existierenden Gefäßen, ist ein wichtiger physiologischer Prozess zur Wundheilung und Wiederherstellung des Blutflusses nach Verletzung. Das Ziel der vorliegenden Arbeit war die genaue Untersuchung der Rolle von microRNAs in angiogenen Prozessen. MicroRNAs sind kleine, einzelsträngige RNA Moleküle, welche die Genexpression durch Bindung an die messenger RNA (mRNA) von Zielgenen und darauffolgenden Degradierung der mRNA oder Repression der Translation regulieren. Die Inhibition der microRNA-prozessierenden Enzyme Dicer und Drosha in Endothelzellen führt zu einer Dysregulation der microRNA Expression und zu einer signifikanten Reduktion der Angiogenese in vitro. Im Gegensatz zu Drosha, dessen Inhibition keinen Effekt auf die in vivo Angiogenese zeigt, führt die Inhibition von Dicer auch in einem in vivo Angiogenese-Modell zu einer deutliche reduzierten Einsprossung von Gefäßen. Zusammenfassend sprechen diese Daten für eine wichtige Rolle von microRNAs in der Endothelzellbiologie. Der zweite Teil der vorliegenden Arbeit beschränkte sich auf die Untersuchung einer, in Endothelzellen hoch exprimierten microRNA, miR-92a. MiR-92a ist ein Mitglied des miR-17-92 Clusters, für den bereits eine Rolle in der Tumorangiogenese beschrieben ist. Die Überexpression der miR-92a in Endothelzellen führt zu einer signifikanten Hemmung der Angiogenese, sowie reduzierter Adhäsion und Migration auf Fibronektin. Zudem wird die Einsprossung von Gefäßen in Matrigel Plugs deutlich gehemmt. In Übereinstimmung hierzu führt die systemische Hemmung der miR-92a mit Hilfe von modifizierten antisense Oligoribonukleotiden (Antagomir-92a) im Mausmodell zu einer Stimulation der Einsprossung von Gefäßen in Matrigel Plugs. In klinisch relevanten Modellen, wie dem Hinterlaufischämie-Modell und dem akuten Myokardinfarkt-Modell, führt die Behandlung mit Antagomir-92a zu einer funktionelle Verbesserung. Immunohistologische Analysen ergaben, dass in den Antagomir-92a behandelten Tieren sowohl die Anzahl der Kapillaren als auch der größeren Gefäße deutlich erhöht ist. Obwohl wir einen direkten Effekt der Antagomir-92a Behandlung auf die Myozyten zum jetzigen Zeitpunkt nicht auschließen können, weisen unsere Daten definitiv auf eine Stimulation der Angiogenese hin. Nachdem wir sowohl in vitro als auch in vivo zeigen konnten, dass miR-92a maßgeblich an angiogenen Prozessen beteiligt ist, stellten wir uns die Frage nach den zugrunde liegenden Zielgenen. Es zeigte sich, dass miR-92a neben einer größeren Anzahl pro-angiogener Faktoren die Integrin Untereinheit a5 sowohl in vitro als auch in vivo reguliert. Desweiteren konnten wir mit Hilfe eines Luciferase Assays die direkte Regulation von Integrin a5 durch miR-92a zeigen. Zusammenfassend deuten diese Daten daraufhin, dass Integrin a5 ein Schlüsselregulator der Antagomir-92a-vermittelten Angiogenese ist und somit unmittelbar an der Verbesserung nach Hinterlaufischämie und akutem Myokardinfarkt beteiligt ist

Design und Synthese von Peptidmimetikabausteinen zur molekularen Erkennung von beta-Faltblattstrukturen

