Lack of compensatory mitophagy in skeletal muscles during sepsis.

Skeletal muscle dysfunction is a major problem in critically ill patients suffering from sepsis. This condition is associated with mitochondrial dysfunction and increased autophagy in skeletal muscles. Autophagy is a proteolytic mechanism involved in eliminating dysfunctional cellular components, including mitochondria. The latter process, referred to as mitophagy, is essential for maintaining mitochondrial quality and skeletal muscle health. Recently, a fluorescent reporter system called mito-QC (i.e. mitochondrial quality control) was developed to specifically quantify mitophagy levels. In the present study, we used mito-QC transgenic mice and confocal microscopy to morphologically monitor mitophagy levels during sepsis. To induce sepsis, Mito-QC mice received Escherichia coli lipopolysaccharide (10 mg kg-1 i.p.) or phosphate-buffered saline and skeletal muscles (hindlimb and diaphragm) were excised 48 h later. In control groups, there was a negative correlation between the basal mitophagy level and overall muscle mitochondrial content. Sepsis increased general autophagy in both limb muscles and diaphragm but had no effect on mitophagy levels. Sepsis was associated with a downregulation of certain mitophagy receptors (Fundc1, Bcl2L13, Fkbp8 and Phbb2). The present study suggests that general autophagy and mitophagy can be dissociated from one another, and that the characteristic accumulation of damaged mitochondria in skeletal muscles under the condition of sepsis may reflect a failure of adequate compensatory mitophagy. KEY POINTS: There was a negative correlation between the basal level of skeletal muscle mitophagy and the mitochondrial content of individual muscles. Mitophagy levels in limb muscles and the diaphragm were unaffected by lipopolysaccharide (LPS)-induced sepsis. With the exception of BNIP3 in sepsis, LPS administration induced either no change or a downregulation of mitophagy receptors in skeletal muscles.

MFN2 overexpression in skeletal muscles of young and old mice causes a mild hypertrophy without altering mitochondrial respiration and H2O2 emission.

AIM: Sarcopenia, the aging-related loss of muscle mass and function, is a debilitating process negatively impacting the quality of life of affected individuals. Although the mechanisms underlying sarcopenia are incompletely understood, impairments in mitochondrial dynamics, including mitochondrial fusion, have been proposed as a contributing factor. However, the potential of upregulating mitochondrial fusion proteins to alleviate the effects of aging on skeletal muscles remains unexplored. We therefore hypothesized that overexpressing Mitofusin 2 (MFN2) in skeletal muscle in vivo would mitigate the effects of aging on muscle mass and improve mitochondrial function. METHODS: MFN2 was overexpressed in young (7 mo) and old (24 mo) male mice for 4 months through intramuscular injections of an adeno-associated viruses. The impacts of MFN2 overexpression on muscle mass and fiber size (histology), mitochondrial respiration, and H2O2 emission (Oroboros fluororespirometry), and various signaling pathways (qPCR and western blotting) were investigated. RESULTS: MFN2 overexpression increased muscle mass and fiber size in both young and old mice. No sign of fibrosis, necrosis, or inflammation was found upon MFN2 overexpression, indicating that the hypertrophy triggered by MFN2 overexpression was not pathological. MFN2 overexpression even reduced the proportion of fibers with central nuclei in old muscles. Importantly, MFN2 overexpression had no impact on muscle mitochondrial respiration and H2O2 emission in both young and old mice. MFN2 overexpression attenuated the increase in markers of impaired autophagy in old muscles. CONCLUSION: MFN2 overexpression may be a viable approach to mitigate aging-related muscle atrophy and may have applications for other muscle disorders.

Autophagy ablation in skeletal muscles worsens sepsis-induced muscle wasting, impairs whole-body metabolism, and decreases survival.

