Alessandra Magenta

miR-200c is upregulated by oxidative stress and induces endothelial cell apoptosis and senescence via ZEB1 inhibition

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 μM hydrogen peroxide (H2O2) 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 H2O2 and were partially rescued by miR-200c inhibition. Moreover, miR-200c target ZEB1 messenger RNA and protein were downmodulated by H2O2 and by miR-200c overexpression. ZEB1 knockdown recapitulated miR-200c-induced responses, and expression of a ZEB1 allele non-targeted by miR-200c, prevented miR-200c phenotype. The mechanism of H2O2-mediated miR-200c upregulation involves p53 and retinoblastoma proteins. Acute hindlimb ischemia enhanced miR-200c in wild-type mice skeletal muscle, whereas in p66ShcA −/− mice, which display lower levels of oxidative stress after ischemia, upregulation of miR-200c was markedly inhibited. In conclusion, ROS induce miR-200c and other miR-200 family members; the ensuing downmodulation of ZEB1 has a key role in ROS-induced apoptosis and senescence.

p66ShcA Modulates Tissue Response to Hindlimb Ischemia

Background—Oxidative stress plays a pivotal role in ischemia and ischemia/reperfusion injury. Because p66ShcA-null (p66ShcA−/−) mice exhibit both lower levels of intracellular reactive oxygen species and increased resistance to cell death induced by oxidative stress, we investigated whether tissue damage that follows acute ischemia or ischemia/reperfusion was altered in p66ShcA−/− mice. Methods and Results—Unilateral hindlimb ischemia was induced by femoral artery dissection, and ischemia/reperfusion was induced with an elastic tourniquet. Both procedures caused similar changes in blood perfusion in p66ShcA wild-type (p66ShcAwt) and p66ShcA−/− mice. However, significant differences in tissue damage were found: p66ShcAwt mice displayed marked capillary density decrease and muscle fiber necrosis. In contrast, in p66ShcA−/− mice, minimal capillary density decrease and myofiber death were present. When apoptosis after ischemia was assayed, significantly lower levels of apoptotic endothelial cells and myofibers were found in p66ShcA−/− mice. In agreement with these data, both satellite muscle cells and endothelial cells isolated from p66ShcA−/− mice were resistant to apoptosis induced by simulated ischemia in vitro. Lower apoptosis levels after ischemia in p66ShcA−/− cells correlated with decreased levels of oxidative stress both in vivo and in vitro. Conclusions—p66ShcA plays a crucial role in the cell death pathways activated by acute ischemia and ischemia/reperfusion, indicating p66ShcA as a potential therapeutic target for prevention and treatment of ischemic tissue damage.

Oxidative stress and microRNAs in vascular diseases.

Oxidative stress has been demonstrated to play a causal role in different vascular diseases, such as hypertension, diabetic vasculopathy, hypercholesterolemia and atherosclerosis. Indeed, increased reactive oxygen species (ROS) production is known to impair endothelial and vascular smooth muscle cell functions, contributing to the development of cardiovascular diseases. MicroRNAs (miRNAs) are non-coding RNA molecules that modulate the stability and/or the translational efficiency of target messenger RNAs. They have been shown to be modulated in most biological processes, including in cellular responses to redox imbalance. In particular, miR-200 family members play a crucial role in oxidative-stress dependent endothelial dysfunction, as well as in cardiovascular complications of diabetes and obesity. In addition, different miRNAs, such as miR-210, have been demonstrated to play a key role in mitochondrial metabolism, therefore modulating ROS production and sensitivity. In this review, we will discuss miRNAs modulated by ROS or involved in ROS production, and implicated in vascular diseases in which redox imbalance has a pathogenetic role.

MyoD stimulates RB promoter activity via the CREB/p300 nuclear transduction pathway

ABSTRACT The induction of RB gene transcription by MyoD is a key event in the process of skeletal muscle differentiation, because elevated levels of the retinoblastoma protein are essential for myoblast cell cycle arrest as well as for the terminal differentiation and survival of postmitotic myocytes. We previously showed that MyoD stimulates transcription from the RB promoter independently of direct binding to promoter sequences. Here we demonstrate that stimulation by MyoD requires a cyclic AMP-responsive element (CRE) in the RB promoter, bound by the transcription factor CREB in differentiating myoblasts. We also show that both the CREB protein level and the level of phosphorylation of the CREB protein at Ser-133 rapidly increase at the onset of muscle differentiation and that both remain high throughout the myogenic process. Biochemical and functional evidence indicates that in differentiating myoblasts, MyoD becomes associated with CREB and is targeted to the RB promoter CRE in a complex also containing the p300 transcriptional coactivator. The resulting multiprotein complex stimulates transcription from the RB promoter. These and other observations strongly suggest that MyoD functions by promoting the efficient recruitment of p300 by promoter-bound, phosphorylated CREB.

