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Jiansheng Lin,1,2 Yinna Wang,2 Chunli Lin,2 Ran Li,3 Gaoxiong Wang1,4 1School of Public Health of Fujian Medical University, Fuzhou, People’s Republic of China; 2Microbiology Laboratory, Quanzhou Women’s and Children’s Hospital, Quanzhou, People’s Republic of China; 3Department of Anesthesiology, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, People’s Republic of China; 4Research Administration Office, Quanzhou Women’s and Children’s Hospital, Quanzhou, People’s Republic of ChinaCorrespondence: Ran Li, Department of Anesthesiology, The Second Affiliated Hospital of Fujian Medical University, 34 Zhongshan North Road, Licheng District, Quanzhou, Fujian Province, 350122, People’s Republic of China, Tel +86 595 22791140, Email lir78256@gmail.com Gaoxiong Wang, Research Administration Office, Quanzhou Women’s and Children’s Hospital, 700 Fengze Street, Fengze District, Quanzhou, Fujian Province, 350122, People’s Republic of China, Tel +86 595 22131685, Email wanggaoxiong2013@163.comPurpose: We aimed to evaluate antibiotic resistance and molecular epidemiological characteristics of non-invasive Haemophilus influenzae (H. influenzae) from pneumonia patients and analyze the whole genome of one invasive H. influenzae isolated from blood in pediatric patients.Methods: Antibiotic susceptibility was tested using the turbidimetric method. β-lactamase-producing and serotyping genes were evaluated via multiplex polymerase chain reaction (PCR), and ftsI was amplified using high-fidelity PCR. Lastly, whole genome sequencing (WGS) was conducted using Illumina HiSeq and PacBio sequencing technology.Results: We observed that the ampicillin (AMP) and amoxicillin/clavulanate (AMC) resistance rates of non-invasive H. influenzae were as high as 99.06% (after adjustment) and 49.53%, respectively. The β-lactamase gene of 106 AMP-resistant strains was blaTEM-1. Group III-like mutation accounted for 71.15% of β-lactamase-positive, AMC-resistant (BLPACR) strain mutants. The novel Asn-526→His mutation was present in one β-lactamase-negative AMP-susceptible (BLNAS) strain. Non-invasive H. influenzae strains all belonged to non-typeable H. influenzae (NTHi). In contrast, the invasive H. influenzae 108 isolated from blood in China belonged to H. influenzae type b (Hib). It belonged to sequence typing ST95 and exhibited sensitivity to all 11 antibiotics. Three prophages were identified, and the capb loci of the H. influenzae strain 108 revealed regions I–III exist in duplicate; however, complete deletion of IS1016 was only present in one of the copies.Conclusion: Non-invasive H. influenzae NTHi with β-lactamase-positive was highly prevalent. Notably, group III-like mutations had increased prevalence among BLPACR strains. H. influenzae belonging to Hib and ST95 was first reported to cause sepsis in China.Keywords: group III-like mutations, BLPACR, prevalence, invasive Haemophilus influenzae, genome

Zhenzhu Zheng, Ying Lei, Yinna Wang, Chunli Lin, Jiansheng Lin Department of Laboratory Medicine, Quanzhou Women’s and Children’s Hospital, Quanzhou, People’s Republic of ChinaCorrespondence: Jiansheng LinDepartment of Laboratory Medicine, Quanzhou Women’s and Children’s Hospital, 700 Fengze Street, Fengze District, Quanzhou, Fujian Province, 350122, People’s Republic of ChinaTel +86 0595 22196131Email 63678462@qq.comBackground: The purpose of this study was to evaluate the prevalence of mobile colistin resistance genes (mcr) in Gram-negative bacteria and to analyze the molecular characteristics of mcr-1 positive Salmonella typhimurium strain 75 and Escherichia coli strain 107 from the Quanzhou Women’s and Children’s Hospital in China.Methods: The genes mcr-1 through mcr-9 were screened via multiplex PCR. Antibiotic susceptibility was detected using a GN11 card with the VITEK-2 compact automated system. Whole genomes were sequenced using PacBio’s single molecule real-time (SMRT) technology.Results: In this study, mcr-1 was detected in only four strains, with a positivity rate of 0.65% (4/616). All the four strains were resistant to more than three different kinds of antibiotics. The mcr-1 positive S. typhimurium strain 75 harbored IncHI2 plasmid, which carried mcr-1 gene, while the mcr-1 positive E. coli strain 107 contained four plasmids including one mcr-1 harboring IncHI2 plasmid, one IncFII plasmid and two IncI1-I (Alpha) plasmids. Mobile elements carrying mcr-1 in the 75_plasmid and 107_plasmid-1 were located in the IS1086(ISApl1)-IS30A(ISApl1)-mcr-1-hp and IS1086(ISApl1)-mcr-1-hp regions, respectively. Tn6010 carrying drug efflux pump genes was found in 75_plasmid, while cn_31611_IS26 carrying multi-drug resistance (MDR) genes were found in 107_plasmid-1.Conclusion: This study found that mcr-1 was prevalent at a low frequency in the Quanzhou Women’s and Children’s Hospital. A similar genetic pattern of mcr-1 transmission was found in both E. coli and S. typhimurium.Keywords: mcr-1, Salmonella typhimurium, Escherichia coli, molecular characteristics