Septic patients frequently develop skeletal muscle wasting and weakness, resulting in severe clinical consequences and adverse outcomes. Sepsis triggers sustained induction of autophagy, a key cellular degradative pathway, in skeletal muscles. However, the impact of enhanced autophagy on sepsis-induced muscle dysfunction remains unclear. Using an inducible and muscle-specific Atg7 knockout mouse model (Atg7iSkM-KO), we investigated the functional importance of skeletal muscle autophagy in sepsis using the cecal ligation and puncture model. Atg7iSkM-KO mice exhibited a more severe phenotype in response to sepsis, marked by severe muscle wasting, hypoglycemia, higher ketone levels, and a decreased in survival as compared to mice with intact Atg7. Sepsis and Atg7 deletion resulted in the accumulation of mitochondrial dysfunction, although sepsis did not further worsen mitochondrial dysfunction in Atg7iSkM-KO mice. Overall, our study demonstrates that autophagy inactivation in skeletal muscles triggers significant worsening of sepsis-induced muscle and metabolic dysfunctions and negatively impacts survival.

MYTHO is a novel regulator of skeletal muscle autophagy and integrity.

Autophagy is a critical process in the regulation of muscle mass, function and integrity. The molecular mechanisms regulating autophagy are complex and still partly understood. Here, we identify and characterize a novel FoxO-dependent gene, d230025d16rik which we named Mytho (Macroautophagy and YouTH Optimizer), as a regulator of autophagy and skeletal muscle integrity in vivo. Mytho is significantly up-regulated in various mouse models of skeletal muscle atrophy. Short term depletion of MYTHO in mice attenuates muscle atrophy caused by fasting, denervation, cancer cachexia and sepsis. While MYTHO overexpression is sufficient to trigger muscle atrophy, MYTHO knockdown results in a progressive increase in muscle mass associated with a sustained activation of the mTORC1 signaling pathway. Prolonged MYTHO knockdown is associated with severe myopathic features, including impaired autophagy, muscle weakness, myofiber degeneration, and extensive ultrastructural defects, such as accumulation of autophagic vacuoles and tubular aggregates. Inhibition of the mTORC1 signaling pathway in mice using rapamycin treatment attenuates the myopathic phenotype triggered by MYTHO knockdown. Skeletal muscles from human patients diagnosed with myotonic dystrophy type 1 (DM1) display reduced Mytho expression, activation of the mTORC1 signaling pathway and impaired autophagy, raising the possibility that low Mytho expression might contribute to the progression of the disease. We conclude that MYTHO is a key regulator of muscle autophagy and integrity.

The impact of high-fat feeding and parkin overexpression on skeletal muscle mass, mitochondrial respiration, and H2O2 emission.

Obesity is a major risk factor for developing various health problems, including insulin resistance and type 2 diabetes. Although controversial, accumulation of mitochondrial dysfunction, and notably an increase in mitochondrial reactive oxygen species (ROS) production, was proposed as a key contributor leading to obesity-induced insulin resistance. Here, our goal was to investigate whether Parkin overexpression, a key regulator of the removal of dysfunctional mitochondria through mitophagy, could confer protection against obesity-induced mitochondrial dysfunction. To this end, intramuscular injections of adeno-associated viruses (AAVs) were performed to overexpress Parkin in limb muscle of 6-mo-old mice fed a control diet (CD) or a high-fat diet (HFD) for 12 wk. An AAV-expressing the green fluorescent protein (GFP) was used as control. HFD increased fat mass, altered glycemia, and resulted in insulin resistance. Parkin overexpression resulted in an increase in muscle mass in both CD and HFD mice. In CD mice, Parkin overexpression increased maximal mitochondrial respiration and lowered H2O2 emission. HFD increased mitochondrial respiration and, surprisingly, also lowered H2O2 emission. Parkin overexpression did not significantly impact mitochondrial function in HFD mice. Taken altogether, our results indicate that Parkin overexpression positively impacts muscle and mitochondrial health under basal conditions and challenges the notion that intrinsic mitochondrial dysfunction is involved in the development of insulin resistance caused by high-fat feeding.

Long Non-coding RNA Rhabdomyosarcoma 2-Associated Transcript Regulates Angiogenesis in Endothelial Cells.

