Jasenka Guduric-Fuchs

Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities

BackgroundThe term endothelial progenitor cells (EPCs) is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs) and outgrowth endothelial cells (OECs).MethodsHuman blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types.ResultsOur data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN) with links to immunity and inflammation (TLRs, CD14, HLAs), whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins) are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs.ConclusionsThis study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types

BackgroundMicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported.ResultsSmall RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146a overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cell types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p).ConclusionThe global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.

Myeloid Angiogenic Cells Act as Alternative M2Macrophages and Modulate Angiogenesis throughInterleukin-8

Endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials have shown such cell therapy to be feasible for treating ischemic disease. However, clinical outcomes have been contradictory owing to the diverse range of EPC types used. We recently characterized two EPC subtypes, and identified outgrowth endothelial cells as the only EPC type with true progenitor and endothelial characteristics. By contrast, myeloid angiogenic cells (MACs) were shown to be monocytic cells without endothelial characteristics despite being widely described as “EPCs.” In the current study we demonstrated that although MACs do not become endothelial cells or directly incorporate into a microvascular network, they can significantly induce endothelial tube formation in vitro and vascular repair in vivo. MAC-derived interleukin-8 (IL-8) was identified as a key paracrine factor, and blockade of IL-8 but not vascular endothelial growth factor (VEGF) prevented MAC-induced angiogenesis. Extracellular IL-8 transactivates VEGFR2 and induces phosphorylation of extracellular signal-regulated kinases. Further transcriptomic and immunopheno-typic analysis indicates that MACs represent alternative activated M2 macrophages. Our findings demonstrate an unequivocal role for MACs in angiogenesis, which is linked to paracrine release of cytokines such as IL-8. We also show, for the first time, the true identity of these cells as alternative M2 macrophages with proangiogenic, antiinflammatory and pro-tissue-repair properties.

Prediction of microRNAs affecting mRNA expression during retinal development

BackgroundMicroRNAs (miRNAs) are small RNA molecules (~22 nucleotides) which have been shown to play an important role both in development and in maintenance of adult tissue. Conditional inactivation of miRNAs in the eye causes loss of visual function and progressive retinal degeneration. In addition to inhibiting translation, miRNAs can mediate degradation of targeted mRNAs. We have previously shown that candidate miRNAs affecting transcript levels in a tissue can be deduced from mRNA microarray expression profiles. The purpose of this study was to predict miRNAs which affect mRNA levels in developing and adult retinal tissue and to confirm their expression.ResultsMicroarray expression data from ciliary epithelial retinal stem cells (CE-RSCs), developing and adult mouse retina were generated or downloaded from public repositories. Analysis of gene expression profiles detected the effects of multiple miRNAs in CE-RSCs and retina. The expression of 20 selected miRNAs was confirmed by RT-PCR and the cellular distribution of representative candidates analyzed by in situ hybridization. The expression levels of miRNAs correlated with the significance of their predicted effects upon mRNA expression. Highly expressed miRNAs included miR-124, miR-125a, miR-125b, miR-204 and miR-9. Over-expression of three miRNAs with significant predicted effects upon global mRNA levels resulted in a decrease in mRNA expression of five out of six individual predicted target genes assayed.ConclusionsThis study has detected the effect of miRNAs upon mRNA expression in immature and adult retinal tissue and cells. The validity of these observations is supported by the experimental confirmation of candidate miRNA expression and the regulation of predicted target genes following miRNA over-expression. Identified miRNAs are likely to be important in retinal development and function. Misregulation of these miRNAs might contribute to retinal degeneration and disease. Conversely, manipulation of their expression could potentially be used as a therapeutic tool in the future.

Deep sequencing reveals predominant expression of miR‐21 amongst the small non‐coding RNAs in retinal microvascular endothelial cells

The retinal vascular endothelium is essential for angiogenesis and is involved in maintaining barrier selectivity and vascular tone. The aim of this study was to identify and quantify microRNAs and other small regulatory non‐coding RNAs (ncRNAs) which may regulate these crucial functions. Primary bovine retinal microvascular endothelial cells (RMECs) provide a well‐characterized in vitro system for studying angiogenesis. RNA extracted from RMECs was used to prepare a small RNA library for deep sequencing (Illumina Genome Analyzer). A total of 6.8 million reads were mapped to 250 known microRNAs in miRBase (release 16). In many cases, the most frequent isomiR differed from the sequence reported in miRBase. In addition, five novel microRNAs, 13 novel bovine orthologs of known human microRNAs and multiple new members of the miR‐2284/2285 family were detected. Several ∼30 nucleotide sno‐miRNAs were identified, with the most highly expressed being derived from snoRNA U78. Highly expressed microRNAs previously associated with endothelial cells included miR‐126 and miR‐378, but the most highly expressed was miR‐21, comprising more than one‐third of all mapped reads. Inhibition of miR‐21 with an LNA inhibitor significantly reduced proliferation, migration, and tube‐forming capacity of RMECs. The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs and other small RNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in RMECs. Knockdown of miR‐21 suggests a role for this microRNA in regulation of angiogenesis in the retinal microvasculature. J. Cell. Biochem. 113: 2098–2111, 2012.

