Reduction of Cardiac Fibrosis by Interference With YAP-Dependent Transactivation.

BACKGROUND: Conversion of cardiac stromal cells into myofibroblasts is typically associated with hypoxia conditions, metabolic insults, and/or inflammation, all of which are predisposing factors to cardiac fibrosis and heart failure. We hypothesized that this conversion could be also mediated by response of these cells to mechanical cues through activation of the Hippo transcriptional pathway. The objective of the present study was to assess the role of cellular/nuclear straining forces acting in myofibroblast differentiation of cardiac stromal cells under the control of YAP (yes-associated protein) transcription factor and to validate this finding using a pharmacological agent that interferes with the interactions of the YAP/TAZ (transcriptional coactivator with PDZ-binding motif) complex with their cognate transcription factors TEADs (TEA domain transcription factors), under high-strain and profibrotic stimulation. METHODS: We employed high content imaging, 2-dimensional/3-dimensional culture, atomic force microscopy mapping, and molecular methods to prove the role of cell/nuclear straining in YAP-dependent fibrotic programming in a mouse model of ischemia-dependent cardiac fibrosis and in human-derived primitive cardiac stromal cells. We also tested treatment of cells with Verteporfin, a drug known to prevent the association of the YAP/TAZ complex with their cognate transcription factors TEADs. RESULTS: Our experiments suggested that pharmacologically targeting the YAP-dependent pathway overrides the profibrotic activation of cardiac stromal cells by mechanical cues in vitro, and that this occurs even in the presence of profibrotic signaling mediated by TGF-ß1 (transforming growth factor beta-1). In vivo administration of Verteporfin in mice with permanent cardiac ischemia reduced significantly fibrosis and morphometric remodeling but did not improve cardiac performance. CONCLUSIONS: Our study indicates that preventing molecular translation of mechanical cues in cardiac stromal cells reduces the impact of cardiac maladaptive remodeling with a positive effect on fibrosis.

P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.

The epigenetic enzyme p300/CBP-associated factor (PCAF) belongs to the GCN5-related N-acetyltransferase (GNAT) family together with GCN5. Although its transcriptional and post-translational function is well characterized, little is known about its properties as regulator of cell metabolism. Here, we report the mitochondrial localization of PCAF conferred by an 85 aa mitochondrial targeting sequence (MTS) at the N-terminal region of the protein. In mitochondria, one of the PCAF targets is the isocitrate dehydrogenase 2 (IDH2) acetylated at lysine 180. This PCAF-regulated post-translational modification might reduce IDH2 affinity for isocitrate as a result of a conformational shift involving predictively the tyrosine at position 179. Site-directed mutagenesis and functional studies indicate that PCAF regulates IDH2, acting at dual level during myoblast differentiation: at a transcriptional level together with MyoD, and at a post-translational level by direct modification of lysine acetylation in mitochondria. The latter event determines a decrease in IDH2 function with negative consequences on muscle fiber formation in C2C12 cells. Indeed, a MTS-deprived PCAF does not localize into mitochondria, remains enriched into the nucleus, and contributes to a significant increase of muscle-specific gene expression enhancing muscle differentiation. The role of PCAF in mitochondria is a novel finding shedding light on metabolic processes relevant to early muscle precursor differentiation.-Savoia, M., Cencioni, C., Mori, M., Atlante, S., Zaccagnini, G., Devanna, P., Di Marcotullio, L., Botta, B., Martelli, F., Zeiher, A. M., Pontecorvi, A., Farsetti, A., Spallotta, F., Gaetano, C. P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.

Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells From Type2 Diabetes Patients: Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation.

RATIONALE: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. OBJECTIVE: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. METHODS AND RESULTS: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. CONCLUSIONS: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.

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.

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.

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.

Proliferation of Multiple Cell Types in the Skeletal Muscle Tissue Elicited by Acute p21 Suppression.

Although in the last decades the molecular underpinnings of the cell cycle have been unraveled, the acquired knowledge has been rarely translated into practical applications. Here, we investigate the feasibility and safety of triggering proliferation in vivo by temporary suppression of the cyclin-dependent kinase inhibitor, p21. Adeno-associated virus (AAV)-mediated, acute knockdown of p21 in intact skeletal muscles elicited proliferation of multiple, otherwise quiescent cell types, notably including satellite cells. Compared with controls, p21-suppressed muscles exhibited a striking two- to threefold expansion in cellularity and increased fiber numbers by 10 days post-transduction, with no detectable inflammation. These changes partially persisted for at least 60 days, indicating that the muscles had undergone lasting modifications. Furthermore, morphological hyperplasia was accompanied by 20% increases in maximum strength and resistance to fatigue. To assess the safety of transiently suppressing p21, cells subjected to p21 knockdown in vitro were analyzed for γ-H2AX accumulation, DNA fragmentation, cytogenetic abnormalities, ploidy, and mutations. Moreover, the differentiation competence of p21-suppressed myoblasts was investigated. These assays confirmed that transient suppression of p21 causes no genetic damage and does not impair differentiation. Our results establish the basis for further exploring the manipulation of the cell cycle as a strategy in regenerative medicine.