Smooth muscle cell migration is essential for many diverse biological processes such as pulmonary/cardiovascular development and homeostasis. Abi1 (Abelson interactor 1) is an adapter protein that has been implicated in nonmuscle cell migration. However, the role and mechanism of Abi1 in smooth muscle migration are largely unknown. Here, Abi1 knockdown by shRNA reduced human airway smooth muscle cell migration, which was restored by Abi1 rescue. Abi1 localized at the tip of lamellipodia and its protrusion coordinated with F-actin at the leading cell edge of live cells. In addition, we identified profilin-1 (Pfn-1), a G-actin transporter, as a new partner for Abi1. Abi1 knockdown reduced the recruitment of Pfn-1 to the leading cell edge. Moreover, Abi1 knockdown reduced the localization of the actin-regulatory proteins c-Abl (Abelson tyrosine kinase) and N-WASP (neuronal Wiskott–Aldrich Syndrome Protein) at the cell edge without affecting other migration-related proteins including pVASP (phosphorylated vasodilator stimulated phosphoprotein), cortactin and vinculin. Furthermore, we found that c-Abl and integrin β1 regulated the positioning of Abi1 at the leading edge. Taken together, the results suggest that Abi1 regulates cell migration by affecting Pfn-1 and N-WASP, but not pVASP, cortactin and focal adhesions. Integrin β1 and c-Abl are important for the recruitment of Abi1 to the leading edge.

Summary: Asthma is a complex pulmonary disorder with multiple pathological mechanisms. A key pathological feature of chronic asthma is airway remodeling, which is largely attributed to airway smooth muscle (ASM) hyperplasia that contributes to thickening of the airway wall and further drives asthma pathology. The cellular processes that mediate ASM cell proliferation are not completely elucidated. Using multiple approaches, we demonstrate that the adapter protein Abi1 (Abelson interactor 1) is upregulated in ∼50% of ASM cell cultures derived from patients with asthma. Loss-of-function studies demonstrate that Abi1 regulates the activation of Jak2 (Janus kinase 2) and STAT3 (signal transducers and activators of transcription 3) as well as the proliferation of both nonasthmatic and asthmatic human ASM cell cultures. These findings identify Abi1 as a molecular switch that activates Jak2 kinase and STAT3 in ASM cells and demonstrate that a dysfunctional Abi1-associated pathway contributes to the progression of asthma.

In this article, vibration abatement problems of a riser system with system uncertainty, input deadzone, and output constraint are considered. For obtaining better control precision, a boundary control law is constructed by employing the backstepping method and Lyapunov's theory. The output constraint is guaranteed by utilizing a barrier Lyapunov function. Adaptive neural networks are designed to cope with the uncertainty of the riser and compensate for the effect caused by the asymmetric deadzone nonlinearity. With the designed controller, the output constraint is satisfied, and the system stability is guaranteed through Lyapunov synthesis. In the end, numerical simulation results are provided to display the performance of the developed adaptive neural network boundary control law.

Hemolysis is a pathological component of many diseases and is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether the mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules from the α-granule of platelets, including the glycoprotein thrombospondin-1 (TSP-1). We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet TLR4 signaling and leads to the downstream phosphorylation of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.Key pointsHeme induces platelet mtROS production by inhibiting complex-V activity via TLR4 signaling.Heme stimulated platelet granule secretion is regulated by mtROS.

Background Asthma is a complicated chronic inflammatory disorder characterized by airway inflammation and bronchial hyperresponsiveness. Group 2 innate lymphoid cells (ILC2) are tissue-resident innate effector cells that can mediate airway inflammation and hyperresponsiveness through production of IL-5, IL-13 and VEGFA. ILC2 in asthma patients exhibit an activated phenotype. However, molecular pathways that control ILC2 activation are not well understood. Methods MYC expression was examined in ILC2 sorted from peripheral blood of healthy controls and asthma patients or cultured with or without activating cytokines. CRISPR knockout technique was used to delete c-Myc in primary murine lung ILC2 or an ILC2 cell line. Cell proliferation was examined, gene expression pattern was profiled by genome-wide microarray analysis, and direct gene targets were identified by Chromatin immunoprecipitation (ChIP). ILC2 responses, airway inflammation and airway hyperresponsiveness were examined in Balb/c mice challenged with Alternaria extracts, with or without treatment with JQ1. Results ILC2 from asthma patients expressed increased amounts of MYC. Deletion of c-Myc in ILC2 results in reduced proliferation, decreased cytokine production, and reduced expression of many lymphocyte activation genes. ChIP identified Stat6 as a direct gene target of c-Myc in ILC2. In vivo inhibition of c-Myc by JQ1 treatment repressed ILC2 activity and suppressed Alternaria-induced airway inflammation and AHR. Conclusion c-Myc expression is upregulated during ILC2 activation. c-Myc is essential for ILC2 activation and their in vivo pathogenic effects. These findings suggest that targeting c-Myc may unlock novel strategies to combat asthma or asthma exacerbation.

