Interaction between AIF and CHCHD4 Regulates Respiratory Chain Biogenesis.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.

Global MicroRNA Profiling Uncovers miR-206 as a Negative Regulator of Hematopoietic Commitment in Human Pluripotent Stem Cells.

Although human pluripotent stem cells (hPSCs) can theoretically differentiate into any cell type, their ability to produce hematopoietic cells is highly variable from one cell line to another. The underlying mechanisms of this heterogeneity are not clearly understood. Here, using a whole miRNome analysis approach in hPSCs, we discovered that their hematopoietic competency was associated with the expression of several miRNAs and conversely correlated to that of miR-206 specifically. Lentiviral-based miR-206 ectopic expression in H1 hematopoietic competent embryonic stem (ES) cells markedly impaired their differentiation toward the blood lineage. Integrative bioinformatics identified a potential miR-206 target gene network which included hematopoietic master regulators RUNX1 and TAL1. This work sheds light on the critical role of miR-206 in the generation of blood cells off hPSCs. Our results pave the way for future genetic manipulation of hPSCs aimed at increasing their blood regenerative potential and designing better protocols for the generation of bona fide hPSC-derived hematopoietic stem cells.

Direct and rapid identification of T315I-Mutated BCR-ABL expressing leukemic cells using infrared microspectroscopy.

Despite the major success obtained by the use of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), resistances to therapies occur due to mutations in the ABL-kinase domain of the BCR-ABL oncogene. Amongst these mutations, the "gatekeeper" T315I is a major concern as it renders leukemic cells resistant to all licenced TKI except Ponatinib. We report here that Fourier transform infrared (FTIR) microspectroscopy is a powerful methodology allowing rapid and direct identification of a spectral signature in single cells expressing T315I-mutated BCR-ABL. The specificity of this spectral signature is confirmed using a Dox-inducible T315I-mutated BCR-ABL-expressing human UT-7 cells as well as in murine embryonic stem cells. Transcriptome analysis of UT-7 cells expressing BCR-ABL as compared to BCR-ABL T315I clearly identified a molecular signature which could be at the origin of the generation of metabolic changes giving rise to the spectral signature. Thus, these results suggest that this new methodology can be applied to the identification of leukemic cells harbouring the T315I mutation at the single cell level and could represent a novel early detection tool of mutant clones. It could also be applied to drug screening strategies to target T315I-mutated leukemic cells.

microRNA blood signature for localized radiation injury.

A radiological accident, whether from industrial, medical, or malicious origin, may result in localized exposure to high doses of ionizing radiations, leading to the development of local radiation injury (LRI), that may evolve toward deep ulceration and necrosis of the skin and underlying tissues. Early diagnosis is therefore crucial to facilitate identification and management of LRI victims. Circulating microRNAs (miRNA) have been studied as potential diagnostic biomarkers of several diseases including hematological defects following whole-body irradiation (WBI). This study aims to identify a blood miRNA signature associated with LRI in a preclinical C57BL/6J mouse model of hindlimb irradiation using different 10-MV X-ray doses that lead to injuries of different severities. To this end, we first performed broad-spectrum plasma miRNA profiling, followed by a targeted validation step, on two independent animal cohorts. Using a multivariate sparse partial least square discriminant analysis, we identified a panel of eight circulating miRNAs able to segregate mice according to LRI severity. Interestingly, these miRNAs were previously associated with WBI (miR-150-5p, miR-342-3p, miR-146a-5p), inflammation (miR-18a-5p, miR-148b-3p, miR-532-5p) and skin diseases (miR-139-5p, miR-195-5p). Our results suggest the use of circulating miRNAs as suitable molecular biomarkers for LRI prognosis and diagnosis.

MicroRNA target prediction and validation.

The accurate prediction and validation of microRNA targets is essential to understanding the function of microRNAs. Computational predictions indicate that all human genes may be regulated by microRNAs, with each microRNA possibly targeting thousands of genes. Here we discuss computational and experimental methods for identifying mammalian microRNA targets. We describe microRNA target prediction resources and procedures that are suitable for experiments where more accurate prediction of microRNA targets is more important than detecting all putative targets. We then discuss experimental methods for identifying and validating microRNA target genes, with an emphasis on the target reporter assay as the method of choice for specifically testing functional microRNA target sites.

