MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative gene expression regulators. In the present study, we investigated miRNAs role in endothelial cell response to hypoxia. We found that the expression of miR-210 progressively increased upon exposure to hypoxia. miR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary-like structures on Matrigel and vascular endothelial growth factor-driven cell migration. Conversely, miR-210 blockade via anti-miRNA transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to vascular endothelial growth factor. miR-210 overexpression did not affect endothelial cell growth in both normoxia and hypoxia. However, antimiR-210 transfection inhibited cell growth and induced apoptosis, in both normoxia and hypoxia. We determined that one relevant target of miR-210 in hypoxia was Ephrin-A3 since miR-210 was necessary and sufficient to down-modulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences; indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. We conclude that miR-210 up-regulation is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration, and differentiation.

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...

Background-MicroRNAs are involved in various critical functions, including the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. We hypothesize that microRNA-210 can rescue cardiac function after myocardial infarction by upregulation of angiogenesis and inhibition of cellular apoptosis in the heart. Methods and Results-Using microRNA microarrays, we first showed that microRNA-210 was highly expressed in live mouse HL-1 cardiomyocytes compared with apoptotic cells after 48 hours of hypoxia exposure. We confirmed by polymerase chain reaction that microRNA-210 was robustly induced in these cells. Gain-of-function and loss-of-function approaches were used to investigate microRNA-210 therapeutic potential in vitro. After transduction, microRNA-210 can upregulate several angiogenic factors, inhibit caspase activity, and prevent cell apoptosis compared with control. Afterward, adult FVB mice underwent intramyocardial injections with minicircle vector carrying microRNA-210 precursor, minicircle carrying microRNA-scramble, or sham surgery. At 8 weeks, echocardiography showed a significant improvement of left ventricular fractional shortening in the minicircle vector carrying microRNA-210 precursor group compared with the minicircle carrying microRNA-scramble control. Histological analysis confirmed decreased cellular apoptosis and increased neovascularization. Finally, 2 potential targets of microRNA-210, Efna3 and Ptp1b, involved in angiogenesis and apoptosis were confirmed through additional experimental validation. Conclusion-MicroRNA-210 can improve angiogenesis, inhibit apoptosis, and improve cardiac function in a murine model of myocardial infarction. It represents a potential novel therapeutic approach for treatment of ischemic heart disease. (Circulation. 2010;122[suppl 1]:S124 -S131.)Key Words: gene therapy Ⅲ ischemic heart disease Ⅲ microRNA Ⅲ minicircle vector I schemic heart disease is the number 1 cause of morbidity and mortality in the United States owing to aging, obesity, diabetes, and other comorbid diseases. One potent therapeutic approach for ischemic heart disease is to reduce oxygen consumption, inhibit cardiomyocyte apoptosis, increase coronary flow, and induce revascularization. MicroRNAs (miRNAs), representing approximately 1% of the eukaryotic transcriptome, is an evolutionarily conserved family of noncoding RNAs of 20 to 22 nucleotides that negatively regulate the expression of protein-coding genes through translational inhibition and RNA decay. miRNAs are involved in diverse biological progresses, including cellular differentiation, proliferation, angiogenesis, and apoptosis. 1 To date, 721 miRNAs have been discovered in human and 597 miRNAs in the mouse according to the miRBase Sequence Database Release 14 (www.mirbase.org/). miRNAs can regulate approximately 30% human protein-coding genes. 2 Importantly, the successful suppression of murine liver cancer by systemic delivery of miR-26a suggests the potential of using miRNAs as a novel therapeutic tool. 3 In th...