Background: Long non-coding RNAs (lncRNAs) are non-coding RNAs that have more than 200 nucleotides. They have recently emerged as important regulators of angiogenesis. To identify novel lncRNAs that may be involved in the regulation of angiogenesis, we detected the mRNA of 84 lncRNAs in human umbilical vein endothelial cells (HUVECs) exposed to hypoxia for 24h. One of these, rhabdomyosarcoma 2-associated transcript (RMST), is significantly upregulated by hypoxia. Little is known about the presence and roles of RMST in EC function. Objective: The main objective of the study was to investigate the regulation of RMST in ECs and to determine its role in EC survival, proliferation, migration, and differentiation. Methods: Using qPCR, basal mRNA levels of 10 RMST isoforms in HUVECs were measured. Levels were then measured in response to 24h of hypoxia, 7days of differentiation in a co-culture assay, and exposure to four different angiogenesis factors. Functional roles of RMST in EC survival, migration, and differentiation were quantified by using a loss-of-function approach (transfection with single-stranded antisense LNA GapmeRs). EC survival was measured using cell counts and crystal violet assays. Cell migration and differentiation were measured using scratch wound healing and Matrigel® differentiation assays, respectively. Results: Five RMST isoforms (RMST-202, -203, -204, -206, and -207) were detected in HUVECs and human microvascular endothelial cells (HMEC-1s). Other types of vascular cells, including human aortic valve interstitial cells and human aortic smooth muscle cells, did not display this expression profile. RMST was significantly upregulated in response to 24h of hypoxia and in response to 7days of HUVEC co-culture with human lung fibroblasts. RMST was significantly downregulated by angiopoietin-2 (Ang-2), but not by VEGF, FGF-2, or angiopoietin-1 (Ang-1). Selective knockdown of RMST demonstrated that it promotes EC survival in response to serum deprivation. It is also required for VEGF- and Ang-1-induced EC survival and migration, but not for differentiation. Conclusion: We conclude that RMST is expressed in human ECs and that this expression is upregulated in response to hypoxia and during differentiation into capillary-like structures. We also conclude that RMST plays important roles in EC survival and migration.

ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells.

BACKGROUND: Angiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors. OBJECTIVE: The main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response. METHODS: In silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos). RESULTS: Ang-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively. CONCLUSION: We conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

Roles of miR-640 and Zinc Finger Protein 91 (ZFP91) in Angiopoietin-1-Induced In Vitro Angiogenesis.

Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes angiogenesis. It has been established that regulatory loops exist between angiogenic growth factors and distinct pro or anti-angiogenic miRNAs, but the nature and the roles of Ang-1-regulated miRNAs remain unclear. In this study, we assessed the role of miR-640 in Ang-1-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Exposure to Ang-1 (300 ng/mL) from 6 to 72 h significantly decreased expression of mature miR-640, a response that was mediated by Tie-2 receptors and was also observed in response to Ang-2, the vascular endothelial growth factor, and transforming growth factor ß. Increasing miR-640 levels using a mimic inhibited Ang-1-induced cell migration and capillary-like tube formation whereas inhibition of miR-640 enhanced these responses. Pull down assays of biotinylated miR-640 revealed that miR-640 directly targets Zinc Finger Protein 91 (ZFP91), an atypical E3-ubiquitin ligase. Ang-1 exposure induced ZFP91 expression through down-regulation of miR-640. Silencing of ZFP91 significantly inhibited Ang-1-induced cell migration and tube formation. We conclude that Ang-1 upregulates ZFP91 expression through transcriptional down-regulation of miR-640 and that ZFP91 plays important roles in the promotion of Ang-1-induced endothelial cell migration and differentiation.

Parkin Overexpression Attenuates Sepsis-Induced Muscle Wasting.

