Nitric oxide releasing nanofiber stimulates revascularization in response to ischemia via cGMP-dependent protein kinase.

Nitric oxide (NO) promotes angiogenesis via various mechanisms; however, the effective transmission of NO in ischemic diseases is unclear. Herein, we tested whether NO-releasing nanofibers modulate therapeutic angiogenesis in an animal hindlimb ischemia model. Male wild-type C57BL/6 mice with surgically-induced hindlimb ischemia were treated with NO-releasing 3-methylaminopropyltrimethoxysilane (MAP3)-derived or control (i.e., non-NO-releasing) nanofibers, by applying them to the wound for 20 min, three times every two days. The amount of NO from the nanofiber into tissues was assessed by NO fluorometric assay. The activity of cGMP-dependent protein kinase (PKG) was determined by western blot analysis. Perfusion ratios were measured 2, 4, and 14 days after inducing ischemia using laser doppler imaging. On day 4, Immunohistochemistry (IHC) with F4/80 and gelatin zymography were performed. IHC with CD31 was performed on day 14. To determine the angiogenic potential of NO-releasing nanofibers, aorta-ring explants were treated with MAP3 or control fiber for 20 min, and the sprout lengths were examined after 6 days. As per either LDPI (Laser doppler perfusion image) ratio or CD31 capillary density measurement, angiogenesis in the ischemic hindlimb was improved in the MAP3 nanofiber group; further, the total nitrate/nitrite concentration in the adduct muscle increased. The number of macrophage infiltrations and matrix metalloproteinase-9 (MMP-9) activity decreased. Vasodilator-stimulated phosphoprotein (VASP), one of the major substrates for PKG, increased phosphorylation in the MAP3 group. MAP3 nanofiber or NO donor SNAP (s-nitroso-n-acetyl penicillamine)-treated aortic explants showed enhanced sprouting in an ex vivo aortic ring assay, which was partially abrogated by KT5823, a potent inhibitor of PKG. These findings suggest that the novel NO-releasing nanofiber, MAP3 activates PKG and promotes therapeutic angiogenesis in response to hindlimb ischemia.

Clinical Validation of the Unparalleled Sensitivity of the Novel Allele-Discriminating Priming System Technology-Based EGFR Mutation Assay in Patients with Operable Non-Small Cell Lung Cancer.

PURPOSE: Recently, we developed allele-discriminating priming system (ADPS) technology. This method increases the sensitivity of conventional quantitative polymerase chain reaction up to 100 folds, with limit of detection, 0.01%, with reinforced specificity. This prospective study aimed to develop and validate the accuracy of ADPS epidermal growth factor receptor (EGFR) Mutation Test Kit using clinical specimens. MATERIALS AND METHODS: In total 189 formalin-fixed paraffin-embedded tumor tissues resected from patients with non-small cell lung cancer were used to perform a comparative evaluation of the ADPS EGFR Mutation Test Kit versus the cobas EGFR Mutation Test v2, which is the current gold standard. When the two methods had inconsistent results, next-generation sequencing-based CancerSCAN was utilized as a referee. RESULTS: The overall agreement of the two methods was 97.4% (93.9%-99.1%); the positive percent agreement, 95.0% (88.7%-98.4%); and the negative percent agreement, 100.0% (95.9%-100.0%). EGFR mutations were detected at a frequency of 50.3% using the ADPS EGFR Mutation Test Kit and 52.9% using the cobas EGFR Mutation Test v2. There were 10 discrepant mutation calls between the two methods. CancerSCAN reproduced eight ADPS results. In two cases, mutant allele fraction was ultra-low at 0.02% and 0.06%, which are significantly below the limit of detection of the cobas assay and CancerSCAN. Based on the EGFR genotyping by ADPS, the treatment options could be switched in five patients. CONCLUSION: The highly sensitive and specific ADPS EGFR Mutation Test Kit would be useful in detecting the patients who have lung cancer with EGFR mutation, and can benefit from the EGFR targeted therapy.

Pharmacological inhibition of TBK1/IKKε blunts immunopathology in a murine model of SARS-CoV-2 infection.

TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation.

Nogo receptor-Fc delivered by haematopoietic cells enhances neurorepair in a multiple sclerosis model.

