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.

AimsCirculating microRNAs (miRNAs) may represent a novel class of biomarkers; therefore, we examined whether acute myocardial infarction (MI) modulates miRNAs plasma levels in humans and mice.Methods and resultsHealthy donors (n = 17) and patients (n = 33) with acute ST-segment elevation MI (STEMI) were evaluated. In one cohort (n = 25), the first plasma sample was obtained 517 ± 309 min after the onset of MI symptoms and after coronary reperfusion with percutaneous coronary intervention (PCI); miR-1, -133a, -133b, and -499-5p were ∼15- to 140-fold control, whereas miR-122 and -375 were ∼87–90% lower than control; 5 days later, miR-1, -133a, -133b, -499-5p, and -375 were back to baseline, whereas miR-122 remained lower than control through Day 30. In additional patients (n = 8; four treated with thrombolysis and four with PCI), miRNAs and troponin I (TnI) were quantified simultaneously starting 156 ± 72 min after the onset of symptoms and at different times thereafter. Peak miR-1, -133a, and -133b expression and TnI level occurred at a similar time, whereas miR-499-5p exhibited a slower time course. In mice, miRNAs plasma levels and TnI were measured 15 min after coronary ligation and at different times thereafter. The behaviour of miR-1, -133a, -133b, and -499-5p was similar to STEMI patients; further, reciprocal changes in the expression levels of these miRNAs were found in cardiac tissue 3–6 h after coronary ligation. In contrast, miR-122 and -375 exhibited minor changes and no significant modulation. In mice with acute hind-limb ischaemia, there was no increase in the plasma level of the above miRNAs.ConclusionAcute MI up-regulated miR-1, -133a, -133b, and -499-5p plasma levels, both in humans and mice, whereas miR-122 and -375 were lower than control only in STEMI patients. These miRNAs represent novel biomarkers of cardiac damage.

Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit ؉ stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting ␣ 2 , ␣ 4 , and ␣ 5 integrinmediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit ؉ cells, while cytokine withdrawal or the overexpression of the cyclin-dependent kinase (CDK) inhibitor p16 INK4 restored such differentiation, suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection, plasma SDF-1 levels were up-regulated, while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RNA and protein level was also observed. Finally, using an in vivo assay such as injection of matrigel plugs, we found that SDF IntroductionIt has been shown that endothelial progenitor cells (EPCs) play a role in vascular repair following ischemic injury. 1 EPCs give rise to endothelial-like cells in culture, growing as spindleshaped cells attaching to culture dishes coated with extracellular matrix (ECM) components. 2 However, the mechanisms driving EPC differentiation are largely unknown. Stromal-derived factor 1 (SDF-1) regulates adhesion/chemotaxis of bone marrow hematopoietic progenitor cells through activation/regulation of specific integrin molecules. [3][4][5] This factor is, therefore, suggested to play a major role in successful hematopoietic stem cell (HSC) engraftment in the bone marrow. 6 In vivo gene inactivation of SDF-1 and its receptor C-X-C chemokine receptor 4 in mice led to early embryonic lethality due to abnormal cerebellar, gastrointestinal vasculature, and hematopoiesis development. [7][8][9] A role for SDF-1 in HSC/EPC recruitment from bone marrow (BM) to peripheral blood (PB) has been proposed, based on the evidence that granulocyte colony stimulating factor (G-CSF)-mediated HSC/EPC mobilization causes an imbalance between the expression of BM SDF-1 and CXCR4 in HSCs, 10 and that SDF-1␣ adenovirus gene transfer enhances the number of circulating HSCs/EPCs. [11][12][13] Recently, overexpression of SDF-1 in ischemic tissues has been found to enhance EPC recruitment from PB and to induce neoangiogenesis. 14,15 In this paper, we show that SDF-1 increases EPC number through enhancement of (BM) c-kit ϩ stem cell adhesion onto extracellular matrix components by integrin receptors. Further, we show that treatment of c-kit ϩ cells with mitogenic cytokines abolished SDF-1-mediated EPC differen...

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...