microRNAs in ischaemic cardiovascular diseases.

microRNAs (miRNAs) are non-coding RNA molecules that modulate the stability and/or the translational efficiency of specific messenger RNAs. They have been shown to play a regulatory role in most biological processes and their expression is disrupted in many cardiovascular diseases. This review describes studies performed at Policlinico San Donato-IRCCS in cell cultures, animal models, and patients, showing a penetrant role of miRNAs in cell response to hypoxia and in ischaemic cardiovascular diseases. These experiments indicate miRNA as an emerging class of therapeutic targets.

miR-210 is essential to retinal homeostasis in fruit flies and mice.

BACKGROUND: miR-210 is one of the most evolutionarily conserved microRNAs. It is known to be involved in several physiological and pathological processes, including response to hypoxia, angiogenesis, cardiovascular diseases and cancer. Recently, new roles of this microRNA are emerging in the context of eye and visual system homeostasis. Recent studies in Drosophila melanogaster unveiled that the absence of miR-210 leads to a progressive retinal degeneration characterized by the accumulation of lipid droplets and disruptions in lipid metabolism. However, the possible conservation of miR-210 knock-out effect in the mammalian retina has yet to be explored. RESULTS: We further investigated lipid anabolism and catabolism in miR-210 knock-out (KO) flies, uncovering significant alterations in gene expression within these pathways. Additionally, we characterized the retinal morphology of flies overexpressing (OE) miR-210, which was not affected by the increased levels of the microRNA. For the first time, we also characterized the retinal morphology of miR-210 KO and OE mice. Similar to flies, miR-210 OE did not affect retinal homeostasis, whereas miR-210 KO mice exhibited photoreceptor degeneration. To explore other potential parallels between miR-210 KO models in flies and mice, we examined lipid metabolism, circadian behaviour, and retinal transcriptome in mice, but found no similarities. Specifically, RNA-seq confirmed the lack of involvement of lipid metabolism in the mice's pathological phenotype, revealing that the differentially expressed genes were predominantly associated with chloride channel activity and extracellular matrix homeostasis. Simultaneously, transcriptome analysis of miR-210 KO fly brains indicated that the observed alterations extend beyond the eye and may be linked to neuronal deficiencies in signal detection and transduction. CONCLUSIONS: We provide the first morphological characterization of the retina of miR-210 KO and OE mice, investigating the role of this microRNA in mammalian retinal physiology and exploring potential parallels with phenotypes observed in fly models. Although the lack of similarities in lipid metabolism, circadian behaviour, and retinal transcriptome in mice suggests divergent mechanisms of retinal degeneration between the two species, transcriptome analysis of miR-210 KO fly brains indicates the potential existence of a shared upstream mechanism contributing to retinal degeneration in both flies and mammals.

miR-210 as a therapeutic target in diabetes-associated endothelial dysfunction.

BACKGROUND AND PURPOSE: MicroRNA (miR)-210 function in endothelial cells and its role in diabetes-associated endothelial dysfunction are not fully understood. We aimed to characterize the miR-210 function in endothelial cells and study its therapeutic potential in diabetes. EXPERIMENTAL APPROACH: Two different diabetic mouse models (db/db and Western diet-induced), miR-210 knockout and transgenic mice, isolated vessels and human endothelial cells were used. KEY RESULTS: miR-210 levels were lower in aortas isolated from db/db than in control mice. Endothelium-dependent relaxation (EDR) was impaired in aortas from miR-210 knockout mice, and this was restored by inhibiting miR-210 downstream protein tyrosine phosphatase 1B (PTP1B), mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2), and mitochondrial oxidative stress. Inhibition of these pathways also improved EDR in both diabetic mouse models. High glucose reduced miR-210 levels in endothelial cells and impaired EDR in mouse aortas, effects that were reversed by overexpressing miR-210. However, plasma miR-210 levels were not affected in individuals with type 2 diabetes (T2D) following improved glycaemic status. Of note, genetic overexpression using miR-210 transgenic mice and pharmacological overexpression using miR-210 mimic in vivo ameliorated endothelial dysfunction in both diabetic mouse models by decreasing PTP1B, GPD2 and oxidative stress. Genetic overexpression of miR-210 altered the aortic transcriptome, decreasing genes in pathways involved in oxidative stress. miR-210 mimic restored decreased nitric oxide production by high glucose in endothelial cells. CONCLUSION AND IMPLICATIONS: This study unravels the mechanisms by which down-regulated miR-210 by high glucose induces endothelial dysfunction in T2D and demonstrates that miR-210 serves as a novel therapeutic target.

miR-210 hypoxamiR in Angiogenesis and Diabetes.

