We examined the effect of reactive oxygen species (ROS) on MicroRNAs (miRNAs) expression in endothelial cells in vitro, and in mouse skeletal muscle following acute hindlimb ischemia. Human umbilical vein endothelial cells (HUVEC) were exposed to 200 lM hydrogen peroxide (H 2 O 2 ) for 8 to 24 h; miRNAs profiling showed that miR-200c and the co-transcribed miR-141 increased more than eightfold. The other miR-200 gene family members were also induced, albeit to a lower level. Furthermore, miR-200c upregulation was not endothelium restricted, and occurred also on exposure to an oxidative stress-inducing drug: 1,3-bis(2 chloroethyl)-1nitrosourea (BCNU). miR-200c overexpression induced HUVEC growth arrest, apoptosis and senescence; these phenomena were also induced by Reactive oxygen species (ROS) play a causal role in a variety of cardiovascular diseases, including ischemia, ischemia/ reperfusion (I/R) injury, diabetic vasculopathy and atherosclerosis, and in aging. 1-3 ROS, which include H 2 O 2 , superoxide anion and hydroxyl radicals have been demonstrated to inhibit cell growth and to induce cell death and senescence. 1 MicroRNAs (miRNAs) are small non-coding RNAs, usually 21-23 nucleotides long, which regulate the stability and/or the translational efficiency of target messenger RNAs (mRNAs). 4 They appear to be closely conserved across species and to them have been ascribed diverse functions, including regulation of proliferation, differentiation, senescence and death. 5 The objective of the present work was to establish the effect of ROS on miRNAs expression, and to determine whether miRNAs modulate endothelial cells (EC) response to oxidative stress. In light of the important role that tumor suppressor proteins retinoblastoma (pRb) and p53 have in responses to ROS, we examined their contribution to miRNAs expression on oxidative stress exposure. The retinoblastoma family, which includes pRb, p130 and p107, is an integral part of the mechanism that regulates proliferation and senescence via phosphorylation-sensitive interactions, regulating either positively or negatively E2F transcription factors family. 6 H 2 O 2 causes rapid pRb dephosphorylation by the activity of protein phosphatase 2A 7,8 and successively, by the increase of p53 protein, which in turns upregulates the CDK inhibitor p21 Waf1/Cip1/Sdi1 (p21). 7 The ROS effect on miRNAs expression was also evaluated in vivo, in a mouse model of hindlimb ischemia, both in wild-type (wt) and in p66 ShcA -null (p66 ShcAÀ/À ) mice. The mammalian adaptor protein p66 ShcA regulates ROS metabolism and apoptosis. The cytoplasmic fraction of p66 ShcA is phosphorylated in serine 36 residue in response to several stimuli, including UV and H 2 O 2 . 9 Moreover, a fraction of p66 ShcA is localized in the mitochondria and functions as a redox enzyme that generates ROS; 10 accordingly p66 ShcAÀ/À mice display lower levels of intracellular ROS 9 and decreased oxidative stress levels and tissue damage following ischemia and I/R injury. 2,3 In the present work, we show tha...

The aim of this work was to identify micro-RNAs (miRNAs) involved in the pathological pathways activated in skeletal muscle damage and regeneration by both dystrophin absence and acute ischemia. Eleven miRNAs were deregulated both in MDX mice and in Duchenne muscular dystrophy patients (DMD signature). Therapeutic interventions ameliorating the mdx-phenotype rescued DMD-signature alterations. The significance of DMD-signature changes was characterized using a damage/regeneration mouse model of hind-limb ischemia and newborn mice. According to their expression, DMD-signature miRNAs were divided into 3 classes. 1) Regeneration miRNAs, miR-31, miR-34c, miR-206, miR-335, miR-449, and miR-494, which were induced in MDX mice and in DMD patients, but also in newborn mice and in newly formed myofibers during postischemic regeneration. Notably, miR-206, miR-34c, and miR-335 were up-regulated following myoblast differentiation in vitro. 2) Degenerative-miRNAs, miR-1, miR-29c, and miR-135a, that were down-modulated in MDX mice, in DMD patients, in the degenerative phase of the ischemia response, and in newborn mice. Their down-modulation was linked to myofiber loss and fibrosis. 3) Inflammatory miRNAs, miR-222 and miR-223, which were expressed in damaged muscle areas, and their expression correlated with the presence of infiltrating inflammatory cells. These findings show an important role of miRNAs in physiopathological pathways regulating muscle response to damage and regeneration.

