PMP2 regulates myelin thickening and ATP production during remyelination.

It is well established that axonal Neuregulin 1 type 3 (NRG1t3) regulates developmental myelin formation as well as EGR2-dependent gene activation and lipid synthesis. However, in peripheral neuropathy disease context, elevated axonal NRG1t3 improves remyelination and myelin sheath thickness without increasing Egr2 expression or activity, and without affecting the transcriptional activity of canonical myelination genes. Surprisingly, Pmp2, encoding for a myelin fatty acid binding protein, is the only gene whose expression increases in Schwann cells following overexpression of axonal NRG1t3. Here, we demonstrate PMP2 expression is directly regulated by NRG1t3 active form, following proteolytic cleavage. Then, using a transgenic mouse model overexpressing axonal NRG1t3 (NRG1t3OE) and knocked out for PMP2, we demonstrate that PMP2 is required for NRG1t3-mediated remyelination. We demonstrate that the sustained expression of Pmp2 in NRG1t3OE mice enhances the fatty acid uptake in sciatic nerve fibers and the mitochondrial ATP production in Schwann cells. In sum, our findings demonstrate that PMP2 is a direct downstream mediator of NRG1t3 and that the modulation of PMP2 downstream NRG1t3 activation has distinct effects on Schwann cell function during developmental myelination and remyelination.

DMT1-dependent endosome-mitochondria interactions regulate mitochondrial iron translocation and metastatic outgrowth.

Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1-dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells.

Regulation of DNA damage and transcriptional output in the vasculature through a cytoglobin-HMGB2 axis.

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

Nuclear cytoglobin associates with HMGB2 and regulates DNA damage and genome-wide transcriptional output in the vasculature.

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

A role for cytoglobin in regulating intracellular hydrogen peroxide and redox signals in the vasculature.

The oxidant hydrogen peroxide serves as a signaling molecule that alters many aspects of cardiovascular functions. Recent studies suggest that cytoglobin - a hemoglobin expressed in the vasculature - may promote electron transfer reactions with proposed functions in hydrogen peroxide decomposition. Here, we determined the extent to which cytoglobin regulates intracellular hydrogen peroxide and established mechanisms. We found that cytoglobin decreased the hyperoxidation of peroxiredoxins and maintained the activity of peroxiredoxin 2 following challenge with exogenous hydrogen peroxide. Cytoglobin promoted a reduced intracellular environment and facilitated the reduction of the thiol-based hydrogen peroxide sensor Hyper7 after bolus addition of hydrogen peroxide. Cytoglobin also limited the inhibitory effect of hydrogen peroxide on glycolysis and reversed the oxidative inactivation of the glycolytic enzyme GAPDH. Our results indicate that cytoglobin in cells exists primarily as oxyferrous cytoglobin (CygbFe 2+ -O 2 ) with its cysteine residues in the reduced form. We found that the specific substitution of one of two cysteine residues on cytoglobin (C83A) inhibited the reductive activity of cytoglobin on Hyper7 and GAPDH. Carotid arteries from cytoglobin knockout mice were more sensitive to glycolytic inhibition by hydrogen peroxide than arteries from wildtype mice. Together, these results support a role for cytoglobin in regulating intracellular redox signals associated with hydrogen peroxide through oxidation of its cysteine residues, independent of hydrogen peroxide reaction at its heme center.

Radiation-Induced Macrophage Senescence Impairs Resolution Programs and Drives Cardiovascular Inflammation.