p66ShcA and Oxidative Stress Modulate Myogenic Differentiation and Skeletal Muscle Regeneration after Hind Limb Ischemia

Oxidative stress plays a pivotal role in ischemic injury, and p66ShcAko mice exhibit both lower oxidative stress and decreased tissue damage following hind limb ischemia. Thus, it was investigated whether tissue regeneration following acute hind limb ischemia was altered in p66ShcAko mice. Upon femoral artery dissection, muscle regeneration started earlier and was completed faster than in wild-type (WT) control. Moreover, faster regeneration was associated with decreased oxidative stress. Unlike ischemia, cardiotoxin injury induced similar skeletal muscle damage in both genotypes. However, p66ShcAko mice regenerated faster, in agreement with the regenerative advantage upon ischemia. Since no difference between p66ShcAwt and knock-out (ko) mice was found in blood perfusion recovery after ischemia, satellite cells (SCs), a resident population of myogenic progenitors, were examined. Similar SCs numbers were present in WT and ko mice. However, in vitro cultured p66ShcAko SCs displayed lower oxidative stress levels and higher proliferation rate and differentiated faster than WT. Furthermore, when exposed to sublethal H2O2 doses, p66ShcAko SCs were resistant to H2O2-induced inhibition of differentiation. Finally, myogenic conversion induced by MyoD overexpression was more efficient in p66ShcAko fibroblasts compared with WT. The present work demonstrates that oxidative stress and p66ShcA play a crucial role in the regenerative pathways activated by acute ischemia.

p66ShcA modulates oxidative stress and survival of endothelial progenitor cells in response to high glucose

AIMS A close relationship exists between hyperglycaemia, oxidative stress, and diabetic complications. In fact, high glucose (HG) determines the overproduction of reactive oxygen species (ROS) by the mitochondria. p66ShcA is a gene that regulates the apoptotic responses to oxidative stress. Indeed, p66ShcA knockout (ko) mice display decreased ROS production and increased resistance to ROS-induced cell death in a variety of pathophysiological settings. Reduced endothelial progenitor cell (EPC) number, differentiation, and function are relevant components of the angiogenesis impairment observed in diabetic patients. We examined the role of p66ShcA in the EPC deficit induced by HG. METHODS AND RESULTS Mouse bone marrow-derived c-kit+ cells differentiate in endothelial-like cells when plated on fibronectin (BM-derived EPCs). We found that cell culture in the presence of HG up-regulated p66ShcA protein expression and that HG exposure markedly decreased the number of BM-derived EPCs. Conversely, p66ShcA ko BM-derived EPCs were not sensitive to HG inhibition. Indeed, the resistance of p66ShcA ko BM-derived EPCs to HG was associated with reduced levels of both apoptosis and oxidative stress. To functionally link the HG response to ROS production, p66ShcA ko BM-derived EPCs were reconstituted either with p66ShcA wild-type (wt) or with a p66ShcA allele (p66ShcA qq) that was devoid of its ROS-generating function. We found that only p66ShcA wt and not the qq mutant rescued p66ShcA ko cell sensitivity to HG. One major feature of oxidative stress is its ability to reduce the bio-availability of nitric oxide (NO) that, in turn, plays a crucial role in endothelial differentiation and function. We found that the p66ShcA deletion prevented the HG-induced increase of nitrotyrosine, and that the resistance to HG of p66ShcA ko BM-derived EPCs was prevented by NO synthase inhibition. With a reciprocal approach, the treatment of p66ShcA wt cells with a NO donor prevented the HG-induced deficit. Finally, using a Matrigel plug angiogenesis assay, we demonstrated that p66ShcA ko prevented diabetic impairment of angiogenesis in vivo. CONCLUSION p66ShcA deletion rescues the BM-derived EPCs defect induced by HG, indicating p66ShcA as a potential therapeutic target in diabetic vasculopathy.

Protein Phosphatase 2A Subunit PR70 Interacts with pRb and Mediates Its Dephosphorylation

ABSTRACT The retinoblastoma tumor suppressor protein (pRb) regulates cell proliferation and differentiation via phosphorylation-sensitive interactions with specific targets. While the role of cyclin/cyclin-dependent kinase complexes in the modulation of pRb phosphorylation has been extensively studied, relatively little is known about the molecular mechanisms regulating phosphate removal by phosphatases. Protein phosphatase 2A (PP2A) is constituted by a core dimer bearing catalytic activity and one variable B regulatory subunit conferring target specificity and subcellular localization. We previously demonstrated that PP2A core dimer binds pRb and dephosphorylates pRb upon oxidative stress. In the present study, we identified a specific PP2A-B subunit, PR70, that was associated with pRb both in vitro and in vivo. PR70 overexpression caused pRb dephosphorylation; conversely, PR70 knockdown prevented both pRb dephosphorylation and DNA synthesis inhibition induced by oxidative stress. Moreover, we found that intracellular Ca2+ mobilization was necessary and sufficient to trigger pRb dephosphorylation and PP2A phosphatase activity of PR70 was Ca2+ induced. These data underline the importance of PR70-Ca2+ interaction in the signal transduction mechanisms triggered by redox imbalance and leading to pRb dephosphorylation.