Die Erkennung exponierter beta-Faltblattstrukturen spielt eine wichtige Rolle in Protein-Protein-Wechselwirkungen. Ein fehlendes oder auch ungewolltes Auftreten dieser beta-Faltblatt-Struktur kann verheerende Konsequenzen nach sich ziehen. Das Peptid-Rückgrat von beta-Faltblatt-Strukturen besitzt in regelmäßigen Abständen einen Abschnitt mit je einem Wasserstoffbrücken-Donor und -Akzeptor in der Reihenfolge Donor-Akzeptor-Donor-Akzeptor-usw. In Protein-Protein-Wechselwirkungen dienen diese Wasserstoffbrückenfunktionen zur Bindung zweier beta-Faltblätter. Synthetische Moleküle mit komplementären Wasserstoffbrückenfunktionen und komplementärer Struktur zum beta-Faltblatt versprechen somit eine Inhibitorfunktion für Protein-Protein-Wechselwirkungen oder eine Stabilisierungsfunktion der beta-Faltblatt-Struktur. Diese Arbeit leistet hierzu einen Beitrag durch die Entwicklung und Untersuchung des Bindungsverhaltens eines Dipeptid-Mimetikums. Im ersten Teil dieser Arbeit wurde die Synthese der Methoxypyrrolaminosäure (MOPAS) als Dipeptid-Mimetikum entwickelt und mit der Synthese der Pyrrolaminosäure (PAS) verglichen. Dabei spielt die zusätzliche Alkoxygruppe eine herausragende Rolle bei der Vororientierung im Oligomer des Bausteins. Diese Oligomerisierung wurde im nächsten Abschnitt sowohl an der festen Phase als auch in Lösung untersucht, wobei eine Optimierung der Kupplung des MOPAS-Bausteins in Lösung durch Verwendung des Hydroxybenzotriazol- (OBt-) Aktivesters erreicht werden konnte, mit Kupplungsausbeuten, wie sie in der Standard-Peptid-Kupplung üblich sind. Die anschließenden Bindungsstudien an kurze Di- und Tetrapeptide zeigen, neben der für beta-Faltblatt-bildenden Strukturen üblichen Selbstaggregation, eine vergleichbare Bindungsaffinität an diese Peptide. Dabei ergeben Untersuchungen zur Stöchiometrie eine hohe Wahrscheinlichkeit der erwartenden Bindung zweier MOPAS-Rezeptor-Moleküle an je eine Seite der Peptide bei Bindungskonstanten von bis zu 100 l/mol. Die De-Aggregation eines unlöslichen Proteins durch das Dimer des MOPAS-Bausteins zeigt die mögliche Anwendung bei der De-Aggregation von Amyloid-Plaque. Um bessere Aussagen über die Struktur von Peptiden im gebundenen Zustand treffen zu können, wurde der Rezeptor kovalent mit dem Peptid verknüpft. Dadurch wurde das Wasserstoffbrückenbindungsereignis intramolekular. Beta-Haarnadelschleifen aus D Prolin und Glycin sind dafür ideal und bringen einen Arm mit MOPAS-Einheiten und den anderen peptidischen Arm zusammen, sodass sich Wechselwirkungen ausbilden können. Die Untersuchung der Peptidstruktur ergibt dann die erwartete, induzierte beta-Strang-Konformation im Peptid-Arm. In einem weiteren Abschnitt wurde dann diese kovalente Form der Verknüpfung des Rezeptors und des Peptids durch eine koordinative Bindung ersetzt. So konnte in Zusammenarbeit mit Michael Kruppa ein Rezeptor mit Histidin-bindender Stelle und beta-Faltblatt-bindener Stelle in Form zweier MOPAS-Einheiten synthetisiert werden. Wird an diesen Rezeptor ein Pentapeptid mit endständigem Histidin gebunden, kann dann selbst in einem stark kompetetiven Lösungsmittel wie DMSO die Wechselwirkung zwischen Peptid und Rezeptor gezeigt werden. Auch hier wird die Geometrie des Peptids in der Art verändert, dass sich die Konformation eines beta-Strangs ausbildet. In den letzten beiden Abschnitten konnte der MOPAS-Baustein so verändert werden, dass er den chiralen proteinogenen Aminosäuren noch ähnlicher wird. Am Aminomethyl-Arm war es möglich, eine Isopropylgruppe sowohl mit hohen Ausbeuten als auch mit einer Enantioselektivität >96 % einzuführen. Die Einführung eines Restes am Pyrrolring und somit Bildung eines quartären Kohlenstoffs konnte am MOPAS-Baustein gezeigt werden, wodurch der Weg zur stereoselektiven Synthese damit offen steht

MicroRNA regulation of endothelial homeostasis and commitment—implications for vascular regeneration strategies using stem cell therapies

Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration

Synthesis of new n-ethyl dehydroamino acid derivatives : n-ethyl β,β-dibromo, n-ethyl β-bromo β-substituted, n-ethyl β,β-disubstituted n-protected dehydroamino acid methyl esters

Recently we reported the use of a sequence of alkylation and dehydration methodologies to obtain from the methyl esters of N-(4-nitrophenylsulfonyl)-β-hydroxyamino acids, new non-proteinogenic amino acids, namely, N-ethyl-α,β-dehydroamino acids. Thus, it was possible to obtain for the first time, non-natural amino acids which incorporate both the N-ethyl and α,β-dehydro moieties. Herein, we report the application of this N-alkylation procedure to several methyl esters of β,β-dibromo and β-bromo, β-substituted dehydroamino acids protected with standard amine protecting groups such as tert-butyloxycarbonyl, benzyloxycarbonyl and 4-nitrobenzyloxycarbonyl as well as acyl and sulfonyl groups. The procedure allows the synthesis of the methyl esters of N-protected, N-ethyl, β,β-dibromo and N-ethyl, β-bromo, β-substituted dehydroamino acids in fair to high yields. Some of these N-ethylated dehydroamino acid derivatives were used as substrates in cross coupling reactions to give N-ethyl, β,β-disubstituted dehydroalanine derivatives.The Portuguese Foundation for Science and Technology (FCT) and the Fundo Europeu de Desenvolvimento Regional (FEDER) are thanked for financial support to the Chemistry Centre of University of Minho. The Bruker Avance II+ 400 NMR spectrometer is part of the National NMR Network and was purchased in the framework of the National Program for Scientific Re-equipment, REDE/1517/RMN/2005, with funds from POCI 2010, FEDER and FCT

Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease

Downregulation of miR-92a Is Associated with Aggressive Breast Cancer Features and Increased Tumour Macrophage Infiltration

BACKGROUND: MicroRNAs are small non-coding RNAs involved in the regulation of gene expression on a posttranscriptional level. These regulatory RNAs have been implicated in numerous cellular processes and are further deregulated in different cancer types, including breast cancer. MiR-92a is part of the miR-17∼92 cluster, which was first reported to be linked to tumourigenesis. However, little is known about the expression of miR-92a in breast cancer and potential associations to tumour properties. The expression of miR-92a was therefore characterized in 144 invasive breast cancer samples using in situ hybridization and related to clinico-pathological data as well as to selected key properties of the tumour stroma, including the presence of macrophages (CD68) and cancer activated fibroblasts (alpha-SMA). METHODOLOGY/PRINCIPAL FINDINGS: To measure miR-92a levels, an in situ hybridisation protocol was developed and validated using cell lines and miR-92a inhibitors. The expression in the tumour samples was objectively evaluated using digital image analysis program subtracting background activities. We found that the miR-92a expression varied between tumours and was inversely correlated to tumour grade (r = -0.276, p = 0.003) and recurrence-free survival (p = 0.008) and provided independent prognostic information in multivariate Cox analysis (HR: 0.375, CI: 0.145-0.972, p = 0.043). MiR-92a was moreover inversely correlated to the number of infiltrating macrophages in the tumour stroma (r = -0.357, p<0.001), and downregulation of miR-92a promoted cell migration (p<0.01). CONCLUSIONS/SIGNIFICANCE: This study demonstrates that downregulation of miR-92a in breast cancer is linked to key epithelial and stromal properties as well as clinical outcome

E,Z-Stereodivergent synthesis of N-tosyl α,β-dehydroamino esters via a Mukaiyama-Michael addition

The stereodivergent synthesis of N-tosyl α,β-dehydroamino esters via a Mukaiyama-Michael addition is reported. The reaction of silylketene acetals with N-tosylimines derived from β,γ-unsaturated α-keto esters in dichloromethane provided the corresponding (Z)-α,β-dehydroamino esters while the (E)-isomers were obtained when the reaction was carried out in the presence of 10 mol% copper(II) triflate

MicroRNAs in vascular tissue engineering and post-ischemic neovascularization

Increasing numbers of paediatric patients with congenital heart defects are surviving to adulthood, albeit with continuing clinical needs. Hence, there is still scope for revolutionary new strategies to correct vascular anatomical defects. Adult patients are also surviving longer with the adverse consequences of ischemic vascular disease, especially after acute coronary syndromes brought on by plaque erosion and rupture. Vascular tissue engineering and therapeutic angiogenesis provide new hope for these patients. Both approaches have shown promise in laboratory studies, but have not yet been able to deliver clear evidence of clinical success. More research into biomaterials, molecular medicine and cell and molecular therapies is necessary. This review article focuses on the new opportunities offered by targeting microRNAs for the improved production and greater empowerment of vascular cells for use in vascular tissue engineering or for increasing blood perfusion of ischemic tissues by amplifying the resident microvascular network

MicroRNA-296 is enriched in cancer cells and downregulates p21WAF1 mRNA expression via interaction with its 3′ untranslated region

MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21WAF1 (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3′UTR, and the Hu binding site of p21-3′UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21WAF1 pathway

Decrease of miR-146b-5p in Monocytes during Obesity Is Associated with Loss of the Anti-Inflammatory but Not Insulin Signaling Action of Adiponectin

Background: Low adiponectin, a well-recognized antidiabetic adipokine, has been associated with obesity-related inflammation, oxidative stress and insulin resistance. Globular adiponectin is an important regulator of the interleukin-1 receptor-associated kinase (IRAK)/NFkB pathway in monocytes of obese subjects. It protects against inflammation and oxidative stress by inducing IRAK3. microRNA (miR)-146b-5p inhibits NFkB-mediated inflammation by targeted repression of IRAK1 and TNF receptor-associated factor-6 (TRAF6). Therefore, we measured the expression of miR-146b-5p in monocytes of obese subjects. Because it was low we determined the involvement of this miR in the anti-inflammatory, antioxidative and insulin signaling action of globular adiponectin. Methods: miR-146b-5p expression in monocytes of obese subjects was determined by qRT-PCR. The effect of miR-146b-5p silencing on molecular markers of inflammation, oxidative stress and insulin signaling and the association with globular adiponectin was assessed in human THP-1 monocytes. Results: miR-146b-5p was downregulated in monocytes of obese persons. Low globular adiponectin decreased miR-146b-5p and IRAK3 in THP-1 monocytes, associated with increased mitochondrial reactive oxygen species (ROS). Intracellular ROS and insulin receptor substrate-1 (IRS1) protein were unchanged. Silencing of miR-146b-5p with an antisense inhibitor resulted in increased expression of IRAK1 and TRAF6 leading to more NFkB p65 DNA binding activity and TNFa. As