Myoglobin is a monomeric heme protein expressed ubiquitously in skeletal and cardiac muscle and is traditionally considered to function as an oxygen reservoir for mitochondria during hypoxia. It is now well established that low concentrations of myoglobin are aberrantly expressed in a significant proportion of breast cancer tumors. Despite being expressed only at low levels in these tumors, myoglobin is associated with attenuated tumor growth and a better prognosis in breast cancer patients, but the mechanism of this myoglobin-mediated protection against further cancer growth remains unclear. Herein, we report a signaling pathway by which myoglobin regulates mitochondrial dynamics and thereby decreases cell proliferation. We demonstrate in vitro that expression of human myoglobin in MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells induces mitochondrial hyperfusion by up-regulating mitofusins 1 and 2, the predominant catalysts of mitochondrial fusion. This hyperfusion causes cell cycle arrest and subsequent inhibition of cell proliferation. Mechanistically, increased mitofusin expression was due to myoglobin-dependent free-radical production, leading to the oxidation and degradation of the E3 ubiquitin ligase parkin. We recapitulated this pathway in a murine model in which myoglobin-expressing xenografts exhibited decreased tumor volume with increased mitofusin, markers of cell cycle arrest, and decreased parkin expression. Furthermore, in human triple-negative breast tumor tissues, mitofusin and myoglobin levels were positively correlated. Collectively, these results elucidate a new function for myoglobin as a modulator of mitochondrial dynamics and reveal a novel pathway by which myoglobin decreases breast cancer cell proliferation and tumor growth by up-regulating mitofusin levels.

This article handle the issues of the vibration of a flexible riser system with output constraint and system uncertainty. Combining the Lyapunov’s theory, a boundary neural network (NN) control is constructed with backstepping method. The system uncertainty is handled by using neural networks. With the developed control law, the system stability is ensured, and the output constraint is addressed. After the theoretical and simulation verification of the proposed method, it can gain the effectiveness of control law with no difficulty.

Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine-416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with N-WASP (neuronal Wiskott - Aldrich syndrome Protein), an important actin-regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19 and vimentin phosphorylation at Ser-56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non-histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist-induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N-WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation-dependent regulation of the actin cytoskeleton in smooth muscle.

BACKGROUND Sickle cell disease (SCD) is characterized by hemolysis-associated platelet dysfunction that leads to increased risk of thrombosis and plays a role in the high morbidity and mortality of the disease. The mechanisms by which hemolysis induces platelet activation remain unclear. We recently demonstrated that patients with SCD showed increased platelet mitochondrial reactive oxygen species (mtROS) production that correlates with markers of hemolysis and platelet activation. Experiments in isolated platelets demonstrated that mtROS stimulated platelet activation. However, the role of hemolysis-induced mtROS in thrombus formation in vivo remains unclear. OBJECTIVES Here, we hypothesize that scavenging of mtROS attenuates the propensity for thrombosis in mouse models of hemolysis. METHODS We used models of hemolysate infusion into wildtype mice as well as the Berkley transgenic mouse model of SCD, a chronic mode of hemolysis, to test the effect of hemolysis on platelet mtROS production and thrombosis. RESULTS We show that infusion of hemolysate in wildtype mice induces platelet mtROS production and decreases time to vessel occlusion in a model of ferric chloride-induced carotid artery thrombosis. Increased mtROS and propensity for thrombosis was also observed in the Berkley transgenic mouse model of SCD. Notably, treatment with mtROS scavengers decreased platelet mtROS levels and attenuated the propensity for thrombus formation in both models. CONCLUSIONS These data demonstrate that mtROS significantly contribute to the mechanism of hemolysis-induced thrombosis in vivo and suggest a potential role for mitochondrially targeted antioxidant therapy in hemolysis and SCD-related thrombosis.

It is well established that myoglobin supports mitochondrial respiration through the storage and transport of oxygen as well as through the scavenging of nitric oxide. However, during ischemia/reperfusion (I/R), myoglobin and mitochondria both propagate myocardial injury through the production of oxidants. Nitrite, an endogenous signaling molecule and dietary constituent, mediates potent cardioprotection after I/R and this effect relies on its interaction with both myoglobin and mitochondria. While independent mechanistic studies have demonstrated that nitrite-mediated cardioprotection requires the presence of myoglobin and the post-translational S-nitrosation of critical cysteine residues on mitochondrial complex I, it is unclear whether myoglobin directly catalyzes the S-nitrosation of complex I or whether mitochondrial-dependent nitrite reductase activity contributes to S-nitrosation. Herein, using purified myoglobin and isolated mitochondria, we characterize and directly compare the nitrite reductase activities of mitochondria and myoglobin and assess their contribution to mitochondrial S-nitrosation. We demonstrate that myoglobin is a significantly more efficient nitrite reductase than isolated mitochondria. Further, deoxygenated myoglobin catalyzes the nitrite-dependent S-nitrosation of mitochondrial proteins. This reaction is enhanced in the presence of oxidized (Fe(3+)) myoglobin and not significantly affected by inhibitors of mitochondrial respiration. Using a Chinese Hamster Ovary cell model stably transfected with human myoglobin, we show that both myoglobin and mitochondrial complex I expression are required for nitrite-dependent attenuation of cell death after anoxia/reoxygenation. These data expand the understanding of myoglobin’s role both as a nitrite reductase to a mediator of S-nitrosation and as a regulator of mitochondrial function, and have implications for nitrite-mediated cardioprotection after I/R.