Nitric oxide, oxidative stress, and p66Shc interplay in diabetic endothelial dysfunction.

Increased oxidative stress and reduced nitric oxide (NO) bioavailability play a causal role in endothelial cell dysfunction occurring in the vasculature of diabetic patients. In this review, we summarized the molecular mechanisms underpinning diabetic endothelial and vascular dysfunction. In particular, we focused our attention on the complex interplay existing among NO, reactive oxygen species (ROS), and one crucial regulator of intracellular ROS production, p66Shc protein.

Cyclin D1 degradation enhances endothelial cell survival upon oxidative stress

The understanding of endothelial cell responses to oxidative stress may provide insights into aging mechanisms and into the pathogenesis of numerous cardiovascular diseases. In this study, we examined the regulation and the functional role of cyclin D1, a crucial player in cell proliferation and survival. On H2O2 treatment, endothelial cells showed a rapid down‐modulation of cyclin D1. Other D‐cyclins were similarly regulated, and this decrease was also observed after exposure to other oxidative stress‐inducing stimuli, namely 1,3‐bis (2 chloroethyl)‐1 nitrosourea treatment and ischemia. H2O2 treatment induced cyclin D1 ubiquitination followed by proteasome degradation. Phospholipase C inhibition prevented cyclin D1 degradation, and its activation triggered cyclin D1 down‐modulation in the absence of oxidative stress. Activated phospholipase C generates inositol‐1,4,5‐trisphosphate (IP3) and Ca2+ release from internal stores. We found that both IP3‐receptor inhibition and intracellular Ca2+ chelation prevented cyclin D1 degradation induced by oxidative stress. Furthermore, Ca2+ increase was transduced by Ca2+/calmodulin‐dependent protein kinase (CaMK). In fact, H2O2 stimulated CaMK activity, CaMK inhibitors prevented H2O2‐induced cyclin D1 down‐modulation, and CaMK overexpression induced cyclin D1 degradation. Finally, overriding of cyclin D1 down‐modulation via its forced overexpression or via CaMK inhibition increased cell sensitivity to H2O2‐induced apoptotic cell death. Thus, cyclin D1 degradation enhances endothelial cell survival on oxidative stress.—Fasanaro, P., Magenta, A., Zaccagnini, G., Cicchillitti, L., Fucile, S., Eusebi, F., Biglioli, P., Capogrossi, M. C., Martelli, F. Cyclin D1 degradation enhances endothelial cell survival upon oxidative stress. FASEB J. 20, E503–E515 (2006)

Circulating miR-33a and miR-33b are up-regulated in familial hypercholesterolaemia in paediatric age

Hypercholesterolaemia is one of the major causes of CVD (cardiovascular disease). It is associated with enhanced oxidative stress, leading to increased lipid peroxidation which in turn determines endothelial dysfunction and susceptibility to coronary vasoconstriction and atherosclerosis. Different miRNAs are involved in the pathogenesis of CVD and play an important role in inflammatory process control, therefore, together with atherogenic factors, they can stimulate atherosclerotic degeneration of the vessel walls of arteries. miR-33a and miR-33b play a pivotal role in a variety of biological processes including cholesterol homoeostasis, HDL (high-density lipoprotein)-cholesterol formation, fatty acid oxidation and insulin signalling. Our study aimed to determine whether circulating miR-33a and miR-33b expression was altered in familial hypercholesterolaemic children. Total RNA was extracted from plasma, and miR-33a and miR-33b were measured by quantitative real-time PCR. We found that miR-33a and miR-33b were significantly up-regulated in the plasma of 28 hypercholesterolaemic children compared with 25 healthy subjects (4.49±0.27-fold increase, P<0.001, and 3.21±0.39-fold increase, P<0.05 respectively), and for both miRNAs, a positive correlation with total cholesterol, LDL (low-density lipoprotein)-cholesterol, LDL-cholesterol/HDL-cholesterol ratio, apolipoprotein B, CRP (C-reactive protein) and glycaemia was found. OLS (ordinary least squares) regression analysis revealed that miR-33a was significantly affected by the presence of FH (familial hypercholesterolaemia), glycaemia and CRP (P<0.001, P<0.05 and P<0.05 respectively). The same analysis showed that miR-33b was significantly related to FH and CRP (P<0.05 and P<0.05 respectively). Although it is only explorative, the present study could be the first to point to the use of miR-33a and miR-33b as early biomarkers for cholesterol levels in childhood, once validated in independent larger cohorts.