Nogo receptor 1 is the high affinity receptor for the potent myelin-associated inhibitory factors that make up part of the inflammatory extracellular milieu during experimental autoimmune encephalomyelitis. Signalling through the Nogo receptor 1 complex has been shown to be associated with axonal degeneration in an animal model of multiple sclerosis, and neuronal deletion of this receptor homologue, in a disease specific manner, is associated with preserving axons even in the context of neuroinflammation. The local delivery of Nogo receptor(1-310)-Fc, a therapeutic fusion protein, has been successfully applied as a treatment in animal models of spinal cord injury and glaucoma. As multiple sclerosis and experimental autoimmune encephalomyelitis exhibit large numbers of inflammatory cell infiltrates within the CNS lesions, we utilized transplantable haematopoietic stem cells as a cellular delivery method of the Nogo receptor(1-310)-Fc fusion protein. We identified CNS-infiltrating macrophages as the predominant immune-positive cell type that overexpressed myc-tagged Nogo receptor(1-310)-Fc fusion protein at the peak stage of experimental autoimmune encephalomyelitis. These differentiated phagocytes were predominant during the extensive demyelination and axonal damage, which are associated with the engulfment of the protein complex of Nogo receptor(1-310)-Fc binding to myelin ligands. Importantly, mice transplanted with haematopoietic stem cells transduced with the lentiviral vector carrying Nogo receptor(1-310)-Fc and recovered from the peak of neurological decline during experimental autoimmune encephalomyelitis, exhibiting axonal regeneration and eventual remyelination in the white matter tracts. There were no immunomodulatory effects of the transplanted, genetically modified haematopoietic stem cells on immune cell lineages of recipient female mice induced with experimental autoimmune encephalomyelitis. We propose that cellular delivery of Nogo receptor(1-310)-Fc fusion protein through genetically modified haematopoietic stem cells can modulate multifocal experimental autoimmune encephalomyelitis lesions and potentiate neurological recovery.

CDCP1 Expression Is a Potential Biomarker of Poor Prognosis in Resected Stage I Non-Small-Cell Lung Cancer.

BACKGROUND: Although early-stage lung cancer has increased owing to the introduction of screening programs, high recurrence rate remains a critical concern. We aimed to explore biomarkers related to the prognosis of surgically resected non-small-cell lung cancer (NSCLC). METHODS: In this retrospective study, we collected medical records of patients with NSCLC and matched tissue microarray blocks from surgical specimens. Semiquantitative immunohistochemistry was performed for measuring the expression level of fibroblast activation protein-alpha (FAP-α), Jagged-1 (JAG1), and CUB-domain-containing protein 1 (CDCP1). RESULTS: A total of 453 patients who underwent complete resection between January 2011 and February 2012 were enrolled; 55.2% patients had stage I NSCLC, and 31.1% presented squamous cell carcinoma. Disease stage was a significant risk factor for recurrence and death, and age ≥ 65 years and male sex were associated with poor overall survival. FAP-a and JaG1 were not related to survivals, while CDCP1-expressing patients exhibited poor disease-free and overall survival. Moreover, CDCP1 expression in stage I NSCLC was significantly associated with recurrence. CONCLUSIONS: Old age, male sex, and high pathological stage were poor prognostic factors in patients with NSCLC who underwent surgical resection. Furthermore, CDCP1 expression could serve as a biomarker for poor prognosis in stage I NSCLC.

Earlier-Phased Cancer Immunity Cycle Strongly Influences Cancer Immunity in Operable Never-Smoker Lung Adenocarcinoma.

Exome and transcriptome analyses of clinically homogeneous early-stage never-smoker female patients with lung adenocarcinoma were performed to understand tumor-T cell interactions and immune escape points. Using our novel gene panels of eight functional categories in the cancer-immunity cycle, three distinct subgroups were identified in this immune checkpoint blockade-refractory cohort by defective gene expression in two major domains, i.e., type I interferon production/signaling pathway and antigen-presenting machinery. Our approach could play a critical role in understanding immune evasion mechanisms, developing a method for effective selection of rare immune checkpoint blockade responders, and finding new treatment strategies.

PDX models of human lung squamous cell carcinoma: consideration of factors in preclinical and co-clinical applications.

BACKGROUND: Treatment of human lung squamous cell carcinoma (LUSC) using current targeted therapies is limited because of their diverse somatic mutations without any specific dominant driver mutations. These mutational diversities preventing the use of common targeted therapies or the combination of available therapeutic modalities would require a preclinical animal model of this tumor to acquire improved clinical responses. Patient-derived xenograft (PDX) models have been recognized as a potentially useful preclinical model for personalized precision medicine. However, whether the use of LUSC PDX models would be appropriate enough for clinical application is still controversial. METHODS: In the process of developing PDX models from Korean patients with LUSC, the authors investigated the factors influencing the successful initial engraftment of tumors in NOD scid gamma mice and the retainability of the pathological and genomic characteristics of the parental patient tumors in PDX tumors. CONCLUSIONS: The authors have developed 62 LUSC PDX models that retained the pathological and genomic features of parental patient tumors, which could be used in preclinical and co-clinical studies. Trial registration Tumor samples were obtained from 139 patients with LUSC between November 2014 and January 2019. All the patients provided signed informed consents. This study was approved by the institutional review board (IRB) of Samsung Medical Center (2018-03-110).