It has been reported that actin polymerization is regulated by protein tyrosine phosphorylation in smooth muscle on contractile stimulation. The role of protein serine/threonine phosphorylation in modulating actin dynamics is underinvestigated. SLK (Ste20-like kinase) is a serine/threonine protein kinase that plays a role in apoptosis, cell cycle, proliferation, and migration. The function of SLK in smooth muscle is mostly unknown. Here, SLK knockdown (KD) inhibited acetylcholine (ACh)-induced actin polymerization and contraction without affecting myosin light chain phosphorylation at Ser-19 in human airway smooth muscle. Stimulation with ACh induced paxillin phosphorylation at Ser-272, which was reduced in SLK KD cells. However, SLK did not catalyze paxillin Ser-272 phosphorylation in vitro. But, SLK KD attenuated Plk1 (polo-like kinase 1) phosphorylation at Thr-210. Plk1 mediated paxillin phosphorylation at Ser-272 in vitro. Expression of the nonphosphorylatable paxillin mutant S272A (substitution of alanine at Ser-272) attenuated the agonist-enhanced F-actin/G-actin ratios without affecting myosin light chain phosphorylation. Because N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) phosphorylation at Tyr-256 (an indication of its activation) promotes actin polymerization, we also assessed the role of paxillin phosphorylation in N-WASP activation. S272A paxillin inhibited the ACh-enhanced N-WASP phosphorylation at Tyr-256. Together, these results suggest that SLK regulates paxillin phosphorylation at Ser-272 via Plk1, which modulates N-WASP activation and actin polymerization in smooth muscle. SLK-mediated actin cytoskeletal reorganization may facilitate force transmission between the contractile units and the extracellular matrix.

Nitrite represents an endocrine reserve of bioavailable nitric oxide (NO) that mediates a number of physiological responses including conferral of cytoprotection after ischemia/reperfusion (I/R). It has long been known that nitrite can react with non-heme iron to form dinitrosyliron complexes (DNIC). However, it remains unclear how quickly nitrite-dependent DNIC form in vivo, whether formation kinetics differ from that of NO-dependent DNIC, and whether DNIC play a role in the cytoprotective effects of nitrite. Here we demonstrate that chronic but not acute nitrite supplementation increases DNIC concentration in the liver and kidney of mice. Although DNIC have been purported to have antioxidant properties, we show that the accumulation of DNIC in vivo is not associated with nitrite-dependent cytoprotection after hepatic I/R. Further, our data in an isolated mitochondrial model of anoxia/reoxygenation show that while NO and nitrite demonstrate similar S-nitrosothiol formation kinetics, DNIC formation is significantly greater with NO and associated with mitochondrial dysfunction as well as inhibition of aconitase activity. These data are the first to directly compare mitochondrial DNIC formation by NO and nitrite. This study suggests that nitrite-dependent DNIC formation is a physiological consequence of dietary nitrite. The data presented herein implicate mitochondrial DNIC formation as a potential mechanism underlying the differential cytoprotective effects of nitrite and NO after I/R, and suggest that DNIC formation is potentially responsible for the cytotoxic effects observed at high NO concentrations., Graphical abstract fx1, Highlights • Dietary nitrite results in DNIC formation in many tissues, most notably the liver. • Nitrite-dependent DNIC accumulate within the mitochondrion. • NO generates greater DNIC formation in the mitochondrion than nitrite. • At high concentrations of NO DNIC formation is associated with mitochondrial injury.

We report that MAIT cells repress group 2 innate lymphoid cell activation and restrict allergen-induced airway inflammation and airway hyperresponsiveness.

Introduction We assessed performance and safety of the NuvaRing® Applicator. Methods We randomized women (18–45 years) to insert a placebo ring using the applicator or fingers-only and then vice versa. We assessed outcomes post-insertion and then 24–72 h later. Results Insertion was 100% successful using both methods (applicator, n = 163; fingers-only, n = 162). A total of 8.6% (applicator) and 4.3% (fingers-only) of subjects reported at least 1 treatment-related adverse event (AE); all were mild. Subjects reported 5 applicator-related AEs (vulvovaginal pain, 4; abdominal cramping, 1). There was no vaginal bleeding within 15 h post-applicator use. Ring expulsions were rare (applicator, 1; fingers-only, 2). Conclusion NuvaRing Applicator is effective and well-tolerated (NCT02275546).