Inflammatory cells dynamics control neovascularization and tissue healing after localized radiation induced injury in mice.

Local overexposure to ionizing radiation leads to chronic inflammation, vascular damage and cachexia. Here we investigate the kinetics of inflammatory cells from day (D)1 to D180 after mouse hindlimb irradiation and analyze the role of monocyte (Mo) subsets in tissue revascularization. At D1, we find that Mo and T cells are mobilized from spleen and bone marrow to the blood. New vessel formation during early phase, as demonstrated by ~1.4- and 2-fold increased angiographic score and capillary density, respectively, correlates with an increase of circulating T cells, and Mohi and type 1-like macrophages in irradiated muscle. At D90 vascular rarefaction and cachexia are observed, associated with decreased numbers of circulating Molo and Type 2-like macrophages in irradiated tissue. Moreover, CCR2- and CX3CR1-deficency negatively influences neovascularization. However adoptive transfer of Mohi enhances vessel growth. Our data demonstrate the radiation-induced dynamic inflammatory waves and the major role of inflammatory cells in neovascularization.

HuMSC-EV induce monocyte/macrophage mobilization to orchestrate neovascularization in wound healing process following radiation injury.

This study aims to investigate the mechanisms of human mesenchymal stem cell-derived extracellular vesicles (HuMSC-EV)-induced proangiogenic paracrine effects after radiation injury. HuMSC-EV were locally administered in mice hindlimb following 80-Gy X-ray irradiation and animals were monitored at different time points. HuMSC-EV improved neovascularization of the irradiated tissue, by stimulating angiogenesis, normalizing cutaneous blood perfusion, and increasing capillary density and production of proangiogenic factors. HuMSC-EV also stimulated vasculogenesis by promoting the recruitment and differentiation of bone marrow progenitors. Moreover, HuMSC-EV improved arteriogenesis by increasing the mobilization of monocytes from the spleen and the bone marrow and their recruitment into the muscle, with a pro-inflammatory potential. Importantly, monocyte depletion by clodronate treatment abolished the proangiogenic effect of HuMSC-EV. The critical role of Ly6C(hi) monocyte subset in HuMSC-EV-induced neovascularization process was further confirmed using Ccr2-/- mice. This study demonstrates that HuMSC-derived EV enhances the neovascularization process in the irradiated tissue by increasing the production of proangiogenic factors, promoting the recruitment of vascular progenitor cells, and the mobilization of innate cells to the injured site. These results support the concept that HuMSC-EV might represent a suitable alternative to stem cells for therapeutic neovascularization in tissue repair.

Circulating microvesicles correlate with radiation proctitis complication after radiotherapy.

In a large retrospective study, we assessed the putative use of circulating microvesicles (MVs), as innovative biomarkers of radiation toxicity in a cohort of 208 patients with prostate adenocarcinoma overexposed to radiation. The level of platelet (P)-, monocyte (M)- and endothelial (E)-derived MVs were assessed by flow cytometry. Rectal bleeding toxicity scores were collected at the time of blood sampling and during the routine follow-up and were tested for association with MVs using a multivariate logistic regression. MVs dosimetric correlation was investigated using dose volume histograms information available for a subset of 36 patients. The number of PMVs was significantly increased in patients with highest toxicity grades compared to lower grades. Risk prediction analysis revealed that increased numbers of PMVs, and an increased amount of MMVs relative to EMVs, were associated with worst rectal bleeding grade compared to the time of blood sampling. Moreover, a significant correlation was found between PMV and MMV numbers, with the range of doses up to the median exposure (40 Gy) of bladder/rectum and anterior rectal wall, respectively. MVs could be considered as new biomarkers to improve the identification of patients with high toxicity grade and may be instrumental for the prognosis of radiation therapy complications.

Molecular Changes In Cardiac Tissue As A New Marker To Predict Cardiac Dysfunction Induced By Radiotherapy.

