CircANKRD12 Is Induced in Endothelial Cell Response to Oxidative Stress.

Redox imbalance of the endothelial cells (ECs) plays a causative role in a variety of cardiovascular diseases. In order to better understand the molecular mechanisms of the endothelial response to oxidative stress, the involvement of circular RNAs (circRNAs) was investigated. CircRNAs are RNA species generated by a "back-splicing" event, which is the covalent linking of the 3'- and 5'-ends of exons. Bioinformatics analysis of the transcriptomic landscape of human ECs exposed to H2O2 allowed us to identify a subset of highly expressed circRNAs compared to their linear RNA counterparts, suggesting a potential biological relevance. Specifically, circular Ankyrin Repeat Domain 12 (circANKRD12), derived from the junction of exon 2 and exon 8 of the ANKRD12 gene (hsa_circ_0000826), was significantly induced in H2O2-treated ECs. Conversely, the linear RNA isoform of ANKRD12 was not modulated. An increased circular-to-linear ratio of ANKRD12 was also observed in cultured ECs exposed to hypoxia and in skeletal muscle biopsies of patients affected by critical limb ischemia (CLI), two conditions associated with redox imbalance and oxidative stress. The functional relevance of circANKRD12 was shown by the inhibition of EC formation of capillary-like structures upon silencing of the circular but not of the linear isoform of ANKRD12. Bioinformatics analysis of the circANKRD12-miRNA-mRNA regulatory network in H2O2-treated ECs identified the enrichment of the p53 and Foxo signaling pathways, both crucial in the cellular response to redox imbalance. In keeping with the antiproliferative action of the p53 pathway, circANKRD12 silencing inhibited EC proliferation. In conclusion, this study indicates circANKRD12 as an important player in ECs exposed to oxidative stress.

Hypoxia-induced miR-210 modulates the inflammatory response and fibrosis upon acute ischemia.

Hypoxia-induced miR-210 is a crucial component of the tissue response to ischemia, stimulating angiogenesis and improving tissue regeneration. Previous analysis of miR-210 impact on the transcriptome in a mouse model of hindlimb ischemia showed that miR-210 regulated not only vascular regeneration functions, but also inflammation. To investigate this event, doxycycline-inducible miR-210 transgenic mice (Tg-210) and anti-miR-210 LNA-oligonucleotides were used. It was found that global miR-210 expression decreased inflammatory cells density and macrophages accumulation in the ischemic tissue. To dissect the underpinning cell mechanisms, Tg-210 mice were used in bone marrow (BM) transplantation experiments and chimeric mice underwent hindlimb ischemia. MiR-210 overexpression in the ischemic tissue was sufficient to increase capillary density and tissue repair, and to reduce inflammation in the presence of Wt-BM infiltrating cells. Conversely, when Tg-210-BM cells migrated in a Wt ischemic tissue, dysfunctional angiogenesis, inflammation, and impaired tissue repair, accompanied by fibrosis were observed. The fibrotic regions were positive for α-SMA, Vimentin, and Collagen V fibrotic markers and for phospho-Smad3, highlighting the activation of TGF-ß1 pathway. Identification of Tg-210 cells by in situ hybridization showed that BM-derived cells contributed directly to fibrotic areas, where macrophages co-expressing fibrotic markers were observed. Cell cultures of Tg-210 BM-derived macrophages exhibited a pro-fibrotic phenotype and were enriched with myofibroblast-like cells, which expressed canonical fibrosis markers. Interestingly, inhibitors of TGF-ß type-1-receptor completely abrogated this pro-fibrotic phenotype. In conclusion, a context-dependent regulation by miR-210 of the inflammatory response was identified. miR-210 expression in infiltrating macrophages is associated to improved angiogenesis and tissue repair when the ischemic recipient tissue also expresses high levels of miR-210. Conversely, when infiltrating an ischemic tissue with mismatched miR-210 levels, macrophages expressing high miR-210 levels display a pro-fibrotic phenotype, leading to impaired tissue repair, fibrosis, and dysfunctional angiogenesis.