Nitrite acts as an endocrine source of bioactive nitric oxide, impacting vascular reactivity, angiogenesis and cytoprotection. Nitrite has recently been shown to have a metabolic role although its effects and mechanisms of action in the obese insulin-resistant state are unknown. We examined glucose tolerance and insulin secretion using the frequently sampled intravenous glucose tolerance test and insulin sensitivity using the hyperinsulinaemic euglycaemic clamp in obese male oblep mice administered nitrite (100 mg kg−1 day−1) or saline (control) for 7 days and compared responses to the known insulin-sensitizing effects of rosiglitazone (6 mg kg−1 day−1). Under weight-matched conditions, nitrite lowered blood pressure relative to saline and rosiglitazone, whereas only rosiglitazone was effective at reducing hepatic glucose output and basal blood glucose. Both nitrite and rosiglitazone produced improvements, relative to saline, in glucose tolerance (12,524 ± 602, 12,811 ± 692 vs.14,428 ± 335 mg (dl min)−1, respectively; P

The tyrosine kinase c-Abl participates in the regulation of various cellular functions including cell proliferation, adhesion, migration, smooth muscle contraction and cancer progression. However, knowledge regarding transcriptional regulation of c-Abl is surprisingly limited. Sp1 is a founding member of the Sp1 transcription factor family that has been implicated in housekeeping gene expression, tumor cell proliferation and differentiation. Here, we show that knockdown and rescue of Sp1 affected growth factor-mediated c-Abl expression in cells. c-Abl promoter activity was also affected by Sp1 knockdown. This is the first evidence to suggest that Sp1 is an important transcription factor to regulate c-Abl expression. In addition, Sp1 phosphorylation at Thr-453 and Thr-739 has been proposed to regulate its activity in Drosophila cells. We unexpectedly found that growth factors did not induce Sp1 phosphorylation at these two residues. In contrast, growth factor stimulation upregulated Sp1 expression. Intriguingly, inhibition of ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) reduced expression of Sp1 and c-Abl. Furthermore, c-Abl knockdown diminished ERK1/2 phosphorylation and Sp1 expression. Taken together, these studies suggest that Sp1 can modulate c-Abl expression at transcription level. Conversely, c-Abl affects ERK1/2 activation and Sp1 expression in cells.

Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS- S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature.

Aims Acute kidney injury (AKI) is a common complication among patients hospitalized for acute heart failure (AHF), and is associated with increased mortality. The goal of this study was to derive and validate a prediction score for AKI in AHF patients. Methods The hospital medical records of 1709 patients with AHF were reviewed. AKI was defined as an increase in serum creatinine (SCr) of ≥26.4 μmol/L or ≥50% within 48 h. A multivariate logistic regression analysis was undertaken to develop a new prediction score. The area under the receiver operating characteristic (ROC) curve and the Hosmer-Lemeshow goodness-of-fit statistic test were calculated to assess the discrimination and calibration of the prediction score, respectively. Results Acute kidney injury developed in 32.2% of patients with AHF. Factors independently associated with the risk of AKI included: ≥70 years of age, ≥3 previous hospital admissions for AHF, systolic blood pressure

Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiological responses including modulation of ischemia/reperfusion injury, glucose tolerance, and vascular remodeling. Although the exact molecular mechanisms underlying nitrite’s actions are unknown, the current paradigm suggests that these effects depend on the hypoxic reduction of nitrite to nitric oxide (NO). Mitochondrial biogenesis is a fundamental mechanism of cellular adaptation and repair. However, the effect of nitrite on mitochondrial number has not been explored. Herein, we report that nitrite stimulates mitochondrial biogenesis through a mechanism distinct from that of NO. We demonstrate that nitrite significantly increases cellular mitochondrial number by augmenting the activity of adenylate kinase, resulting in AMP kinase phosphorylation, downstream activation of sirtuin-1, and deacetylation of PGC1α, the master regulator of mitochondrial biogenesis. Unlike NO, nitrite-mediated biogenesis does not require the activation of soluble guanylate cyclase and results in the synthesis of more functionally efficient mitochondria. Further, we provide evidence that nitrite mediates biogenesis in vivo. In a rat model of carotid injury, 2 weeks of continuous oral nitrite treatment postinjury prevented the hyperproliferative response of smooth muscle cells. This protection was accompanied by a nitrite-dependent upregulation of PGC1α and increased mitochondrial number in the injured artery. These data are the first to demonstrate that nitrite mediates differential signaling compared to NO. They show that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro and suggest that nitrite-mediated biogenesis may play a protective role in the setting of vascular injury.