The contribution of radiotherapy, per se, to late cardiotoxicity remains controversial. To clarify its impact on the development of early cardiac dysfunction, we developed an experimental model in which the hearts of rats were exposed, in a fractionated plan, to clinically relevant doses of ionizing radiation for oncological patients that undergo thoracic radiotherapy. Rat hearts were exposed to daily doses of 0.04, 0.3, and 1.2 Gy for 23 days, achieving cumulative doses of 0.92, 6.9, and 27.6 Gy, respectively. We demonstrate that myocardial deformation, assessed by global longitudinal strain, was impaired (a relative percentage reduction of >15% from baseline) in a dose-dependent manner at 18 months. Moreover, by scanning electron microscopy, the microvascular density in the cardiac apex was significantly decreased exclusively at 27.6 Gy dosage. Before GLS impairment detection, several tools (qRT-PCR, mass spectrometry, and western blot) were used to assess molecular changes in the cardiac tissue. The number/expression of several genes, proteins, and KEGG pathways, related to inflammation, fibrosis, and cardiac muscle contraction, were differently expressed in the cardiac tissue according to the cumulative dose. Subclinical cardiac dysfunction occurs in a dose-dependent manner as detected by molecular changes in cardiac tissue, a predictor of the severity of global longitudinal strain impairment. Moreover, there was no dose threshold below which no myocardial deformation impairment was detected. Our findings i) contribute to developing new markers and exploring non-invasive magnetic resonance imaging to assess cardiac tissue changes as an early predictor of cardiac dysfunction; ii) should raise red flags, since there is no dose threshold below which no myocardial deformation impairment was detected and should be considered in radiation-based imaging and -guided therapeutic cardiac procedures; and iii) highlights the need for personalized clinical approaches.

mimiRNA: a microRNA expression profiler and classification resource designed to identify functional correlations between microRNAs and their targets.

MOTIVATION: microRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by inhibiting target mRNA genes. Their tissue- and disease-specific expression patterns have immense therapeutic and diagnostic potential. To understand these patterns, a reliable compilation of miRNA and mRNA expression data is required to compare multiple tissue types. Moreover, with the appropriate statistical tools, such a resource could be interrogated to discover functionally related miRNA-mRNA pairs. RESULTS: We have developed mimiRNA, an online resource that integrates expression data from 1483 samples and permits visualization of the expression of 635 human miRNAs across 188 different tissues or cell types. mimiRNA incorporates a novel sample classification algorithm, ExParser, that groups identical miRNA or mRNA experiments from separate sources. This enables mimiRNA to provide reliable expression profiles and to discover functional relations between miRNAs and mRNAs such as miRNA targets. Additionally, mimiRNA incorporates a decision tree algorithm to discover distinguishing miRNA features between two tissue or cell types. We validate the efficacy of our resource on independent experimental data and through biologically relevant analyses. AVAILABILITY: http://mimirna.centenary.org.au. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Micro-RNA response to imatinib mesylate in patients with chronic myeloid leukemia.

BACKGROUND: Micro-RNAs (miRNAs) control gene expression by destabilizing targeted transcripts and inhibiting their translation. Aberrant expression of miRNAs has been described in many human cancers, including chronic myeloid leukemia. Current first-line therapy for newly diagnosed chronic myeloid leukemia is imatinib mesylate, which typically produces a rapid hematologic response. However the effect of imatinib on miRNA expression in vivo has not been thoroughly examined. DESIGN AND METHODS: Using a TaqMan Low-Density Array system, we analyzed miRNA expression in blood samples from newly diagnosed chronic myeloid leukemia patients before and within the first two weeks of imatinib therapy. Quantitative real-time PCR was used to validate imatinib-modulated miRNAs in sequential primary chronic myeloid leukemia samples (n=11, plus 12 additional validation patients). Bioinformatic target gene prediction analysis was performed based on changes in miRNA expression. RESULTS: We observed increased expression of miR-150 and miR-146a, and reduced expression of miR-142-3p and miR-199b-5p (3-fold median change) after two weeks of imatinib therapy. A significant correlation (P<0.05) between the Sokal score and pre-treatment miR-142-3p levels was noted. Expression changes in the same miRNAs were consistently found in an additional cohort of chronic myeloid leukemia patients, as compared to healthy subjects. Peripheral blood cells from chronic phase and blast crisis patients displayed a 30-fold lower expression of miR-150 compared to normal samples, which is of particular interest since c-Myb, a known target of miR-150, was recently shown to be necessary for Bcr-Abl-mediated transformation. CONCLUSIONS: We found that imatinib treatment of chronic myeloid leukemia patients rapidly normalizes the characteristic miRNA expression profile, suggesting that miRNAs may serve as a novel clinically useful biomarker in this disease.