Significance: microRNA-210 (miR-210) is the master hypoxia-inducible miRNA (hypoxamiR) since it has been found to be significantly upregulated under hypoxia in a wide range of cell types. Recent advances: Gene ontology analysis of its targets indicates that miR-210 modulates several aspects of cellular response to hypoxia. Due to its high pleiotropy, miR-210 not only plays a protective role by fine-tuning mitochondrial metabolism and inhibiting red-ox imbalance and apoptosis, but it can also promote cell proliferation, differentiation, and migration, substantially contributing to angiogenesis. Critical issues: As most miRNAs, modulating different gene pathways, also miR-210 can potentially lead to different and even opposite effects, depending on the physio-pathological contexts in which it acts. Future direction: The use of miRNAs as therapeutics is a fast growing field. This review aimed at highlighting the role of miR-210 in angiogenesis in the context of ischemic cardiovascular diseases and diabetes in order to clarify the molecular mechanisms underpinning miR-210 action. Particular attention will be dedicated to experimentally validated miR-210 direct targets involved in cellular processes related to angiogenesis and diabetes mellitus, such as mitochondrial metabolism, redox balance, apoptosis, migration, and adhesion. Antioxid. Redox Signal. 36, 685-706.

HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment.

The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.

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.

GMP-based CD133(+) cells isolation maintains progenitor angiogenic properties and enhances standardization in cardiovascular cell therapy.

The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133(+) cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133(+) cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133(+) cells. Validation of CD133(+) cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as 'proofs of principle' of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133(+) cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133(+) cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies.

Common micro-RNA signature in skeletal muscle damage and regeneration induced by Duchenne muscular dystrophy and acute ischemia.

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.

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.

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.

Oxidative Stress-Induced miR-200c Disrupts the Regulatory Loop Among SIRT1, FOXO1, and eNOS.

AIMS: Reactive oxygen species (ROS) play a pivotal role in different pathologic conditions, including ischemia, diabetes, and aging. We previously showed that ROS enhance miR-200c expression, causing endothelial cell (EC) apoptosis and senescence. Herein, we dissect the interaction among miR-200c and three strictly related proteins that modulate EC function and ROS production: sirtuin 1 (SIRT1), endothelial nitric oxide synthase (eNOS), and forkhead box O1 (FOXO1). Moreover, the role of miR-200c on ROS modulation was also investigated. RESULTS: We demonstrated that miR-200c directly targets SIRT1, eNOS, and FOXO1; via this mechanism, miR-200c decreased NO and increased the acetylation of SIRT1 targets, that is, FOXO1 and p53. FOXO1 acetylation inhibited its transcriptional activity on target genes, that is, SIRT1 and the ROS scavengers, catalase and manganese superoxide dismutase. In keeping, miR-200c increased ROS production and induced p66Shc protein phosphorylation in Ser-36; this mechanism upregulated ROS and inhibited FOXO1 transcription, reinforcing this molecular circuitry. These in vitro results were validated in three in vivo models of oxidative stress, that is, human skin fibroblasts from old donors, femoral arteries from old mice, and a murine model of hindlimb ischemia. In all cases, miR-200c was higher versus control and its targets, that is, SIRT1, eNOS, and FOXO1, were downmodulated. In the mouse hindlimb ischemia model, anti-miR-200c treatment rescued these targets and improved limb perfusion. Innovation and Conclusion: miR-200c disrupts SIRT1/FOXO1/eNOS regulatory loop. This event promotes ROS production and decreases NO, contributing to endothelial dysfunction under conditions of increased oxidative stress such as aging and ischemia. Antioxid. Redox Signal. 27, 328-344.

p66ShcA modulates tissue response to hindlimb ischemia.

BACKGROUND: Oxidative stress plays a pivotal role in ischemia and ischemia/reperfusion injury. Because p66(ShcA)-null (p66(ShcA)-/-) 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 p66(ShcA)-/- 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 p66(ShcA) wild-type (p66(ShcA)wt) and p66(ShcA)-/- mice. However, significant differences in tissue damage were found: p66(ShcA)wt mice displayed marked capillary density decrease and muscle fiber necrosis. In contrast, in p66(ShcA)-/- 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 p66(ShcA)-/- mice. In agreement with these data, both satellite muscle cells and endothelial cells isolated from p66(ShcA)-/- mice were resistant to apoptosis induced by simulated ischemia in vitro. Lower apoptosis levels after ischemia in p66(ShcA)-/- cells correlated with decreased levels of oxidative stress both in vivo and in vitro. CONCLUSIONS: p66(ShcA) plays a crucial role in the cell death pathways activated by acute ischemia and ischemia/reperfusion, indicating p66(ShcA) as a potential therapeutic target for prevention and treatment of ischemic tissue damage.