Summary Recent evidence suggests that molecular hydrogen has therapeutic value for disease states that involve inflammation. We hypothesized that drinking hydrogen-rich water (HW) daily would protect cardiac and aortic allograft recipients from inflammation-associated deterioration. Heterotopic heart transplantation with short-course tacrolimus immunosuppression and orthotopic aortic transplantation were performed in allogeneic rat strains. HW was generated either by bubbling hydrogen gas through tap water (Bu-HW) or via chemical reaction using a magnesium stick [Mg + 2H2O → Mg (OH)2 + H2] immersed in tap water (Mg-HW). Recipients were given either regular water (RW), Mg-HW, Bu-HW, or Mg-HW that had been subsequently degassed (DW). Graft survival was assessed by daily palpation for a heartbeat. Drinking Mg-HW or Bu-HW was remarkably effective in prolonging heart graft survival and reducing intimal hyperplasia in transplanted aortas as compared with grafts treated with RW or DW. Furthermore, T cell proliferation was significantly inhibited in the presence of hydrogen in vitro, accompanied by less production of interleukin-2 and interferon-γ. Hydrogen treatment was also associated with increased graft ATP levels and increased activity of the enzymes in mitochondrial respiratory chain. Drinking HW prolongs survival of cardiac allografts and reduces intimal hyperplasia of aortic allografts.

Caveolin-1 (Cav-1)−/− mice develop mild pulmonary hypertension as they age. In this study, we sought to determine the effect of chronic hypoxia, an established model of pulmonary hypertension, on young Cav-1−/− mice with no measurable signs of pulmonary hypertension. Exposure of Cav-1−/− mice to chronic hypoxia resulted in an initial rise in right ventricular (RV) systolic pressure (RVSP) similar to wild-type (WT) mice. By three weeks RVSP decreased in the Cav-1−/− mice, whereas it was maintained in WT mice. The drop in RVSP in Cav-1−/− mice was accompanied by decreased cardiac output, increased RV hypertrophy, RV interstitial fibrosis, decreased RV sarco(endo)plasmic reticulum Ca2+-ATPase 2a mRNA and decreased RV function compared with WT mice. Importantly, minimal differences were noted in pulmonary vascular remodeling between WT and Cav-1−/− mice, and left ventricular function was normal in hypoxic Cav-1−/− mice. Mechanistically, increased endothelial nitric oxide synthase uncoupling and increased tyrosine nitration of protein kinase G were detected in the RV of Cav-1−/− mice. These hemodynamic, histological, and molecular changes were prevented in Cav-1−/− mice expressing an endothelial-specific Cav-1 transgene or by nitric oxide synthase inhibition. These data suggest that, in Cav-1−/− mice, increased oxidative/nitrosative stress due to endothelial nitric oxide synthase uncoupling modifies the response of the RV to pressure overload, accelerating the deterioration of RV function.

Nitrite, a dietary constituent and endogenous signaling molecule previously thought to be a biologically inert product of endogenous nitric oxide oxidation, has recently been shown to regulate a myriad of biological processes. For example, nitrite has been shown to inhibit smooth muscle proliferation and attenuate restenosis after vascular injury. However, the mechanism of nitrite-dependent inhibition of smooth muscle proliferation remains elusive. Nitrite is an established regulator of mitochondrial morphology and function, and mitochondrial dynamics, the active formation (fusion) or fragmentation (fission) of cellular mitochondrial networks, has been previously shown to regulate cell cycle progression. Thus, we hypothesized that nitrite modulates mitochondrial dynamics and function to inhibit cell cycle progression and attenuate smooth muscle cell proliferation. Using rat aortic smooth muscle cells (RASMCs) we demonstrate that nitrite inhibits RASMC proliferation induced by platelet derived growth factor (PDGF) in a concentration dependent manner. This phenomenon is associated with mitochondrial fusion dependent on the upregulation of mitofusin-1 (Mfn1). Further, nitrite treatment upregulates the cyclin dependent kinase inhibitor p21, an effect that is abolished in Mfn1 deficient RASMCs. Ongoing studies are focused on determining the mechanism by which nitrite upregulates Mfn1 thereby stimulating mitochondrial fusion while increasing p21 expression. These data have important implications for dietary and pharmacological modulation of vascular health and uncover a novel potential physiological mechanism for the regulation of smooth muscle cell number.

Vascular disease, a significant cause of morbidity and mortality in the developed world, results from vascular injury. Following vascular injury, damaged or dysfunctional endothelial cells and activated SMCs engage in vasoproliferative remodeling and the formation of flow-limiting intimal hyperplasia (IH). We hypothesized that vascular injury results in decreased bioavailability of NO secondary to dysregulated arginine-dependent NO generation. Furthermore, we postulated that nitrite-dependent NO generation is augmented as an adaptive response to limit vascular injury/proliferation and can be harnessed for its protective effects. Here we report that sodium nitrite (intraperitoneal, inhaled, or oral) limited the development of IH in a rat model of vascular injury. Additionally, nitrite led to the generation of NO in vessels and SMCs, as well as limited SMC proliferation via p21Waf1/Cip1 signaling. These data demonstrate that IH is associated with increased arginase-1 levels, which leads to decreased NO production and bioavailability. Vascular injury also was associated with increased levels of xanthine oxidoreductase (XOR), a known nitrite reductase. Chronic inhibition of XOR and a diet deficient in nitrate/nitrite each exacerbated vascular injury. Moreover, established IH was reversed by dietary supplementation of nitrite. The vasoprotective effects of nitrite were counteracted by inhibition of XOR. These data illustrate the importance of nitrite-generated NO as an endogenous adaptive response and as a pathway that can be harnessed for therapeutic benefit.