Conserved expression patterns predict microRNA targets.

microRNAs (miRNAs) are major regulators of gene expression and thereby modulate many biological processes. Computational methods have been instrumental in understanding how miRNAs bind to mRNAs to induce their repression but have proven inaccurate. Here we describe a novel method that combines expression data from human and mouse to discover conserved patterns of expression between orthologous miRNAs and mRNA genes. This method allowed us to predict thousands of putative miRNA targets. Using the luciferase reporter assay, we confirmed 4 out of 6 of our predictions. In addition, this method predicted many miRNAs that act as expression enhancers. We show that many miRNA enhancer effects are mediated through the repression of negative transcriptional regulators and that this effect could be as common as the widely reported repression activity of miRNAs. Our findings suggest that the indirect enhancement of gene expression by miRNAs could be an important component of miRNA regulation that has been widely neglected to date.

Early and Late Protective Effect of Bone Marrow Mononuclear Cell Transplantation on Radiation-Induced Vascular Dysfunction and Skin Lesions.

Skin lesions caused by accidental exposure to radiation or by radiotherapy are a major clinical challenge. We evaluated the effect of bone marrow mononuclear cells (BMMNC) on collagen remodeling and vascular function in radiation-induced skin lesions in the acute and late phases in mice. We studied the effect of BMMNC transplantation in a mouse model of cutaneous radiation injury combining local skin gamma-irradiation and biopsy punch wound. Mice were first irradiated, punched and then BMMNC were intramuscularly administered. Seven days after injury, BMMNC promoted wound healing by (i) increasing re-epithelialization, tissue collagen density and mRNA levels of collagens 1A1, 1A2, and 3A1, and (ii) inhibiting the radiation-induced vascular activation and limiting interactions between leukocytes and the vascular endothelium compared with control. Importantly, BMMNC did not amplify the inflammatory response despite the infiltration of neutrophils and macrophages associated with the expression of IL-6 and MCP-1 mRNAs in the tissue. Remarkably, the beneficial effects of BMMNC therapy on matrix remodeling were maintained for 2 months. Furthermore, BMMNC injection restored vascular function in skin tissue by increasing vascular density and vascular permeability. This therapeutic strategy based on BMMNC injection protects against radiation-induced skin lesions by preventing vascular dysfunction and unfavorable remodeling in the acute and late phases.

Identifying microRNA determinants of human myelopoiesis.

Myelopoiesis involves differentiation of hematopoietic stem cells to cellular populations that are restricted in their self-renewal capacity, beginning with the common myeloid progenitor (CMP) and leading to mature cells including monocytes and granulocytes. This complex process is regulated by various extracellular and intracellular signals including microRNAs (miRNAs). We characterised the miRNA profile of human CD34+CD38+ myeloid progenitor cells, and mature monocytes and granulocytes isolated from cord blood using TaqMan Low Density Arrays. We identified 19 miRNAs that increased in both cell types relative to the CMP and 27 that decreased. miR-125b and miR-10a were decreased by 10-fold and 100-fold respectively in the mature cells. Using in vitro granulopoietic differentiation of human CD34+ cells we show that decreases in both miR-125b and miR-10a correlate with a loss of CD34 expression and gain of CD11b and CD15 expression. Candidate target mRNAs were identified by co-incident predictions between the miRanda algorithm and genes with increased expression during differentiation. Using luciferase assays we confirmed MCL1 and FUT4 as targets of miR-125b and the transcription factor KLF4 as a target of miR-10a. Together, our data identify miRNAs with differential expression during myeloid development and reveal some relevant miRNA-target pairs that may contribute to physiological differentiation.