Radiation is associated with tissue damage and increased risk of atherosclerosis, but there are currently no treatments and a very limited mechanistic understanding of how radiation impacts tissue repair mechanisms. We uncovered that radiation significantly delayed temporal resolution programs that were associated with decreased efferocytosis in vivo. Resolvin D1 (RvD1), a known proresolving ligand, promoted swift resolution and restored efferocytosis in sublethally irradiated mice. Irradiated macrophages exhibited several features of senescence, including increased expression of p16INK4A and p21, heightened levels of SA-ß-gal, COX-2, several proinflammatory cytokines/chemokines, and oxidative stress (OS) in vitro, and when transferred to mice, they exacerbated inflammation in vivo. Mechanistically, heightened OS in senescent macrophages led to impairment in their ability to carry out efficient efferocytosis, and treatment with RvD1 reduced OS and improved efferocytosis. Sublethally irradiated Ldlr -/- mice exhibited increased plaque necrosis, p16INK4A cells, and decreased lesional collagen compared with nonirradiated controls, and treatment with RvD1 significantly reduced necrosis and increased lesional collagen. Removal of p16INK4A hematopoietic cells during advanced atherosclerosis with p16-3MR mice reduced plaque necrosis and increased production of key intraplaque-resolving mediators. Our results demonstrate that sublethal radiation drives macrophage senescence and efferocytosis defects and suggest that RvD1 may be a new therapeutic strategy to limit radiation-induced tissue damage.

SDH Subunit C Regulates Muscle Oxygen Consumption and Fatigability in an Animal Model of Pulmonary Emphysema.

Patients with pulmonary emphysema often develop locomotor muscle dysfunction, which is independently associated with disability and higher mortality in that population. Muscle dysfunction entails reduced force generation capacity, which partially depends on fibers' oxidative potential, yet very little mechanistic research has focused on muscle respiration in pulmonary emphysema. Using a recently established animal model of pulmonary emphysema-driven skeletal muscle dysfunction, we found downregulation of SDHC (succinate dehydrogenase subunit C) in association with lower oxygen consumption and fatigue tolerance in locomotor muscles. Reduced SDH activity has been previously observed in muscles from patients with pulmonary emphysema, and we found that SDHC is required to support respiration in cultured muscle cells. Moreover, in vivo gain of SDH function in emphysema animals' muscles resulted in better oxygen consumption rate and fatigue tolerance. These changes correlated with a larger number of relatively more oxidative type 2-A and 2X fibers and a reduced amount of 2B fibers. Our data suggest that SDHC is a key regulator of respiration and fatigability in pulmonary emphysema-driven skeletal muscles, which could be impactful in developing strategies aimed at attenuating this comorbidity.

Emerging perspectives on cytoglobin, beyond NO dioxygenase and peroxidase.

Cytoglobin is an evolutionary ancient hemoglobin with poor functional annotation. Rather than constrained to penta coordination, cytoglobin's heme iron may exist either as a penta or hexacoordinated arrangement when exposed to different intracellular environments. Two cysteine residues at the surface of the protein form an intramolecular disulfide bond that regulates iron coordination, ligand binding, and peroxidase activity. Overall, biochemical results do not support a role for cytoglobin as a direct antioxidant enzyme that scavenges hydrogen peroxide because the rate of the reaction of cytoglobin with hydrogen peroxide is several orders of magnitude slower than metal and thiol-based peroxidases. Thus, alternative substrates such as fatty acids have been suggested and regulation of nitric oxide bioavailability through nitric oxide dioxygenase and nitrite reductase activities has received experimental support. Cytoglobin is broadly expressed in connective, muscle, and nervous tissues. Rational for differential cellular distribution is poorly understood but inducibility in response to hypoxia is one of the most established features of cytoglobin expression with regulation through the transcription factor hypoxia-inducible factor (HIF). Phenotypic characterization of cytoglobin deletion in the mouse have indicated broad changes that include a heightened inflammatory response and fibrosis, increase tumor burden, cardiovascular dysfunction, and hallmarks of senescence. Some of these changes might be reversed upon inhibition of nitric oxide synthase. However, subcellular and molecular interactions have been seldom characterized. In addition, specific molecular mechanisms of action are still lacking. We speculate that cytoglobin functionality will extend beyond nitric oxide handling and will have to encompass indirect regulatory antioxidant and redox sensing functions.