Can We Design a Nogo Receptor-Dependent Cellular Therapy to Target MS?

The current landscape of therapeutics designed to treat multiple sclerosis (MS) and its pathological sequelae is saturated with drugs that modify disease course and limit relapse rates. While these small molecules and biologicals are producing profound benefits to patients with reductions in annualized relapse rates, the repair or reversal of demyelinated lesions with or without axonal damage, remains the principle unmet need for progressive forms of the disease. Targeting the extracellular pathological milieu and the signaling mechanisms that drive neurodegeneration are potential means to achieve neuroprotection and/or repair in the central nervous system of progressive MS patients. The Nogo-A receptor-dependent signaling mechanism has raised considerable interest in neurological disease paradigms since it can promulgate axonal transport deficits, further demyelination, and extant axonal dystrophy, thereby limiting remyelination. If specific therapeutic regimes could be devised to directly clear the Nogo-A-enriched myelin debris in an expedited manner, it may provide the necessary CNS environment for neurorepair to become a clinical reality. The current review outlines novel means to achieve neurorepair with biologicals that may be directed to sites of active demyelination.

Preconditioning with far-infrared irradiation enhances proliferation, cell survival, and migration of rat bone marrow-derived stem cells via CXCR4-ERK pathways.

Far-infrared radiation (FIR) has been shown to exert positive effects on the cardiovascular system. However, the biological effects of FIR on bone marrow-derived stem cells (BMSCs) are not understood. In the present study, BMSCs were isolated from rat femur bone marrow and cultured in vitro. To investigate the effects of an FIR generator with an energy flux of 0.13 mW/cm2 on rat BMSCs, survival of BMSCs was measured by crystal violet staining, and cell proliferation was additionally measured using Ez-Cytox cell viability, EdU, and Brd U assays. FIR preconditioning was found to significantly increase BMSC proliferation and survival against H2O2. The scratch and transwell migration assays showed that FIR preconditioning resulted in an increase in BMSC migration. qRT-PCR and Western blot analyses demonstrated that FIR upregulated Nanog, Sox2, c-Kit, Nkx2.5, and CXCR4 at both the mRNA and protein levels. Consistent with these observations, PD98059 (an ERK inhibitor) and AMD3100 (a CXCR4 inhibitor) prevented the activation of CXCR4/ERK and blocked the cell proliferation and migration induced by FIR. Overall, these findings provide the first evidence that FIR confers a real and significant benefit on the preconditioning of BMSCs, and might lead to novel strategies for improving BMSC therapy for cardiac ischemia.

Cardioprotective effects of PKG activation by soluble GC activator, BAY 60-2770, in ischemia-reperfusion-injured rat hearts.

Soluble guanylate cyclase (sGC) has been suggested as a therapeutic target for cardiac ischemia-reperfusion (IR) injury. Until now, the molecular mechanism of BAY 60-2770, a sGC activator, in cardiac IR injury has not been assessed. To identify the cardioprotective effects of BAY 60-2770 in IR-injured rat hearts, IR injury was established by occlusion of LAD for 40 min and reperfusion for 7 days, and the effects of BAY 60-2770 on myocardial protection were assessed by echocardiography and TTC staining. 5 nM and 5 µM of BAY 60-2770 were perfused into isolated rat hearts in a Langendorff system. After 10- or 30-min reperfusion with BAY 60-2770, cGMP and cAMP concentrations and PKG activation status were examined. Hearts were also perfused with 1 µM KT5823 or 100 µM 5-HD in conjunction with 5 nM Bay 60-2770 to evaluate the protective role of PKG. Mitochondrial oxidative stress was investigated under hypoxia-reoxygenation in H9c2 cells. In IR-injured rat hearts, BAY 60-2770 oral administration reduced infarct size by TTC staining and improved left ventricular function by echocardiography. Tissue samples from BAY 60-2770-perfused hearts had approximately two-fold higher cGMP levels. BAY 60-2770 increased PKG activity in the myocardium, and the reduced infarct area by BAY 60-2770 was abrogated by KT-5823 in isolated myocardium. In H9c2 cardiac myoblasts, hypoxia-reoxygenation-mediated mitochondrial ROS generation was diminished with BAY 60-2770 treatment, but was recovered by pretreatment with KT-5823. BAY 60-2770 demonstrated a protective effect against cardiac IR injury via mitoKATP opening and decreased mitoROS by PKG activation. BAY 60-2770 has a protective effect against cardiac IR injury via mitoKATP opening and decreased mitoROS by PKG activation. These results demonstrated that BAY 60-2770 may be used as a therapeutic agent for cardiac IR injury.