Background Recent advances in novel medical gases, including hydrogen and carbon monoxide (CO), have demonstrated significant opportunities for therapeutic use. This study was designed to evaluate the effects of inhaled hydrogen or CO, or both, on cold ischemia/reperfusion (I/R) injury of the myocardium. Methods Syngeneic heterotopic heart transplantation was performed in rats after 6 or 18 hours of cold ischemia in Celsior solution. Survival, morphology, apoptosis and marker gene expression were assessed in the grafts after in vivo inhalation of hydrogen (1% to 3%), CO (50 to 250 ppm), both or neither. Both donors and recipients were treated for 1 hour before and 1 hour after reperfusion. Results After 6-hour cold ischemia, inhalation of hydrogen (>2%) or CO (250 ppm) alone attenuated myocardial injury. Prolonged cold ischemia for 18 hours resulted in severe myocardial injury, and treatment with hydrogen or CO alone failed to demonstrate significant protection. Dual treatment with hydrogen and CO significantly attenuated I/R graft injury, reducing the infarcted area and decreasing in serum troponin I and creatine phosphokinase (CPK). Hydrogen treatment alone significantly reduced malondialdehyde levels and serum high-mobility group box 1 protein levels as compared with air-treated controls. In contrast, CO only marginally prevented lipid peroxidation, but it suppressed I/R-induced mRNA upregulation for several pro-inflammatory mediators and reduced graft apoptosis. Conclusions Combined therapy with hydrogen and CO demonstrated enhanced therapeutic efficacy via both anti-oxidant and anti-inflammatory mechanisms, and may be a clinically feasible approach for preventing cold I/R injury of the myocardium.

Reactive oxygen species (ROS) contribute to the development of interstitial fibrosis and tubular atrophy seen in chronic allograft nephropathy (CAN). As molecular hydrogen gas can act as a scavenger of ROS, we tested the effect of treatment with hydrogen water (HW) in a model of kidney transplantation, in which allografts from Lewis rats were orthotopically transplanted into Brown Norway recipients that had undergone bilateral nephrectomy. Molecular hydrogen was dissolved in water and recipients were given HW from day 0 until day 150. Rats that were treated with regular water (RW) gradually developed proteinuria and their creatinine clearance declined, ultimately leading to graft failure secondary to CAN. In contrast, treatment with HW improved allograft function, slowed the progression of CAN, reduced oxidant injury and inflammatory mediator production, and improved overall survival. Inflammatory signaling pathways, such as mitogen-activated protein kinases, were less activated in renal allografts from HW-treated rats as compared with RW-treated rats. Hence, oral HW is an effective antioxidant and antiinflammatory agent that prevented CAN, improved survival of rat renal allografts, and may be of therapeutic value in the setting of transplantation.

Our study reports that staurosporine induces apoptosis in cultured rat hepatocytes in a dose- and time-dependent fashion. Staurosporine induced apparent cleavage of caspase-8, caspase-9, and caspase-3. The release of cytochrome c from mitochondria, and Bid activation were also detected in staurosporine-treated primary hepatocytes. These results suggest that mitochondria-mediated cell death signaling may be involved in staurosporine-induced hepatocyte apoptosis. Bcl-xL overexpression protected from “loss of” mitochondrial transmembrane potential and prevented staurosporine-induced caspase-3 and caspase-8 cleavage. Overexpression of constitutively active ERK and PKB inhibited staurosporine-induced caspase-3 activation and hepatocyte death. PI3K inhibitor (LY294002) and ERK inhibitor (PD98059) significantly reversed the protective effects of Bcl-xL on staurosporine-induced hepatocyte death. Our data suggest that Bcl-xL prevents staurosporine-induced hepatocyte apoptosis by modulating protein kinase B and p44/42 mitogen-activated protein kinase activity and disrupts mitochondria death signaling. J. Cell. Physiol. 215: 676–683, 2008. © 2007 Wiley-Liss, Inc.

Nitrite acts as an endocrine source of bioactive nitric oxide, impacting vascular reactivity, angiogenesis and cytoprotection. Nitrite has recently been shown to have a metabolic role although its effects and mechanisms of action in the obese insulin-resistant state are unknown. We examined glucose tolerance and insulin secretion using the frequently sampled intravenous glucose tolerance test and insulin sensitivity using the hyperinsulinaemic euglycaemic clamp in obese male ob(lep) mice administered nitrite (100 mg kg(-1) day(-1) ) or saline (control) for 7 days and compared responses to the known insulin-sensitizing effects of rosiglitazone (6 mg kg(-1) day(-1) ). Under weight-matched conditions, nitrite lowered blood pressure relative to saline and rosiglitazone, whereas only rosiglitazone was effective at reducing hepatic glucose output and basal blood glucose. Both nitrite and rosiglitazone produced improvements, relative to saline, in glucose tolerance (12,524 ± 602, 12,811 ± 692 vs.14,428 ± 335 mg (dl min)(-1) , respectively; P0.05) and insulin sensitivity (8.6 ± 0.7, 7.9 ± 0.3 vs. 6.6 ± 0.5 mg kg(-1) min(-1) , respectively; P0.001), but there was no effect on insulin secretion. Nitrite exhibited an uncoupling of mitochondrial respiration and a decrease in ATP generation in muscle that was independent of mitochondrial biogenesis or activation of uncoupling proteins. There was no insulin-stimulated phosphorylation of Akt, but nitrite increased the phosphorylation of AMP-activated protein kinase. We conclude that nitrite improves two key components of the metabolic syndrome, blood pressure and insulin sensitivity, independent of weight and with effectiveness comparable to rosiglitazone.