Hypoxia-Induced miR-210 Is Necessary for Vascular Regeneration upon Acute Limb Ischemia.

Critical limb ischemia is the most serious form of peripheral artery disease, characterized by severe functional consequences, difficult clinical management and reduced life expectancy. The goal of this study was to investigate the miR-210 role in the neo-angiogenic response after acute limb ischemia. Complementary approaches were used in a mouse model of hindlimb ischemia: miR-210 loss-of-function was obtained by administration of LNA-oligonucleotides anti-miR-210; for miR-210 gain-of-function, a doxycycline-inducible miR-210 transgenic mouse was used. We tested miR-210 ability to stimulate vascular regeneration following ischemia. We found that miR-210 was necessary and sufficient to stimulate blood perfusion recovery, as well as arteriolar and capillary density increase, in the ischemic muscle. To clarify the molecular events underpinning miR-210 pro-angiogenic action, the transcriptomic changes in ischemic muscles upon miR-210 blocking were analyzed. We found that miR-210 impacted the transcriptome significantly, regulating pathways and functions linked to vascular regeneration. In agreement with a pro-angiogenic role, miR-210 also improved cardiac function and left ventricular remodeling after myocardial infarction. Moreover, miR-210 blocking decreased capillary density in a Matrigel plug assay, indicating that miR-210 is necessary for angiogenesis independently of ischemia. Collectively, these data indicate that miR-210 plays a pivotal role in promoting vascular regeneration.

Dysregulation of Circular RNAs in Myotonic Dystrophy Type 1.

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by "back-splicing", which is the linking covalently of 3'- and 5'-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients.

Noncoding RNAs in the Vascular System Response to Oxidative Stress.

SIGNIFICANCE: Redox homeostasis plays a pivotal role in vascular cell function and its imbalance has a causal role in a variety of vascular diseases. Accordingly, the response of mammalian cells to redox cues requires precise transcriptional and post-transcriptional modulation of gene expression patterns. Recent Advances: Mounting evidence shows that nonprotein-coding RNAs (ncRNAs) are important for the functional regulation of most, if not all, cellular processes and tissues. Not surprisingly, a prominent role of ncRNAs has been identified also in the vascular system response to oxidative stress. CRITICAL ISSUES: The highly heterogeneous family of ncRNAs has been divided into several groups. In this article we focus on two classes of regulatory ncRNAs: microRNAs and long ncRNAs (lncRNAs). Although knowledge in many circumstances, and especially for lncRNAs, is still fragmentary, ncRNAs are clinically interesting because of their diagnostic and therapeutic potential. We outline ncRNAs that are regulated by oxidative stress as well as ncRNAs that modulate reactive oxygen species production and scavenging. More importantly, we describe the role of these ncRNAs in vascular physiopathology and specifically in disease conditions wherein oxidative stress plays a crucial role, such as hypoxia and ischemia, ischemia reperfusion, inflammation, diabetes mellitus, and atherosclerosis. FUTURE DIRECTIONS: The therapeutic potential of ncRNAs in vascular diseases and in redox homeostasis is discussed.

Circular RNAs: Methodological challenges and perspectives in cardiovascular diseases.

Circular RNAs are generated by back-splicing of precursor-mRNAs. Although they have been known for many years, only recently we have started to appreciate their widespread expression and their regulatory functions in a variety of biological processes. Not surprisingly, circular RNA dysregulation and participation in the pathogenic mechanisms have started to emerge in many instances, including cardiovascular diseases. Detection, differential expression analysis and validation are the three critical points for the characterization of any RNA, and circular RNAs are no exception. Their characteristics, however, generate several problems that are yet to be completely addressed, and literature still lacks comprehensive definitions of well-defined best practices. We present a map of the current knowledge regarding circular RNAs and the critical issues limiting our understanding of their regulation and function. The goal was to provide the readers with the tools to critically decide which of the many approaches available is most suitable to their experimental plan. Although particularly focused on cardiovascular diseases, most critical issues concerning circular RNAs are common to many other fields of investigation.

miR-210 Enhances the Therapeutic Potential of Bone-Marrow-Derived Circulating Proangiogenic Cells in the Setting of Limb Ischemia.