miR-210 is a key player of cell response to hypoxia, modulating cell survival, VEGF-driven endothelial cell migration, and the ability of endothelial cells to form capillary-like structures. A crucial step in understanding microRNA (miRNA) function is the identification of their targets. However, only few miR-210 targets have been identified to date. Here, we describe an integrated strategy for large-scale identification of new miR-210 targets by combining transcriptomics and proteomics with bioinformatic approaches. To experimentally validate candidate targets, the RNA-induced silencing complex (RISC) loaded with miR-210 was purified by immunoprecipitation along with its mRNA targets. The complex was significantly enriched in mRNAs of 31 candidate targets, such as BDNF, GPD1L, ISCU, NCAM, and the non-coding RNA Xist. A subset of the newly identified targets was further confirmed by 3-untranslated region (UTR) reporter assays, and hypoxia induced down-modulation of their expression was rescued blocking miR-210, providing support for the approach validity. In the case of 9 targets, such as PTPN1 and P4HB, miR-210 seed-pairing sequences localized in the coding sequence or in the 5-UTR, in line with recent data extending miRNA targeting beyond the "classic" 3-UTR recognition. Finally, Gene Ontology analysis of the targets highlights known miR-210 impact on cell cycle regulation and differentiation, and predicts a new role of this miRNA in RNA processing, DNA binding, development, membrane trafficking, and amino acid catabolism. Given the complexity of miRNA actions, we view such a multiprong approach as useful to adequately describe the multiple pathways regulated by miR-210 during physiopathological processes.miRNAs are 21-23-nucleotide non-protein coding RNA molecules that regulate the stability and/or the translational efficiency of target messenger RNAs (1-3). Mature miRNAs are loaded into the RNA-induced silencing complex (RISC) 3 and mediate the translational inhibition of target mRNA, albeit a few opposing examples have been described as well (4 -6). The rules that guide miRNA-mRNA interaction are very complex and still under investigation. However, the current paradigm states that a Watson-Crick pairing between the mRNA and the 5Ј-region of the miRNA centered on nucleotides 2-7, termed "seed sequence," is required for miRNA-mediated inhibition (7). RISC-miRNA complexes can move the mRNAs they bind to the P-bodies, which are specialized cytoplasmic compartments where translational repression and mRNA turnover is thought to occur (8). Because P-bodies contain many enzymes involved in mRNA exonucleolitic degradation, miRNAs may also have a secondary quantitative inhibitory effect on mRNAs. A role for miRNAs in mRNA destabilization is also suggested by studies reporting robust correlations between the levels of miRNAs and the message of multiple predicted or validated targets (9 -11).miR-210 is currently regarded as "master miRNA" of hypoxic response, because it was found up-regulated by hypoxia in all the ce...

Summary Multiple studies have consistently established that miR (microRNA)-210 induction is a feature of the hypoxic response in both normal and transformed cells. Here, we discuss the emerging biochemical functions of this miRNA and anticipate potential clinical applications. miR-210 is a robust target of hypoxia-inducible factor, and its overexpression has been detected in a variety of cardiovascular diseases and solid tumors. High levels of miR-210 have been linked to an in vivo hypoxic signature and associated with adverse prognosis in cancer patients. A wide spectrum of miR-210 targets have been identified, with roles in mitochondrial metabolism, angiogenesis, DNA repair, and cell survival. Such targets may broadly affect the evolution of tumors and other pathological settings, such as ischemic disorders. Harnessing the knowledge of miR-210’s actions may lead to novel diagnostic and therapeutic approaches.

Increased morbidity and mortality associated with ischemic heart failure (HF) in type 2 diabetic patients requires a deeper understanding of the underpinning pathogenetic mechanisms. Given the implication of microRNAs (miRNAs) in HF, we investigated their regulation and potential role. miRNA expression profiles were measured in left ventricle biopsies from 10 diabetic HF (D-HF) and 19 nondiabetic HF (ND-HF) patients affected by non–end stage dilated ischemic cardiomyopathy. The HF groups were compared with each other and with 16 matched nondiabetic, non-HF control subjects. A total of 17 miRNAs were modulated in D-HF and/or ND-HF patients when compared with control subjects. miR-216a, strongly increased in both D-HF and ND-HF patients, negatively correlated with left ventricular ejection fraction. Six miRNAs were differently expressed when comparing D-HF and ND-HF patients: miR-34b, miR-34c, miR-199b, miR-210, miR-650, and miR-223. Bioinformatic analysis of their modulated targets showed the enrichment of cardiac dysfunctions and HF categories. Moreover, the hypoxia-inducible factor pathway was activated in the noninfarcted, vital myocardium of D-HF compared with ND-HF patients, indicating a dysregulation of the hypoxia response mechanisms. Accordingly, miR-199a, miR-199b, and miR-210 were modulated by hypoxia and high glucose in cardiomyocytes and endothelial cells cultured in vitro. In conclusion, these findings show a dysregulation of miRNAs in HF, shedding light on the specific disease mechanisms differentiating diabetic patients.