Thymine DNA glycosylase is a key regulator of CaMKIIγ expression and vascular smooth muscle phenotype.

Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multigene family with isoform-specific regulation of vascular smooth muscle (VSM) functions. In previous studies, we found that vascular injury resulted in VSM dedifferentiation and reduced expression of the CaMKIIγ isoform in medial wall VSM. Smooth muscle knockout of CaMKIIγ enhanced injury-induced VSM neointimal hyperplasia, whereas CaMKIIγ overexpression inhibited VSM proliferation and neointimal formation. In this study, we evaluated DNA cytosine methylation/demethylation as a mechanism for regulating CaMKII isoform expression in VSM. Inhibition of cytosine methylation with 5-Aza-2'-deoxycytidine significantly upregulated CaMKIIγ expression in cultured VSM cells and inhibited CaMKIIγ downregulation in organ-cultured aorta ex vivo. With the use of methylated cytosine immunoprecipitation, the rat Camk2g promoter was found hypomethylated in differentiated VSM, whereas injury- or cell culture-induced VSM dedifferentiation coincided with Camk2g promoter methylation and decreased expression. We report for the first time that VSM cell phenotype switching is accompanied by marked induction of thymine DNA glycosylase (TDG) protein and mRNA expression in injured arteries in vivo and in cultured VSM synthetic phenotype cells. Silencing Tdg in VSM promoted expression of CaMKIIγ and differentiation markers, including myocardin, and inhibited VSM cell proliferation and injury-induced neointima formation. This study indicates that CaMKIIγ expression in VSM is regulated by cytosine methylation/demethylation and that TDG is an important determinant of this process and, more broadly, VSM phenotype switching and function.NEW & NOTEWORTHY Expression of the calcium calmodulin-dependent protein kinase II-γ isoform (CaMKIIγ) is associated with differentiated vascular smooth muscle (VSM) and negatively regulates proliferation in VSM synthetic phenotype (VSMSyn) cells. This study demonstrates that thymine DNA glycosylase (TDG) plays a key role in regulating CaMKIIγ expression in VSM through promoter cytosine methylation/demethylation. TDG expression is strongly induced in VSMSyn cells and plays key roles in negatively regulating CaMKIIγ expression and more broadly VSM phenotype switching.

Vascular smooth muscle-MAPK14 is required for neointimal hyperplasia by suppressing VSMC differentiation and inducing proliferation and inflammation.

Injury-induced stenosis is a serious vascular complication. We previously reported that p38α (MAPK14), a redox-regulated p38MAPK family member was a negative regulator of the VSMC contractile phenotype in vitro. Here we evaluated the function of VSMC-MAPK14 in vivo in injury-induced neointima hyperplasia and the underlying mechanism using an inducible SMC-MAPK14 knockout mouse line (iSMC-MAPK14-/-). We show that MAPK14 expression and activity were induced in VSMCs after carotid artery ligation injury in mice and ex vivo cultured human saphenous veins. While the vasculature from iSMC-MAPK14-/- mice was indistinguishable from wildtype littermate controls at baseline, these mice exhibited reduced neointima formation following carotid artery ligation injury. Concomitantly, there was an increased VSMC contractile protein expression in the injured vessels and a decrease in proliferating cells. Blockade of MAPK14 through a selective inhibitor suppressed, while activation of MAPK14 by forced expression of an upstream MAPK14 kinase promoted VSMC proliferation in cultured VSMCs. Genome wide RNA array combined with VSMC lineage tracing studies uncovered that vascular injury evoked robust inflammatory responses including the activation of proinflammatory gene expression and accumulation of CD45 positive inflammatory cells, which were attenuated in iSMC-MAPK14-/- mice. Using multiple pharmacological and molecular approaches to manipulate MAPK14 pathway, we further confirmed the critical role of MAPK14 in activating proinflammatory gene expression in cultured VSMCs, which occurs in a p65/NFkB-dependent pathway. Finally, we found that NOX4 contributes to MAPK14 suppression of the VSMC contractile phenotype. Our results revealed that VSMC-MAPK14 is required for injury-induced neointima formation, likely through suppressing VSMC differentiation and promoting VSMC proliferation and inflammation. Our study will provide mechanistic insights into therapeutic strategies for mitigation of vascular stenosis.