Enhanced-autophagy by exenatide mitigates doxorubicin-induced cardiotoxicity.

OBJECTIVES: Exenatide is a glucagon-like peptide-1 analogue that mitigates myocardial injury caused by ischemia-reperfusion injury via the survival signaling pathway. We hypothesized that exenatide would provide a protective effect in doxorubicin-induced cardiotoxicity. METHODS: H9c2 cardiomyocytes were pre-treated with exenatide followed by doxorubicin (DOX), and cell viability and intracellular reactive oxygen species (ROS) were subsequently measured. In order to determine the role of autophagy, we performed western blot as well as TUNEL and autophagosome staining. Additionally, rats were treated with exenatide 1h prior to every DOX treatment. Left ventricular (LV) function and performance were then assessed by echocardiography. Myocardial and serum ROS was measured with DHE fluorescence and ROS/RNS assay. RESULTS: DOX-induced caspase-3 activation decreased after pre-treatment with exenatide both in vivo and in vitro. Oxidative stress was attenuated by exenatide in H9c2 cells, as well as in cardiac tissue and serum. The number of autophagosomes and autophagic markers were further increased by exenatide in the DOX-treated H9c2 cells, which mediated AMPK activation. Suppression of the autophagosome abolished exenatide-induced anti-apoptotic effect. Echocardiography showed that pre-treatment with exenatide significantly improved LV dysfunction that is induced by DOX treatment. Exenatide inhibits the DOX-induced production of intracellular ROS and apoptosis in the myocardium. The autophagic markers increased in exenatide pre-treated cardiac tissue. CONCLUSION: Exenatide reduces DOX-induced apoptosis of cardiomyocytes by upregulating autophagy and improving cardiac dysfunction. These novel results highlight the therapeutic potential of exenatide to prevent doxorubicin cardiotoxicity.

Factors affecting the accuracy of chest compression depth estimation.

OBJECTIVE: We aimed to estimate the accuracy of visual estimation of chest compression depth and identify potential factors affecting accuracy. METHODS: This simulation study used a basic life support mannequin, the Ambu man. We recorded chest compression with 7 different depths from 1 to 7 cm. Each video clip was recorded for a cycle of compression. Three different viewpoints were used to record the video. After filming, 25 clips were randomly selected. Health care providers in an emergency department were asked to estimate the depth of compressions while watching the selected video clips. Examiner determinants such as experience and cardiopulmonary resuscitation training and environment determinants such as the location of the camera (examiner) were collected and analyzed. An estimated depth was considered correct if it was consistent with the one recorded. A multivariate analysis predicting the accuracy of compression depth estimation was performed. RESULTS: Overall, 103 subjects were enrolled in the study; 42 (40.8%) were physicians, 56 (54.4%) nurses, and 5 (4.8%) emergency medical technicians. The mean accuracy was 0.89 (standard deviation, 0.76). Among examiner determinants, only subjects' occupation and clinical experience showed significant association with outcome (P=0.03 and P=0.08, respectively). All environmental determinants showed significant association with the outcome (all P<0.001). Multivariate analysis showed that accuracy rate was significantly associated with occupation, camera position, and compression depth. CONCLUSIONS: The accuracy rate of chest compression depth estimation was 0.89 and was significantly related with examiner's occupation, camera view position, and compression depth.

STAT3 transcriptional factor activated by reactive oxygen species induces IL6 in starvation-induced autophagy of cancer cells.

Autophagy is one of the survival processes of cancer cells, especially in stressful conditions such as starvation, hypoxia and chemotherapeutic agents. However, its roles in tumor survival have not yet been fully elucidated. Here, we found for the first time that JAK2/STAT3 was activated in HeLa cells when they were starved or treated with rapamycin. STAT3 activation was associated with autophagic processes, because it was completely inhibited by 3-methyladenine, partially inhibited by knockdown of molecules associated with autophagic processes and blocked by antioxidants, DPI, a Nox inhibitor and knockdown of p22 phox, indicating that ROS generated by Nox that was activated during autophagic processes activated JAK2/STAT3 pathway. Activated STAT3 directly bound to IL6 promoter and increased IL6 mRNA and protein secretion. Finally, the conditioned media, which included IL6, from starved HeLa cells promoted cancer cell survival in both normal and starved conditions, confirmed by clonogenic, proliferation and cell death assays. These data together indicate that the autophagic process in cancer cells can contribute to their survival by JAk2/STAT3 activation and subsequent secretion of growth factors.