Mesenchymal cells generated from patients with myelodysplastic syndromes are devoid of chromosomal clonal markers and support short- and long-term hematopoiesis in vitro.

Myelodysplastic syndromes (MDS) are clonal malignant stem cell disorders characterized by inefficient hematopoiesis. The role of the marrow microenvironment in the pathogenesis of the disease has been controversial and no study has been performed so far to characterize mesenchymal cells (MC) from MDS patients and to analyse their ability to support hematopoiesis. To this end, we have isolated and characterized MC at diagnostic marrow samples (n=12) and have purified their CD34+CD38- and CD34+CD38+ counterparts (n=7) before using MC as a short- and long-term hematopoietic support. We show that MC can be readily isolated from MDS marrow and exhibit a major expansion potential as well as an intact osteoblastic differentiation ability. They do not harbor the abnormal marker identified by FISH in the hematopoietic cells and they stimulate the growth of autologous clonogenic cells. Conversely, highly purified stem cells and their cytokine-expanded progeny harbor the clonal marker with variable frequencies, and both normal and abnormal long-term culture-initiating cell-derived progeny can be effectively supported by autologous MC. Thus, we demonstrate that MDS marrow is an abundant source of MC appearing both cytogenetically and functionally noninvolved by the malignant process and able to support hematopoiesis, suggesting their possible usefulness in future cell therapy approaches.

Extracellular Vesicles and Vascular Injury: New Insights for Radiation Exposure.

This article reviews our current knowledge about cell-derived extracellular vesicles (EVs), including microparticles and exosomes, and their emergence as mediators of a new important mechanism of cell-to-cell communication. Particular emphasis has been given to the increasing involvement of EVs in the field of radiation-induced vascular injury. Although EVs have been considered for a long time as cell "dust", they in fact precisely reflect the physiological state of the cells. The role of microparticles and exosomes in mediating vascular dysfunction suggests that they may represent novel pathways in short- or long-distance paracrine intercellular signaling in vascular environment. In this article, the mechanisms involved in the biogenesis of microparticles and exosomes, their composition and participation in the pathogenesis of vascular dysfunction are discussed. Furthermore, this article highlights the concept of EVs as potent vectors of biological information and protagonists of an intercellular communication network. Special emphasis is made on EV-mediated microRNA transfer and on the principal consequences of such signal exchange on vascular injury and radiation-induced nontargeted effect. The recent progress in elucidating the biology of EVs has provided new insights for the field of radiation, advancing their use as diagnostic biomarkers or in therapeutic interventions.

Enhanced cloning efficiency of mouse bone marrow macrophage progenitors correlates with increased content of CSF-1 receptor of their progeny at low oxygen tension.

Mononuclear phagocytes are located in every tissue of metazoan organisms. In this extravascular space, they are designated as macrophages and are known to sense and process many signals including the local oxygen tension (PO2), which ranges from 150 mmHg at the lung apices to around 40 mmHg in mixed venous blood and most organs, and to less than 10 mmHg in tissues where long-term and dynamic remodeling processes occur. Most tissue macrophages survive and maintain their differentiated status within an environment bathed by colony-stimulating factor (CSF)-1 through the CSF-1 receptor, encoded by the Csf1r gene. In order to investigate the mRNA expression profile of macrophages as a function of PO2, we developed an in vitro model in which monocyte-derived macrophages were generated from mouse bone marrow progenitor cells grown and maintained under low (36 mmHg) or atmospheric (142 mmHg) PO2, in the presence of L929-conditioned medium (L-CM) as a source of CSF-1. We show that CSF-1-reactive C57BL/6 bone marrow cells displayed an increased cloning efficiency under a PO2 of 36, compared with 142 mmHg. Furthermore, we provide evidence of the overexpression of both CSF-1 receptor protein and mRNA by mouse monocyte-derived macrophages generated from bone marrow under low PO2.