Vimentin expression is required for the development of EMT-related renal fibrosis following unilateral ureteral obstruction in mice.

Most renal transplants ultimately fail secondary to chronic allograft nephropathy (CAN). Vimentin (vim) is a member of the intermediate filament family of proteins and has been shown to be important in the development of CAN. One of the pathways leading to chronic renal fibrosis after transplant is thought to be epithelial to mesenchymal transition (EMT). Even though vim expression is one of the main steps of EMT, it is unknown whether vim expression is required for EMT leading to renal fibrosis and allograft loss. To this end, the role of vim in renal fibrosis was determined via unilateral ureteral obstruction (UUO) in vim knockout mice (129 svs6 vim -/-). Following UUO, kidneys were recovered and analyzed via Western blotting, immunofluorescence, and transcriptomics. Cultured human proximal renal tubular (HK-2) cells were subjected to lentiviral-driven inhibition of vim expression and then treated with transforming growth factor (TGF)-ß to undergo EMT. Immunoblotting as well as wound healing assays were used to determine development of EMT. Western blotting analyses of mice undergoing UUO reveal increased levels of vim soon after UUO. As expected, interstitial collagen deposition increased in control mice following UUO but decreased in vim -/- kidneys. Immunofluorescence analyses also revealed altered localization of ß-catenin in vim -/- mice undergoing UUO without significant changes in mRNA levels. However, RNA sequencing revealed a decrease in ß-catenin-dependent genes in vim -/- kidneys. Finally, vim-silenced HK-2 cell lines undergoing EMT were shown to have decreased cellular migration during wound healing. We conclude that vim inhibition decreases fibrosis following UUO by possibly altering ß-catenin localization and downstream signaling.

A Brief Overview of Nitric Oxide and Reactive Oxygen Species Signaling in Hypoxia-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is characterized by increased vasoconstriction and smooth muscle cell hyperplasia driving pathological vascular remodeling of arterial vessels. In this short review, we discuss the primary source of reactive oxygen species (ROS) and nitric oxide (NO) relevant to PH and the mechanism by which dysregulation of their production contributes to PH. Specifically, hypoxia-induced PH is associated with diminished endothelial nitric oxide synthase (eNOS)-derived NO production and increased production of superoxide (O2•-) through eNOS uncoupling and defective mitochondrial respiration. This drives the inhibition of the NO/soluble guanylate cyclase (sGC) pathway and activation of the transcription factor hypoxia-inducible factor-1α (HIF-1α) with consequential dysregulation of the pulmonary vasculature. Therapeutics aimed at increasing NO or cGMP bioavailabilities are amenable to hypoxia disease-induced PH. Similarly, strategies targeting HIF-1α are now considered. Overall, pulmonary hypertension including hypoxia-induced PH offers unique opportunities for the rational development of therapeutics centered on modulating redox signaling.

Cytoglobin Promotes Cardiac Progenitor Cell Survival against Oxidative Stress via the Upregulation of the NFκB/iNOS Signal Pathway and Nitric Oxide Production.

Human cardiac stem/progenitor cells (hCPCs) may serve in regenerative medicine to repair the infarcted heart. However, this approach is severely limited by the poor survival of donor cells. Recent studies suggest that the mammalian globin cytoglobin (CYGB) regulates nitric oxide (NO) metabolism and cell death. In the present study, we found that CYGB is expressed in hCPCs. Through molecular approaches aimed at increasing or decreasing CYGB expression in hCPCs, we found that CYGB functions as a pro-survival factor in response to oxidative stress. This was associated with the upregulation of primary antioxidant systems such as peroxiredoxins-1, heme oxygenase-1, and anti-apoptotic factors, including BCL2, BCL-XL, and MCL1. Most significantly, we established that CYGB increased the expression of NFкB-dependent genes including iNOS, and that iNOS-dependent NO production was required for a feedforward loop that maintains CYGB expression. Our study delineates for the first time a role for a globin in regulating hCPC survival and establishes mechanistic insights in the function of CYGB. It provides a rationale for the exploration of the CYGB pathway as a molecular target that can be used to enhance the effectiveness of cardiac stem/progenitor cell therapy for ischemic heart disease.