Cyclophilin A (CyPA) is a ubiquitously distributed protein belonging to the immunophilin family. CyPA has peptidyl prolyl cis-trans isomerase (PPIase) activity, which regulates protein folding and trafficking. Although CyPA was initially believed to function primarily as an intracellular protein, recent studies have revealed that it can be secreted by cells in response to inflammatory stimuli. Current research in animal models and humans has provided compelling evidences supporting the critical function of CyPA in several human diseases. This review discusses recently available data about CyPA in cardiovascular diseases, viral infections, neurodegeneration, cancer, rheumatoid arthritis, sepsis, asthma, periodontitis and aging. It is believed that further elucidations of the role of CyPA will provide a better understanding of the molecular mechanisms underlying these diseases and will help develop novel pharmacological therapies.

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.

Abstract-High-mobility group box 1 protein (HMGB1) is a chromatin protein that is released by inflammatory and necrotic cells. Extracellular HMGB1 signals tissue damage, stimulates the secretion of proinflammatory cytokines and chemokines, and modulates stem cell function. The present study examined exogenous HMGB1 effect on mouse left-ventricular function and myocyte regeneration after infarction. Myocardial infarction was induced in C57BL/6 mice by permanent coronary artery ligation. After 4 hours animals were reoperated and 200 ng of purified HMGB1 was administered in the peri-infarcted left ventricle. This intervention resulted in the formation of new myocytes within the infarcted portion of the wall. The regenerative process involved the proliferation and differentiation of endogenous cardiac c-kit ϩ progenitor cells. Circulating c-kit ϩ cells did not significantly contribute to HMGB1-mediated cardiac regeneration. Echocardiographic and hemodynamic parameters at 1, 2, and 4 weeks demonstrated a significant recovery of cardiac performance in HMGB1-treated mice. These effects were not observed in infarcted hearts treated either with the unrelated protein glutathione S-transferase or a truncated form of HMGB1. Thus, HMGB1 appears to be a potent inducer of myocardial regeneration following myocardial infarction. (Circ Res. 2005;97:e73-e83.)

Background-Experimental interleukin-1 receptor antagonist gene overexpression has shown that interleukin-1 receptor antagonist is cardioprotective during global cardiac ischemia. The aim of the present study was to test the impact of an exogenous recombinant human interleukin-1 receptor antagonist (anakinra) in experimental acute myocardial infarction. Methods and Results-Two animal studies were conducted: one of immediate anakinra administration during ischemia in the mouse and one of delayed anakinra administration 24 hours after ischemia in the rat. Seventy-eight Institute of Cancer Research mice and 20 Wistar rats underwent surgical coronary artery ligation (or sham operation) and were treated with either anakinra 1 mg/kg or NaCl 0.9% (saline). Treatment was administered during surgery and then daily for 6 doses in the mice and starting on day 2 daily for 5 doses in the rats. Twenty-eight mice underwent infarct size assessment 24 hours after surgery, 6 saline-treated mice and 22 mice treated with increasing doses of anakinra (1 mg/kg [nϭ6], 10 mg/kg [nϭ6], and 100 mg/kg [nϭ10]); 6 mice were euthanized at 7 days for protein expression analysis. The remaining animals underwent transthoracic echocardiography before surgery and 7 days later just before death. Cardiomyocyte apoptosis was measured in the peri-infarct regions. The antiapoptotic effect of anakinra was tested in a primary rat cardiomyocyte culture during simulated ischemia and in vitro on caspase-1 and -9 activities. At 7 days, 15 of the 16 mice (94%) treated with anakinra were alive versus 11 of the 20 mice (55%) treated with saline (Pϭ0.013).No differences in infarct size at 24 hours compared with saline were observed with the 1-and 10-mg/kg doses, whereas a 13% reduction in infarct size was found with the 100-mg/kg dose (Pϭ0.015). Treatment with anakinra was associated with a significant reduction in cardiomyocyte apoptosis in both the immediate and delayed treatment groups (3.1Ϯ0.2% versus 0.5Ϯ0.3% [PϽ0.001] and 4.2Ϯ0.4% versus 1.1Ϯ0.2% [PϽ0.001], respectively). Compared with saline-treated animals, anakinra-treated mice and rats showed signs of more favorable ventricular remodeling. In vitro, anakinra significantly prevented apoptosis induced by simulated ischemia and inhibited caspase-1 and -9 activities. Conclusions-Administration of anakinra within 24 hours of acute myocardial infarction significantly ameliorates the remodeling process by inhibiting cardiomyocyte apoptosis in 2 different experimental animal models of AMI. This may open the door for using anakinra to prevent postischemic cardiac remodeling and heart failure.