[Quality of life in low income Korean aged].

PURPOSE: The purpose of this study was to identify the quality of life and its predictors in low income Korean aged. METHODS: This was a predictive correlational study. An accessible sample from the population of people who were 65 and over and were supported by the basic livelihood security system was 1,040. Quota sampling with strata of state division in the nation was chosen. Quality of life and its predictors in the subjects were measured. RESULTS: The mean quality of life in the subjects was 47.0+/-10.7. Predictors of this study significantly explained 54.3% of the total variance of quality of life. Depression was the most significant predictor of quality of life. Health problems, district, social support, leisure activity, and health behavior had effects on quality of life. CONCLUSION: This finding indicates that quality of life in lower income Korean aged is different from other populations by economic status. Demographics, health status and social status were predictors of quality of life in the aged with a small income.

[Psychometric evaluation of the Korean social support questionnaire].

PURPOSE: Research related to social support in Korea has been hampered by paucity of measurement tools reflecting Korean culture. The aim of the study was to develop Korean social support questionnaire (KSSQ) based on the Korean social support pyramid and to test psychometric properties of the KSSQ. METHODS: The questionnaire was administered to 701 subjects and 658 college students. Psychometric analyses included factor analyses, expert validity, criterion-related validity, internal consistency, and test-retest reliability. RESULTS: A principal components analysis support for construct validity, eliciting a three factor solution accounting for 65.46% of variance in scores. Concurrent and discriminant validity supported criterion-related validity. Internal consistency of reliability was support with Cronbach's alpha of .97-.98 for the entire scale. Test-retest reliability was .76. CONCLUSION: This initial testing of KSSQ to measure Korean social support demonstrates evidence of reliability and validity. Assessment of known-group validity and norm establishment of KSSQ are suggested to provide further sound psychometric properties and practical measurement tools.

Ets1 transcription factor mediates gastrin-releasing peptide-induced IL-8 regulation in neuroblastoma cells.

Angiogenesis plays a critical role in tumor progression in various cancers, including neuroblastoma. We have previously shown that gastrin-releasing peptide (GRP) stimulates neuroblastoma growth and that its cell surface receptors, gastrin-releasing peptide receptors (GRP-R), are overexpressed in advanced-stage human neuroblastomas; however, the effects of GRP on angiogenesis are not clearly elucidated. Interleukin (IL) 8, a proinflammatory chemokine, plays an important role during tumor angiogenesis. Ets transcription factors, such as oncoproteins, cause tumor development and are also known to induce IL-8 expression. In the present study, we found an increased expression of Ets1 in more undifferentiated human neuroblastomas. Stable transfection of SK-N-SH human neuroblastoma cells with Ets1 plasmid resulted in increased IL-8 luciferase activity and IL-8 secretion into cell culture media. Conversely, silencing of Ets1 resulted in a significant decrease in IL-8 secretion in SK-N-SH cells. Moreover, exogenous GRP treatment increased Ets1 (T38) phosphorylation and Ets1 nuclear accumulation, and enhanced Ets1 binding to its DNA consensus sequence, resulting in the stimulation of IL-8 mRNA expression and protein secretion. Our findings demonstrate that GRP upregulates proangiogenic IL-8 expression in an Ets1-dependent manner, suggesting a critical role of this process during GRP-induced neuroblastoma angiogenesis and metastasis.

MYCN silencing induces differentiation and apoptosis in human neuroblastoma cells.

MYCN amplification strongly correlates with unfavorable outcomes in patients with neuroblastoma. The aim of this study was to investigate the role of MYCN in neuroblastoma cell differentiation and apoptosis. We used the technique of RNA interference to inhibit MYCN gene expression in neuroblastoma cells with variable expression of MYCN. Our results showed that inhibition of MYCN gene expression in MYCN amplified cells induced apoptosis and suppressed cell growth; neuronal differentiation also occurred after MYCN gene silencing. Moreover, N-myc downregulation was associated with decreased Bcl-xL protein levels and caspase-3 activation. These data show that small interfering RNA directed to MYCN, which plays a crucial role in neuroblastoma cell survival, may provide a potential novel therapeutic option for aggressive neuroblastomas.