The Hemoglobin Homolog Cytoglobin in Smooth Muscle Inhibits Apoptosis and Regulates Vascular Remodeling.

OBJECTIVE: The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury. APPROACH AND RESULTS: We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used 2 different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost after VSM cell dedifferentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its reexpression and decreased neointima formation. Similarly, 4 weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, terminal uridine nick-end labeling staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be reexpressed after VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed after antioxidant treatment or NOS2 (nitric oxide synthase 2) inhibition. CONCLUSIONS: These results indicate that CYGB is expressed in vessels primarily in differentiated medial VSM cells where it regulates neointima formation and inhibits apoptosis after injury.

Dual Function for Mature Vascular Smooth Muscle Cells During Arteriovenous Fistula Remodeling.

BACKGROUND: The arteriovenous fistula (AVF) is the preferred form of hemodialysis access for patients with chronic kidney disease. However, AVFs are associated with significant problems including high incidence of both early and late failures, usually attributed to inadequate venous arterialization and neointimal hyperplasia, respectively. Understanding the cellular basis of venous remodeling in the setting of AVF could provide targets for improving AVF patency rates. METHODS AND RESULTS: A novel vascular smooth muscle cell (VSMC) lineage tracing reporter mouse, Myh11-Cre/ERT2-mTmG, was used to track mature VSMCs in a clinically relevant AVF mouse model created by a jugular vein branch end to carotid artery side anastomosis. Prior to AVF surgery, differentiated medial layer VSMCs were labeled with membrane green fluorescent protein (GFP) following tamoxifen induction. Four weeks after AVF surgery, we observed medial VSMC layer thickening in the middle region of the arterialized vein branch. This thickened medial VSMC layer was solely composed of differentiated VSMCs that were GFP+/MYH11+/Ki67-. Extensive neointimal hyperplasia occurred in the AVF region proximal to the anastomosis site. Dedifferentiated VSMCs (GFP+/MYH11-) were a major cellular component of the neointima. Examination of failed human AVF samples revealed that the processes of VSMC phenotypic modulation and intimal hyperplasia, as well as medial VSMC layer thickening, also occurred in human AVFs. CONCLUSIONS: We demonstrated a dual function for mature VSMCs in AVF remodeling, with differentiated VSMCs contributing to medial wall thickening towards venous maturation and dedifferentiated VSMCs contributing to neointimal hyperplasia. These results provide valuable insights into the mechanisms underlying venous adaptations during AVF remodeling.

Selective expression of TSPAN2 in vascular smooth muscle is independently regulated by TGF-ß1/SMAD and myocardin/serum response factor.