Therapies based on circulating proangiogenic cells (PACs) have shown promise in ischemic disease models but require further optimization to reach the bedside. Ischemia-associated hypoxia robustly increases microRNA-210 (miR-210) expression in several cell types, including endothelial cells (ECs). In ECs, miR-210 represses EphrinA3 (EFNA3), inducing proangiogenic responses. This study provides new mechanistic evidences for a role of miR-210 in PACs. PACs were obtained from either adult peripheral blood or cord blood. miR-210 expression was modulated with either an inhibitory complementary oligonucleotide (anti-miR-210) or a miRNA mimic (pre-miR-210). Scramble and absence of transfection served as controls. As expected, hypoxia increased miR-210 in PACs. In vivo, migration toward and adhesion to the ischemic endothelium facilitate the proangiogenic actions of transplanted PACs. In vitro, PAC migration toward SDF-1α/CXCL12 was impaired by anti-miR-210 and enhanced by pre-miR-210. Moreover, pre-miR-210 increased PAC adhesion to ECs and supported angiogenic responses in co-cultured ECs. These responses were not associated with changes in extracellular miR-210 and were abrogated by lentivirus-mediated EFNA3 overexpression. Finally, ex-vivo pre-miR-210 transfection predisposed PACs to induce post-ischemic therapeutic neovascularization and blood flow recovery in an immunodeficient mouse limb ischemia model. In conclusion, miR-210 modulates PAC functions and improves their therapeutic potential in limb ischemia.

High-throughput analysis of the RNA-induced silencing complex in myotonic dystrophy type 1 patients identifies the dysregulation of miR-29c and its target ASB2.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2.

Central role of the p53 pathway in the noncoding-RNA response to oxidative stress.

Oxidative stress plays a fundamental role in many conditions. Specifically, redox imbalance inhibits endothelial cell (EC) growth, inducing cell death and senescence. We used global transcriptome profiling to investigate the involvement of noncoding-RNAs in these phenotypes. By RNA-sequencing, transcriptome changes were analyzed in human ECs exposed to H2O2, highlighting a pivotal role of p53-signaling. Bioinformatic analysis and validation in p53-silenced ECs, identified several p53-targets among both mRNAs and long noncoding-RNAs (lncRNAs), including MALAT1 and NEAT1. Among microRNAs (miRNAs), miR-192-5p was the most induced by H2O2 treatment, in a p53-dependent manner. Down-modulated mRNA-targets of miR-192-5p were involved in cell cycle, DNA repair and stress response. Accordingly, miR-192-5p overexpression significantly decreased EC proliferation, inducing cell death. A central role of the p53-pathway was also confirmed by the analysis of differential exon usage: Upon H2O2 treatment, the expression of p53-dependent 5'-isoforms of MDM2 and PVT1 increased selectively. The transcriptomic alterations identified in H2O2-treated ECs were also observed in other physiological and pathological conditions where redox control plays a fundamental role, such as ECs undergoing replicative senescence, skeletal muscles of critical limb-ischemia patients and the peripheral-blood mononuclear cells of long-living individuals. Collectively, these findings indicate a prominent role of noncoding-RNAs in oxidative stress response.

Increased BACE1-AS long noncoding RNA and ß-amyloid levels in heart failure.