Sepsis elicits skeletal muscle weakness and fiber atrophy. The accumulation of injured mitochondria and depressed mitochondrial functions are considered as important triggers of sepsis-induced muscle atrophy. It is unclear whether mitochondrial dysfunctions in septic muscles are due to the inadequate activation of quality control processes. We hypothesized that overexpressing Parkin, a protein responsible for the recycling of dysfunctional mitochondria by the autophagy pathway (mitophagy), would confer protection against sepsis-induced muscle atrophy by improving mitochondrial quality and content. Parkin was overexpressed for four weeks in the limb muscles of four-week old mice using intramuscular injections of adeno-associated viruses (AAVs). The cecal ligation and perforation (CLP) procedure was used to induce sepsis. Sham operated animals were used as controls. All animals were studied for 48 h post CLP. Sepsis resulted in major body weight loss and myofiber atrophy. Parkin overexpression prevented myofiber atrophy in CLP mice. Quantitative two-dimensional transmission electron microscopy revealed that sepsis is associated with the accumulation of enlarged and complex mitochondria, an effect which was attenuated by Parkin overexpression. Parkin overexpression also prevented a sepsis-induced decrease in the content of mitochondrial subunits of NADH dehydrogenase and cytochrome C oxidase. We conclude that Parkin overexpression prevents sepsis-induced skeletal muscle atrophy, likely by improving mitochondrial quality and contents.

Differential regulation of myofibrillar proteins in skeletal muscles of septic mice.

Sepsis elicits skeletal muscle atrophy as a result of decreased total protein synthesis and/or increased total protein degradation. It is unknown how and whether sepsis differentially affects the expression of specific myofibrillar proteins in respiratory and limb muscles. In this study, we measured the effects of sepsis myofibrillar mRNAs and their corresponding protein levels in the diaphragm (DIA) and tibialis anterior (TA) muscles in a murine cecal ligation and perforation (CLP) model of sepsis. Male mice (C57/BL6j) underwent CLP-induced sepsis. Sham-operated mice were subjected to the same surgical procedures, except for CLP. Mice were euthanized 24, 48, or 96 h postsurgery. Transcript and protein levels of autophagy-related genes, ubiquitin E3 ligases, and several myofibrillar genes were quantified. Sepsis elicited transient fiber atrophy in the DIA and prolonged atrophy in the TA. Atrophy was coincident with increased autophagy and ubiquitin E3 ligase expression. Myosin heavy chain isoforms decreased at 24 h in the DIA and across the time-course in the TA, myosin light chain isoforms decreased across the time-course in both muscles, and troponins T and C as well as tropomyosin decreased after 24 and 48 h in both the DIA and TA. α-Actin and troponin I were unaffected by sepsis. Sepsis-induced decreases in myofibrillar protein levels coincided with decreased mRNA expressions of these proteins, suggesting that transcriptional inhibition is involved. We hypothesize that sepsis-induced muscle atrophy is mediated by decreased transcription and enhanced degradation of specific myofibrillar proteins, including myosin heavy and light chains, troponin C, troponin T, and tropomyosin.

