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...

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