AIMS: Antisense long noncoding RNAs (ncRNAs) are transcripts emerging from the opposite strand of a coding-RNA region and their role in heart failure (HF) is largely unknown. Additionally, HF and Alzheimer's disease (AD) share several non-genetic effectors and risk factors. We investigated the regulation of the ß-secretase-1 (BACE1) gene and of its antisense transcript BACE1-AS in ischaemic HF. METHODS AND RESULTS: BACE1 and BACE1-AS expression was measured in left ventricle biopsies from 18 patients affected by non-end stage ischaemic HF and 17 matched controls. The levels of both transcripts were increased in HF patients. Likewise, both transcripts increased also in a mouse model of ischaemic HF, and their expression was directly correlated. BACE1-AS was expressed by all cardiac cell types and BACE1-AS up- or down-modulation in cultured cardiomyocytes and endothelial cells induced a concordant regulation of the cognate BACE1 transcript. Interestingly, BACE1 increase also induced the intracellular accumulation of its product ß-amyloid. In keeping with these findings, higher BACE1 protein and ß-amyloid peptide levels were also observed in HF. Moreover, increased ß-amyloid 1-40 was also found in the plasma of HF patients. Transcriptomic changes of BACE1-AS overexpressing and ß-amyloid 1-40 treated cells were largely overlapping and indicated changes of relevant biological process such as 'cell cycle and proliferation', 'apoptosis', and 'DNA repair' as well as 'TGFß-, TNFα-, p38-, EGFR-signalling', suggesting a potential maladaptive role of the BACE1-AS/BACE1/ß-amyloid axis. Accordingly, the administration of ß-amyloid peptides decreased the cell viability in endothelial cells and in both human IPS-derived and mouse cardiomyocytes. Moreover, both ß-amyloid treatment and BACE1-AS overexpression increased endothelial cell apoptosis, and this effect was prevented by BACE1 silencing. CONCLUSION: Given the neurotoxic role of ß-amyloid in AD, dysregulation of the BACE1/BACE1-AS/ß-amyloid axis might be relevant in HF pathogenesis, further implicating ncRNAs in the complex scenario of proteotoxicity in cardiac dysfunction.

Long noncoding RNA dysregulation in ischemic heart failure.

BACKGROUND: Long noncoding RNAs (lncRNAs) are non-protein coding transcripts regulating a variety of physiological and pathological functions. However, their implication in heart failure is still largely unknown. The aim of this study is to identify and characterize lncRNAs deregulated in patients affected by ischemic heart failure. METHODS: LncRNAs were profiled and validated in left ventricle biopsies of 18 patients affected by non end-stage dilated ischemic cardiomyopathy and 17 matched controls. Further validations were performed in left ventricle samples derived from explanted hearts of end-stage heart failure patients and in a mouse model of cardiac hypertrophy, obtained by transverse aortic constriction. Peripheral blood mononuclear cells of heart failure patients were also analyzed. LncRNA distribution in the heart was assessed by in situ hybridization. Function of the deregulated lncRNA was explored analyzing the expression of the neighbor mRNAs and by gene ontology analysis of the correlating coding transcripts. RESULTS: Fourteen lncRNAs were significantly modulated in non end-stage heart failure patients, identifying a heart failure lncRNA signature. Nine of these lncRNAs (CDKN2B-AS1/ANRIL, EGOT, H19, HOTAIR, LOC285194/TUSC7, RMRP, RNY5, SOX2-OT and SRA1) were also confirmed in end-stage failing hearts. Intriguingly, among the conserved lncRNAs, h19, rmrp and hotair were also induced in a mouse model of heart hypertrophy. CDKN2B-AS1/ANRIL, HOTAIR and LOC285194/TUSC7 showed similar modulation in peripheral blood mononuclear cells and heart tissue, suggesting a potential role as disease biomarkers. Interestingly, RMRP displayed a ubiquitous nuclear distribution, while H19 RNA was more abundant in blood vessels and was both cytoplasmic and nuclear. Gene ontology analysis of the mRNAs displaying a significant correlation in expression with heart failure lncRNAs identified numerous pathways and functions involved in heart failure progression. CONCLUSIONS: These data strongly suggest lncRNA implication in the molecular mechanisms underpinning HF.