Oxidants Regulated Diaphragm Proteolysis during Mechanical Ventilation in Rats.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: Diaphragm dysfunction and atrophy develop during controlled mechanical ventilation. Although oxidative stress injures muscle during controlled mechanical ventilation, it is unclear whether it causes autophagy or fiber atrophy. WHAT THIS ARTICLE TELLS US THAT IS NEW: Pretreatment of rats undergoing 24 h of mechanical ventilation with N-acetylcysteine prevents decreases in diaphragm contractility, inhibits the autophagy and proteasome pathways, but has no influence on the development of diaphragm fiber atrophy. BACKGROUND: Diaphragm dysfunction and atrophy develop during prolonged controlled mechanical ventilation. Fiber atrophy has been attributed to activation of the proteasome and autophagy proteolytic pathways. Oxidative stress activates the proteasome during controlled mechanical ventilation, but it is unclear whether it also activates autophagy. This study investigated whether pretreatment with the antioxidant N-acetylcysteine affects controlled mechanical ventilation-induced diaphragm contractile dysfunction, fiber atrophy, and proteasomal and autophagic pathway activation. The study also explored whether proteolytic pathway activity during controlled mechanical ventilation is mediated by microRNAs that negatively regulate ubiquitin E3 ligases and autophagy-related genes. METHODS: Three groups of adult male rats were studied (n = 10 per group). The animals in the first group were anesthetized and allowed to spontaneously breathe. Animals in the second group were pretreated with saline before undergoing controlled mechanical ventilation for 24 h. The animals in the third group were pretreated with N-acetylcysteine (150 mg/kg) before undergoing controlled mechanical ventilation for 24 h. Diaphragm contractility and activation of the proteasome and autophagy pathways were measured. Expressions of microRNAs that negatively regulate ubiquitin E3 ligases and autophagy-related genes were measured with quantitative polymerase chain reaction. RESULTS: Controlled mechanical ventilation decreased diaphragm twitch force from 428 ± 104 g/cm (mean ± SD) to 313 ± 50 g/cm and tetanic force from 2,491 ± 411 g/cm to 1,618 ± 177 g/cm. Controlled mechanical ventilation also decreased diaphragm fiber size, increased expression of several autophagy genes, and augmented Atrogin-1, MuRF1, and Nedd4 expressions by 36-, 41-, and 8-fold, respectively. Controlled mechanical ventilation decreased the expressions of six microRNAs (miR-20a, miR-106b, miR-376, miR-101a, miR-204, and miR-93) that regulate autophagy genes. Pretreatment with N-acetylcysteine prevented diaphragm contractile dysfunction, attenuated protein ubiquitination, and downregulated E3 ligase and autophagy gene expression. It also reversed controlled mechanical ventilation-induced microRNA expression decreases. N-Acetylcysteine pretreatment had no affect on fiber atrophy. CONCLUSIONS: Prolonged controlled mechanical ventilation activates the proteasome and autophagy pathways in the diaphragm through oxidative stress. Pathway activation is accomplished, in part, through inhibition of microRNAs that negatively regulate autophagy-related genes.

Negative regulation of angiogenesis by novel micro RNAs.

Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes survival, migration, and differentiation of endothelial cells (ECs). Recent studies have identified several microRNA (miRNA) families that either promote or inhibit angiogenesis. To date, the nature and functional importance of miRNAs in Ang-1-induced angiogenesis are unknown. Microarray screening of known miRNAs in human umbilical vein endothelial cells (HUVECs) revealed that the expressions of miR-103b, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p significantly decrease following exposure to Ang-1 for 24 h. Exposure to the angiogenesis factors angiopoietin-2 (Ang-2), vascular endothelial growth factor, fibroblast growth factor 2, and transforming growth factor ß also inhibits miR-103b expression, but exerts varying effects on the other miRNAs. By overexpressing miR-103b, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p with selective mimics, we demonstrated that the pro-survival effects of Ang-1 are eliminated, Caspase-3 activity increases, and cell migration, proliferation, and capillary-like tube formation decreases. Conversely, transfection with selective miRNA inhibitors increases cell survival, inhibits Caspase-3 activity, and stimulates migration, proliferation and tube formation. miRNet miRNA-target gene network analyses revealed that miR-103, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p directly interact with 47, 95, 165, 108, 49, and 16 gene targets, respectively. Since many of these genes are positive regulators of angiogenic processes, we conclude that these miRNAs function as anti-angiogenic miRNAs and that their downregulation may be essential for Ang-1-induced angiogenesis to occur.

Differential Regulation of the Autophagy and Proteasome Pathways in Skeletal Muscles in Sepsis.