Background/Aim: Vascular calcification is common and contributes to increased cardiovascular mortality in hemodialysis (HD) patients. In this prospective study, we aimed to investigate the associations of serum S100A12 in the presence of severe coronary artery calcification (CAC) and the progression of CAC in HD patients. Methods: Sixty maintenance HD patients and 30 controls were enrolled. Serum S100A12 levels were measured using ELISA. CAC scores (CACs) were measured twice at a 4-year interval using multislice spiral CT. The HD patients were classified as rapid progressors or slow progressors according to the change in the CACs across these 2 measurements (ΔCACs). Results: The incidences of rapid progression of CAC in patients with baseline CACs ≤10, CACs >10 and CACs >400 were 12.5, 40.0 and 64.3%, respectively. Both baseline and 4-year serum S100A12 levels were significantly higher in the rapid progressors than in the slow progressors (medians of 45.6 vs. 30.2 ng/ml, p < 0.001 and 62.3 vs. 39.4 ng/ml, p = 0.002, respectively). The serum S100A12 levels were significantly correlated with baseline CACs (r = 0.466, p < 0.001), 4-year CACs (r = 0.440, p < 0.001) and ΔCACs (r = 0.392, p < 0.001). Importantly, the ΔCACs were significantly correlated with ΔS100A12 levels (r = 0.396, p < 0.001). Logistic regression analysis revealed that the serum S100A12 level was as an independent determinant of the presence of severe CAC and that the increment in the serum S100A12 level was a factor that was significantly independently associated with the progression of CAC. Conclusions: Serum S100A12 levels were significantly associated with the presence of severe CAC, and the increment in serum S100A12 levels was an independent determinant of the progression of CAC.

We examined the regulation of Fas-associating death domain (FADD) protein as an important adaptor molecule in apoptosis signaling and hypothesized that the regulation of FADD could contribute to hepatocyte death. FADD/mediator of receptor-induced toxicity (MORT1) is required for activation of several signaling pathways of cell death. In this study we report the interesting and unexpected result that actinomycin D increased the expression of FADD protein, and we demonstrate that other cellular stresses like ultraviolet irradiation or heat shock could also increase FADD levels in hepatocytes. In cells treated with actinomycin D, FADD levels were elevated homogeneously in the cytoplasm. The increase in cytoplasmic FADD protein by actinomycin D or FADD overexpression alone both correlated with cell death, and specific antisense inhibition of FADD expression consistently diminished approximately 30% of the cell death induced by actinomycin D. These data indicate that FADD protein expression can increase rapidly in hepatocytes exposed to broadly cytotoxic agents.

Cytochrome P-450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) exert diverse biological activities, which include potent vasodilatory, anti-inflammatory, antiapoptotic, and antioxidatant effects, and cardiovascular protection. Liver has abundant epoxygenase expression and high levels of EET production; however, the roles of epoxygenases in liver diseases remain to be elucidated. In this study, we investigated the protection against high-fat diet-induced nonalcoholic fatty liver disease (NAFLD) in mice with endothelial-specific CYP2J2 overexpression (Tie2-CYP2J2-Tr). After 24 wk of high-fat diet, Tie2-CYP2J2-Tr mice displayed attenuated NAFLD compared with controls. Tie2-CYP2J2-Tr mice showed significantly decreased plasma triglyceride levels and liver lipid accumulation, improved liver function, reduced inflammatory responses, and less increase in hepatic oxidative stress than wild-type control mice. These effects were associated with inhibition of NF-κB/JNK signaling pathway activation and enhancement of the antioxidant defense system in Tie2-CYP2J2-Tr mice in vivo. We also demonstrated that 14,15-EET treatment protected HepG2 cells against palmitic acid-induced inflammation and oxidative stress. 14,15-EET attenuated palmitic acid-induced changes in NF-κB/JNK signaling pathways, malondialdehyde generation, glutathione levels, reactive oxygen species production, and NADPH oxidase and antioxidant enzyme expression in HepG2 cells in vitro. Together, these results highlight a new role for CYP epoxygenase-derived EETs in lipotoxicity-related inflammation and oxidative stress and reveal a new molecular mechanism underlying EETs-mediated anti-inflammatory and antioxidant effects that could aid in the design of new therapies for the prevention and treatment of NAFLD.