Transcription factor NF-Y induces apoptosis in cells expressing wild-type p53 through E2F1 upregulation and p53 activation.

The CCAAT-binding transcription factor NF-Y plays a central role in regulating cellular proliferation by controlling the expression of genes required for cell-cycle progression such as cyclin A, cyclin B1, cyclin B2, cdc25A, cdc25C, and cdk1. Here we show that unrestricted NF-Y activity leads to apoptosis in an E2F1- and wild-type p53 (wtp53)-dependent manner. Unrestricted NF-Y activity induced an increase in E2F1 mRNA and protein levels. Furthermore, NF-Y directly bound the E2F1 promoter and this correlated with the appearance of open chromatin marks. The ability of NF-Y to induce apoptosis was impaired in cells lacking E2F1 and wtp53. Moreover, NF-Y overexpression elicited phosphorylation of wt p53Ser18 in an E2F1-dependent manner. Our findings establish that NF-Y acts upstream of E2F1 in p53-mediated apoptosis.

NF-Y dependent epigenetic modifications discriminate between proliferating and postmitotic tissue.

The regulation of gene transcription requires posttranslational modifications of histones that, in concert with chromatin remodeling factors, shape the structure of chromatin. It is currently under intense investigation how this structure is modulated, in particular in the context of proliferation and differentiation. Compelling evidence suggests that the transcription factor NF-Y acts as a master regulator of cell cycle progression, activating the transcription of many cell cycle regulatory genes. However, the underlying molecular mechanisms are not yet completely understood. Here we show that NF-Y exerts its effect on transcription through the modulation of the histone "code". NF-Y colocalizes with nascent RNA, while RNA polymerase II is I phosphorylated on serine 2 of the YSPTSPS repeats within its carboxyterminal domain and histones are carrying modifications that represent activation signals of gene expression (H3K9ac and PAN-H4ac). Comparing postmitotic muscle tissue from normal mice and proliferating muscles from mdx mice, we demonstrate by chromatin immunoprecipitation (ChIP) that NF-Y DNA binding activity correlates with the accumulation of acetylated histones H3 and H4 on promoters of key cell cycle regulatory genes, and with their active transcription. Accordingly, p300 is recruited onto the chromatin of NF-Y target genes in a NF-Y-dependent manner, as demonstrated by Re-ChIP. Conversely, the loss of NF-Y binding correlates with a decrease of acetylated histones, the recruitment of HDAC1, and a repressed heterochromatic state with enrichment of histones carrying modifications known to mediate silencing of gene expression (H3K9me3, H3K27me2 and H4K20me3). As a consequence, NF-Y target genes are downregulated in this context. In conclusion, our data indicate a role of NF-Y in modulating the structure and transcriptional competence of chromatin in vivo and support a model in which NF-Y-dependent histone "code" changes contribute to the proper discrimination between proliferating and postmitotic cells in vivo and in vitro.

Requirement for down-regulation of the CCAAT-binding activity of the NF-Y transcription factor during skeletal muscle differentiation.

NF-Y is composed of three subunits, NF-YA, NF-YB, and NF-YC, all required for DNA binding. All subunits are expressed in proliferating skeletal muscle cells, whereas NF-YA alone is undetectable in terminally differentiated cells in vitro. By immunohistochemistry, we show that the NF-YA protein is not expressed in the nuclei of skeletal and cardiac muscle cells in vivo. By chromatin immunoprecipitation experiments, we demonstrate herein that NF-Y does not bind to the CCAAT boxes of target promoters in differentiated muscle cells. Consistent with this, the activity of these promoters is down-regulated in differentiated muscle cells. Finally, forced expression of the NF-YA protein in cells committed to differentiate leads to an impairment in the down-regulation of cyclin A, cyclin B1, and cdk1 expression and is accompanied by a delay in myogenin expression. Thus, our results indicate that the suppression of NF-Y function is of crucial importance for the inhibition of several cell cycle genes and the induction of the early muscle-specific program in postmitotic muscle cells.

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

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.