OBJECTIVES: Skeletal muscle fiber atrophy develops in response to severe sepsis, but it is unclear as to how the proteolytic pathways that are involved in its development are differentially regulated. We investigated the link between sepsis-induced fiber atrophy and activation of the proteasome and autophagy pathways and whether the degree of activation is more severe and sustained in limb muscles than it is in the diaphragm. DESIGN: Randomized controlled experiment. SETTING: Animal research laboratory. SUBJECTS: Adult male C57/BL6 mice. INTERVENTIONS: Two groups of animals were studied. The sepsis group was subjected to a cecal ligation and perforation technique, whereas the control (sham) group was subjected to abdominal surgery without cecal ligation and perforation. Measurements for both groups were performed 24, 48, and 96 hours after the surgical procedure. MEASUREMENTS AND MAIN RESULTS: Atrophy was quantified in the diaphragm and tibialis anterior by measuring fiber diameter. Autophagy was evaluated using electron microscopic detection of autophagosomes and by measuring LC3B protein lipidation and autophagy-related protein expressions. Proteasomal degradation was quantified by measuring chymotrypsin-like activity of the 26S proteasome and messenger RNA expressions of muscle-specific E3 ligases. Sepsis triggered transient fiber atrophy in the diaphragm that lasted for 24 hours and prolonged atrophy in the tibialis anterior that persisted for 96 hours. The autophagy and proteasome pathways were activated in both muscles at varying intensities over the time course of sepsis. Activation was more pronounced in the tibialis anterior than in the diaphragm. Sepsis inhibited the V-Akt thymoma viral oncogene homolog 1 and complex 1 of the mammalian target of rapamycin pathways and stimulated the AMP-activated protein kinase pathway in both muscles. CONCLUSIONS: Sepsis triggers more severe and sustained muscle fiber atrophy in limb muscles when compared with respiratory muscle. This response is associated with enhanced proteasomal and autophagic proteolytic pathway activities and is triggered by inhibition of the AKT and complex 1 of the mammalian target of rapamycin pathways and activation of the AMPK pathway.

NOX2, NOX4, and mitochondrial-derived reactive oxygen species contribute to angiopoietin-1 signaling and angiogenic responses in endothelial cells.

Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes survival, migration, and differentiation of endothelial cells. Several studies have linked reactive oxygen species (ROS) to Ang-1 signaling and distinct angiogenic responses, but the molecular sources of these ROS have never been clearly identified. In this study, we have identified source-specific contributions of ROS to Ang-1/Tie 2 signaling and angiogenic responses in human umbilical vein endothelial cells (HUVECs), specifically the differential contributions of mitochondrial ROS (mtROS) and ROS from two isoforms of NADPH oxidase (NOX2, NOX4). We demonstrate that: 1) Ang-1 induces significant increases in mtROS production under normal conditions but does not when cells are pre-incubated with mitochondrial antioxidants; 2) Ang-1 induces rapid Tie-2-dependent increases in cytosolic ROS production but does not when NOX2 and NOX4 are knocked down; 3) Ang-1 induces simultaneous increases in phosphorylation of AKT, ERK1/2, p38, and SAPK/JNK proteins within a few minutes of exposure, but this response is strongly and selectively attenuated when NOX2 and NOX4 are knocked down or cells are pre-treated with mitochondrial antioxidants; 4) Ang-1 exerts a strong effect on HUVEC survival in serum-deprived medium and enhances cell migration and capillary tube formation, but the survival response is inhibited by NOX2 knockdown and the migration and tube formation responses are entirely absent with NOX4 knockdown or pre-treatment with mitochondrial antioxidants. We conclude that Ang-1 triggers NOX2, NOX4, and the mitochondria to release ROS and that ROS derived from these sources play distinct roles in the regulation of the Ang-1/Tie 2 signaling pathway and pro-angiogenic responses.

Failed reinnervation in aging skeletal muscle.

BACKGROUND: Skeletal muscle displays a marked accumulation of denervated myofibers at advanced age, which coincides with an acceleration of muscle atrophy. METHODS: In this study, we evaluated the hypothesis that the accumulation of denervated myofibers in advanced age is due to failed reinnervation by examining muscle from young adult (YA) and very old (VO) rats and from a murine model of sporadic denervation secondary to neurotrypsin over-expression (Sarco mouse). RESULTS: Both aging rat muscle and Sarco mouse muscle exhibited marked fiber-type grouping, consistent with repeating cycles of denervation and reinnervation, yet in VO muscle, rapsyn at the endplate increased and was associated with only a 10 % decline in acetylcholine receptor (AChR) intensity, whereas in Sarco mice, there was a decline in rapsyn and a 25 % decrease in AChR intensity. Transcripts of muscle-specific kinase (21-fold), acetylcholine receptor subunits α (68-fold), ε (threefold) and γ (47-fold), neural cell adhesion molecule (66-fold), and runt-related transcription factor 1 (33-fold) were upregulated in VO muscle of the rat, consistent with the marked persistent denervation evidenced by a large proportion of very small fibers (>20 %). In the Sarco mice, there were much smaller increases in denervation transcripts (0-3.5-fold) and accumulation of very small fibers (2-6 %) compared to the VO rat, suggesting a reduced capacity for reinnervation in aging muscle. Despite the marked persistent denervation in the VO rat muscle, transcripts of neurotrophins involved in promoting axonal sprouting following denervation exhibited no increase, and several miRNAs predicted to suppress neurotrophins were elevated in VO rat. CONCLUSIONS: Our results support the hypothesis that the accumulation of denervated fibers with aging is due to failed reinnervation and that this may be affected by a limited neurotrophin response that mediates axonal sprouting following denervation.