Tetraspanins (TSPANs) comprise a large family of 4-transmembrane domain proteins. The importance of TSPANs in vascular smooth muscle cells (VSMCs) is unexplored. Given that TGF-ß1 and myocardin (MYOCD) are potent activators for VSMC differentiation, we screened for TGF-ß1 and MYOCD/serum response factor (SRF)-regulated TSPANs in VSMC by using RNA-seq analyses and RNA-arrays. TSPAN2 was found to be the only TSPAN family gene induced by TGF-ß1 and MYOCD, and reduced by SRF deficiency in VSMCs. We also found that TSPAN2 is highly expressed in smooth muscle-enriched tissues and down-regulated in in vitro models of VSMC phenotypic modulation. TSPAN2 expression is attenuated in mouse carotid arteries after ligation injury and in failed human arteriovenous fistula samples after occlusion by dedifferentiated neointimal VSMC. In vitro functional studies showed that TSPAN2 suppresses VSMC proliferation and migration. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that TSPAN2 is regulated by 2 parallel pathways, MYOCD/SRF and TGF-ß1/SMAD, via distinct binding elements within the proximal promoter. Thus, we identified the first VSMC-enriched and MYOCD/SRF and TGF-ß1/SMAD-dependent TSPAN family member, whose expression is intimately associated with VSMC differentiation and negatively correlated with vascular disease. Our results suggest that TSPAN2 may play important roles in vascular disease.-Zhao, J., Wu, W., Zhang, W., Lu, Y. W., Tou, E., Ye, J., Gao, P., Jourd'heuil, D., Singer, H. A., Wu, M., Long, X. Selective expression of TSPAN2 in vascular smooth muscle is independently regulated by TGF-ß1/SMAD and myocardin/serum response factor.

MYOSLID Is a Novel Serum Response Factor-Dependent Long Noncoding RNA That Amplifies the Vascular Smooth Muscle Differentiation Program.

OBJECTIVE: Long noncoding RNAs (lncRNA) represent a growing class of noncoding genes with diverse cellular functions. We previously reported on SENCR, an lncRNA that seems to support the vascular smooth muscle cell (VSMC) contractile phenotype. However, information about the VSMC-specific lncRNAs regulated by myocardin (MYOCD)/serum response factor, the master switch for VSMC differentiation, is unknown. APPROACH AND RESULTS: To define novel lncRNAs with functions related to VSMC differentiation, we performed RNA sequencing in human coronary artery SMCs that overexpress MYOCD. Several novel lncRNAs showed altered expression with MYOCD overexpression and one, named MYOcardin-induced Smooth muscle LncRNA, Inducer of Differentiation (MYOSLID), was activated by MYOCD and selectively expressed in VSMCs. MYOSLID was a direct transcriptional target of both MYOCD/serum response factor and transforming growth factor-ß/SMAD pathways. Functional studies revealed that MYOSLID promotes VSMC differentiation and inhibits VSMC proliferation. MYOSLID showed reduced expression in failed human arteriovenous fistula samples compared with healthy veins. Although MYOSLID did not affect gene expression of transcription factors, such as serum response factor and MYOCD, its depletion in VSMCs disrupted actin stress fiber formation and blocked nuclear translocation of MYOCD-related transcription factor A (MKL1). Finally, loss of MYOSLID abrogated transforming growth factor-ß1-induced SMAD2 phosphorylation. CONCLUSIONS: We have demonstrated that MYOSLID, the first human VSMC-selective and serum response factor/CArG-dependent lncRNA, is a novel modulator in amplifying the VSMC differentiation program, likely through feed-forward actions of both MKL1 and transforming growth factor-ß/SMAD pathways.

The neuroprotective properties of the superoxide dismutase mimetic tempol correlate with its ability to reduce pathological glutamate release in a rodent model of stroke.

The contribution of oxidative stress to ischemic brain damage is well established. Nevertheless, for unknown reasons, several clinically tested antioxidant therapies have failed to show benefits in human stroke. Based on our previous in vitro work, we hypothesized that the neuroprotective potency of antioxidants is related to their ability to limit the release of the excitotoxic amino acids glutamate and aspartate. We explored the effects of two antioxidants, tempol and edaravone, on amino acid release in the brain cortex, in a rat model of transient occlusion of the middle cerebral artery (MCAo). Amino acid levels were quantified using a microdialysis approach, with the probe positioned in the ischemic penumbra as verified by a laser Doppler technique. Two-hour MCAo triggered a dramatic increase in the levels of glutamate, aspartate, taurine, and alanine. Microdialysate delivery of 10mM tempol reduced the amino acid release by 60-80%, whereas matching levels of edaravone had no effect. In line with these data, an intracerebroventricular injection of tempol but not edaravone (500 nmol each, 15 min before MCAo) reduced infarction volumes by ~50% and improved neurobehavioral outcomes. In vitro assays showed that tempol was superior at removing superoxide anion, whereas edaravone was more potent at scavenging hydrogen peroxide, hydroxyl radical, and peroxynitrite. Overall, our data suggest that the neuroprotective properties of tempol are probably related to its ability to reduce tissue levels of the superoxide anion and pathological glutamate release and, in such a way, limit progression of brain infarction within ischemic penumbra. These new findings may be instrumental in developing new antioxidant therapies for treatment of stroke.