Small RNAs from plants, bacteria and fungi within the order Hypocreales are ubiquitous in human plasma

BackgroundThe human microbiome plays a significant role in maintaining normal physiology. Changes in its composition have been associated with bowel disease, metabolic disorders and atherosclerosis. Sequences of microbial origin have been observed within small RNA sequencing data obtained from blood samples. The aim of this study was to characterise the microbiome from which these sequences are derived.ResultsAbundant non-human small RNA sequences were identified in plasma and plasma exosomal samples. Assembly of these short sequences into longer contigs was the pivotal novel step in ascertaining their origin by BLAST searches. Most reads mapped to rRNA sequences. The taxonomic profiles of the microbes detected were very consistent between individuals but distinct from microbiomes reported at other sites. The majority of bacterial reads were from the phylum Proteobacteria, whilst for 5 of 6 individuals over 90% of the more abundant fungal reads were from the phylum Ascomycota; of these over 90% were from the order Hypocreales. Many contigs were from plants, presumably of dietary origin. In addition, extremely abundant small RNAs derived from human Y RNAs were detected.ConclusionsA characteristic profile of a subset of the human microbiome can be obtained by sequencing small RNAs present in the blood. The source and functions of these molecules remain to be determined, but the specific profiles are likely to reflect health status. The potential to provide biomarkers of diet and for the diagnosis and prognosis of human disease is immense.

Mutational Analysis of MIR184 in Sporadic Keratoconus and Myopia

PURPOSE A mutation miR-184(+57C>T) in the seed region of miR-184 (encoded by MIR184 [MIM*613146]) results in familial severe keratoconus combined with early-onset anterior polar cataract and endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning (EDICT) syndrome (MIM#614303). In order to investigate the phenotypic spectrum resulting from MIR184 mutation, MIR184 was sequenced in a keratoconus cohort of mixed ethnicity and a Chinese axial myopia cohort. METHODS Sequencing of MIR184 was performed in 780 unrelated keratoconus patients and 96 unrelated Han southern Chinese subjects with axial myopia. Effects of identified mutations on RNA secondary structure were predicted computationally using mFold and RNAFold algorithms. MIR184 amplicons from patients harboring mutations were cloned and transfected into human embryonic kidney 293T (HEK293T) cells, and mature mutant miR-184 expression was analyzed by stem-loop real-time quantitative PCR (RT-qPCR). RESULTS Two novel heterozygous substitution mutations in MIR184 were identified in the two patients with isolated keratoconus: miR-184(+8C>A) and miR-184(+3A>G). Computational modeling predicted that these mutations would alter the miR-184 stem-loop stability and secondary structure. Ex vivo miR-184 expression analysis demonstrated that miR-184(+8C>A) almost completely repressed the expression of miR-184 (P = 0.022), and miR-184(+3A>G) reduced the expression of miR-184 by approximately 40% (P = 0.002). There was no significant association of rs41280052, which lies within the stem-loop of miR-184, with keratoconus. No MIR184 mutations were detected in the axial myopia cohort. CONCLUSIONS Two novel heterozygous substitution mutations in MIR184 were identified in two patients with isolated keratoconus: miR-184(+8C>A) and miR-184(+3A>G). Mutations in MIR184 are a rare cause of keratoconus and were found in 2 of 780 (0.25%) cases.

Ex Vivo Expansion of Human Outgrowth Endothelial Cells Leads to IL-8-Mediated Replicative Senescence and Impaired Vasoreparative Function

Harnessing outgrowth endothelial cells (OECs) for vasoreparative therapy and tissue engineering requires efficient ex vivo expansion. How such expansion impacts on OEC function is largely unknown. In this study, we show that OECs become permanently cell‐cycle arrested after ex vivo expansion, which is associated with enlarged cell size, β‐galactosidase activity, DNA damage, tumor suppressor pathway activation, and significant transcriptome changes. These senescence hallmarks were coupled with low telomerase activity and telomere shortening, indicating replicative senescence. OEC senescence limited their regenerative potential by impairing vasoreparative properties in vitro and in vivo. Integrated transcriptome‐proteome analysis identified inflammatory signaling pathways as major mechanistic components of the OEC senescence program. In particular, IL8 was an important facilitator of this senescence; depletion of IL8 in OECs significantly extended ex vivo lifespan, delayed replicative senescence, and enhanced function. While the ability to expand OEC numbers prior to autologous or allogeneic therapy remains a useful property, their replicative senescence and associated impairment of vasorepair needs to be considered. This study also suggests that modulation of the senescence‐associated secretory phenotype could be used to optimize OEC therapy. STEM Cells 2013;31:1657–1668

Distinctive profile of IsomiR expression and novel microRNAs in rat heart left ventricle.

MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486.

Cochlin in the eye: functional implications

Aqueous humor is actively produced in the ciliary epithelium of the anterior chamber and has important functions for the eye. Under normal physiological conditions, the inflow and outflow of the aqueous humor are tightly regulated, but in the pathologic state this balance is lost. Aqueous outflow involves structures of the anterior chamber and experiences most resistance at the level of the trabecular meshwork (TM) that acts as a filter. The modulation of the TM structure regulates the filter and its mechanism remains poorly understood. Proteomic analyses have identified cochlin, a protein of poorly understood function, in the glaucomatous TM but not in healthy control TM from human cadaver eyes. The presence of cochlin has subsequently been confirmed by Western and immunohistochemical analyses. Functionally, cochlin undergoes multimerization induced by shear stress and other changes in the microenvironment. Cochlin along with mucopolysaccharide deposits has been found in the TM of glaucoma patients and in the inner ear of subjects affected by the hearing disorder DNFA9, a late-onset, progressive disease that also involves alterations in fluid shear regimes. In vitro, cochlin induces aggregation of primary TM cells suggesting a role in cell adhesion, possibly in mechanosensation, and in modulation of the TM filter.