Fibulin-5 Regulates Angiopoietin-1/Tie-2 Receptor Signaling in Endothelial Cells.

BACKGROUND: Fibulin-5 is an extracellular matrix glycoprotein that plays critical roles in vasculogenesis and embryonic development. Deletion of Fibulin-5 in mice results in enhanced skin vascularization and upregulation of the angiogenesis factor angiopoietin-1 (Ang-1), suggesting that Fibulin-5 functions as an angiogenesis inhibitor. In this study, we investigate the inhibitory effects of Fibulin-5 on Ang-1/TIE-2 receptor pathway signaling and cell survival in human endothelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant wild-type and RGE-mutant Fibulin-5 proteins were generated through stable transfection of HEK293 and CHO cells, respectively. In vitro solid phase binding assays using pure proteins revealed that wild-type Fibulin-5 does not bind to Ang-1 or TIE-2 proteins but strongly binds to heparin. Binding assays using human umbilical vein endothelial cells (HUVECs) indicated that wild-type Fibulin-5 strongly binds to cells but RGE-mutant Fibulin-5, which is incapable of binding to integrins, does not. Pre-incubation of HUVECs for 1 hr with Fibulin-5 significantly increased caspase 3/7 activity, ERK1/2 phosphorylation, and expressions of the transcription factor early growth response 1 (EGR1) and the dual-specificity phosphatase 5 (DUSP5). Fibulin-5 also strongly attenuated Ang-1-induced TIE-2 and AKT phosphorylation, decreased Ang-1-induced expressions of the transcription factors Inhibitor of DNA Binding 1 (ID1) and Kruppel-like Factor 2 (KLF2), and reversed the inhibitory effect of Ang-1 on serum deprivation-induced cytotoxicity and caspase 3/7 activity. CONCLUSION/SIGNIFICANCE: We conclude that Fibulin-5 strongly binds to the endothelial cell surface through heparin-sulfate proteoglycans and possibly integrins and that it exerts strong anti-angiogenic effects by reducing endothelial cell viability and interfering with the signaling pathways of the Ang-1/TIE-2 receptor axis.

Angiopoietin-1 inhibits toll-like receptor 4 signalling in cultured endothelial cells: role of miR-146b-5p.

AIMS: Bacterial lipopolysaccharides (LPS) induce innate immune inflammatory responses in endothelial cells by activating toll-like receptor 4 (TLR4) signalling. Here, we investigate the effects of angiopoietin-1 (Ang-1) on LPS-induced TLR4 signalling and the role of the miR-146 family of micro RNAs in the effects of Ang-1 on TRL4 signalling. METHODS AND RESULTS: Leucocyte adhesion to human umbilical vein endothelial cells (HUVECs) was detected using fluorescence microscopy. Adhesion molecule, pro-inflammatory cytokine, miR-146a, and miR-146b-5p expressions in HUVECs were quantified using real-time PCR. TLR4 signalling protein levels were measured using immunoblotting. Exposure of HUVECs to LPS for 4-6 h induces robust inflammatory responses, including enhanced leucocyte adhesion, up-regulation of adhesion molecule expression (VCAM1, ICAM1, E-SELECTIN), enhanced cytokine production (TNFα, IL1ß, IL6, and IL8), and increased NFκB luciferase reporter activity. Addition of Ang-1 to the culture medium for 24 h prior to LPS exposure significantly attenuates these responses. Prolonged Ang-1 exposure significantly decreases IRAK1 and TRAF6 protein levels but has no effect on TLR4, MYD88, IRAK4, or TAK1 expressions. Ang-1 triggers significant up-regulation of miR-146b-5p levels but has no effect on miR-146a or miR-146b-3p expressions. Transfection of HUVECs with a miR-146b-5p mimic significantly attenuates LPS-induced inflammatory responses and IRAK1 and TRAF6 expressions. In HUVECs transfected with a miR-146b-5p inhibitor, Ang-1 has no effect on LPS-induced inflammatory responses or IRAK1 and TRAF6 expressions. CONCLUSION: Ang-1 disrupts TLR4 signalling, resulting in inhibition of LPS-induced inflammatory responses in endothelial cells. This inhibition occurs through selective targeting of IRAK1 and TRAF6 proteins by miR-146b-5p.