The effect of omalizumab on small airway inflammation as measured by exhaled nitric oxide in moderate-to-severe asthmatic patients.

Measurement of fractional nitric oxide concentration in exhaled breath (FENO) is a simple, noninvasive method to evaluate eosinophilic airway inflammation. Nitric oxide (NO) arising from peripheral small airways/alveoli (alveolar NO concentration [CalvNO]) can be estimated using multiple flow rates and a two-compartment model of the airways and alveoli. Omalizumab, a monoclonal anti-IgE antibody, is approved for the treatment of allergic asthma and also has been shown to decrease FENO levels. This study investigates the effects of omalizumab, when added to an inhaled corticosteroid (ICS) ± long-acting beta-adrenergic agonist (LABA) treatment, on peripheral small airway/alveolar inflammation reflected by FENO measurements at higher flow rates. We hypothesized that compared with placebo, omalizumab would decrease CalvNO levels in asthmatic patients on ICS ± LABA. Forty-two patients with moderate-to-severe asthma were randomly assigned 2:1 to either omalizumab (n = 29) or placebo treatment (n = 13) for 16 weeks. Selection criteria included moderate-to-severe asthmatic patients on an ICS ± LABA, positive skin test to one or more perennial allergen, screening FENO of >13 ppb, and a baseline IgE of 30-700 IU/mL. FENO measured at multiple flow rates was used to calculate CalvNO over the course of 16 weeks. FENO levels decrease with increasing flow rates (p < 0.05 repeated measures ANOVA) but no differences between the placebo and treatment groups in overall CalvNO levels or in the changes of CalvNO with time were found. Omalizumab did not lower the CalvNO, which could have been caused by the initial low CalvNO in this asthmatic population. The model used may not be completely sufficient and/or sensitive enough to detect small changes in CalvNO.

NADPH oxidase 4 is required for interleukin-1ß-mediated activation of protein kinase Cδ and downstream activation of c-jun N-terminal kinase signaling in smooth muscle.

Reactive oxygen species (ROS) are generated in the vascular wall upon stimulation by proinflammatory cytokines and are important mediators of diverse cellular responses that occur as a result of vascular injury. Members of the NADPH oxidase (NOX) family of proteins have been identified in vascular smooth muscle (VSM) cells as important sources of ROS. In this study, we tested the hypothesis that NOX4 is a proximal mediator of IL-1ß-dependent activation of PKCδ and increases IL-1ß-stimulated c-Jun kinase (JNK) signaling in primary rat aortic VSM cells. We found that stimulation of VSM cells with IL-1ß increased PKCδ activity and intracellular ROS generation. SiRNA silencing of NOX4 but not NOX1 ablated the IL-1ß-dependent increase in ROS production. Pharmacological inhibition of PKCδ activity as well as siRNA depletion of PKCδ or NOX4 blocked the IL-1ß-dependent activation of JNK. Further studies showed that the IL-1ß-dependent upregulation of inducible NO synthase expression was inhibited through JNK inhibition and NOX4 silencing. Taken together, these results indicate that IL-1ß-dependent activation of PKCδ is modulated by NOX4-derived ROS. Our study positions PKCδ as an important redox-sensitive mediator of IL-1ß-dependent signaling and downstream activation of inflammatory mediators in VSM cells.