Early growth response-1 regulates angiopoietin-1-induced endothelial cell proliferation, migration, and differentiation.

OBJECTIVE: Angiopoietin-1 (Ang-1) is an important regulator of angiogenesis in endothelial cells. It promotes migration, proliferation, and differentiation of cells, although the regulating factors involved in these processes remain unclear. In this study, we evaluated the contribution of the transcription factor early growth response-1 (Egr-1) to Ang-1-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Expression of Egr-1 was evaluated with real-time PCR and immunoblotting, whereas Egr-1 DNA binding activity was monitored with electrophoretic mobility shift assays. Cell migration was measured with wound healing and Boyden chamber assays, whereas cell proliferation and differentiation of cells into capillary-like tube structures were monitored with cell counting, BrdU incorporation and Matrigels. To selectively inhibit Egr-1 expression, we used both siRNA oligonucleotides and specific DNAzymes. Egr-1 mRNA expression rose approximately 9-fold within 2 hours of Ang-1 exposure and declined thereafter. Upregulation of Egr-1 expression was accompanied by an increase in nuclear mobilization and augmented DNA binding. These processes were mediated through the Erk1/2, PI-3 kinase/AKT, and mTOR pathways. Knockdown of Egr-1 expression completely abrogated Ang-1-induced endothelial migration and significantly reduced proliferation and capillary-like tube formation of HUVECs that overexpress Ang-1. CONCLUSIONS: Ang-1 triggers significant and transient induction of Egr-1, and Egr-1 contributes to Ang-1-induced endothelial cell migration and proliferation.

Regulation of angiopoietin expression by bacterial lipopolysaccharide.

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-kappaB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.

Regulation of proliferation of skeletal muscle precursor cells by NADPH oxidase.

Skeletal muscle precursor cells are adult stem cells located among muscle fibers. Proliferation, migration, and subsequent differentiation of these cells are critical steps in the repair of muscle injury. We document in this study the roles and mechanisms through which the NAPDH oxidase complex regulates muscle precursor cell proliferation. The NADPH oxidase subunits Nox2, Nox4, p22(phox), p47(phox), and p67(phox) were detected in primary human and murine skeletal muscle precursor cells. In human muscle precursor cells, NADPH oxidase-fusion proteins were localized in the cytosolic and membrane compartments of the cell, except for p47(phox), which was detected in the nucleus. In proliferating subconfluent precursor cells, both Nox2 and Nox4 contributed to O(2)(-) production. However, Nox4 expression was significantly attenuated in differentiated myotubes. Proliferation of precursor cells was significantly reduced by antioxidants (N-acetylcysteine and apocynin), inhibition of p22(phox) expression by using siRNA oligonucleotides, and reduction of Nox4 and p47(phox) activities with dominant-negative vectors and siRNA oligonucleotides resulted in attenuation of activities of the Erk1/2, PI-3 kinase/AKT and NFkappaB pathways and significant reduction in cyclin D1 levels. We conclude that NADPH oxidase is expressed in skeletal muscle precursor cells and that its activity plays an important role in promoting proliferation of these cells.