Stress-induced Cell Research Articles

Effect of hop mixture containing xanthohumol on sleep enhancement in a mouse model and ROS scavenging effect in oxidative stress-induced HT22 cells

Effect of hop mixture containing xanthohumol on sleep enhancement in a mouse model and ROS scavenging effect in oxidative stress-induced HT22 cells

Genomic Aberrations Associated with the Pathophysiological Mechanisms of Neurodevelopmental Disorders.

Genomic studies are increasingly revealing that neurodevelopmental disorders are caused by underlying genomic alterations. Chromosomal microarray testing has been used to reliably detect minute changes in genomic copy numbers. The genes located in the aberrated regions identified in patients with neurodevelopmental disorders may be associated with the phenotypic features. In such cases, haploinsufficiency is considered to be the mechanism, when the deletion of a gene is related to neurodevelopmental delay. The loss-of-function mutation in such genes may be evaluated using next-generation sequencing. On the other hand, the patients with increased copy numbers of the genes may exhibit different clinical symptoms compared to those with loss-of-function mutation in the genes. In such cases, the additional copies of the genes are considered to have a dominant negative effect, inducing cell stress. In other cases, not the copy number changes, but mutations of the genes are responsible for causing the clinical symptoms. This can be explained by the dominant negative effects of the gene mutations. Currently, the diagnostic yield of genomic alterations using comprehensive analysis is less than 50%, indicating the existence of more subtle alterations or genomic changes in the untranslated regions. Copy-neutral inversions and insertions may be related. Hence, better analytical algorithms specialized for the detection of such alterations are required for higher diagnostic yields.

Low Concentration Ethanol Prevents Oxidative Stress- Induced Cardiac Cells Injury Under HyperglycemicConditions by Inhibiting the SAPK/JNK Signaling Pathway and ROS Generation

The purpose of this study was to determine the effects and mechanisms of ethanol on oxidative stress-induced cardiac H9c2 cells mitochondrial injury under hyperglycemic conditions. Under hyperglycemic conditions, ethanol pretreatment (10-100 μM) prevented H2O2-induced mitochondria swelling, as well as decreased cell viability and Respiratory Control Ratio (RCR) in the H9c2 cells. It also prevented TMRE fluorescence intensity loss and DCF fluorescence intensity increase under hyperglycemic conditions. These effects of ethanol were reversed by the SAPK/JNK agonist, anisomycin. Finally, treatment of H9c2 cells with 33mM glucose significantly enhanced Akt and ERK phosphorylation, which was not affected by ethanol. However, ethanol decreased the phosphorylation of SAPK/JNK under hyperglycemic conditions. Collectively, these findings indicate that under hyperglycemic conditions, that ethanol prevents oxidative stress-induced mitochondrial injury in cardiac H9c2 cells by preventing ROS generation via inhibiting the SAPK/JNK signaling pathway.

From bench to bar side: Evaluating the red wine storage lesion.

Vitally essential red fluids like packed cells and red wine are seriously influenced in quality when stored over prolonged periods. In the case of red cell concentrates, the resulting storage lesion has particular significance in perioperative medicine. We hypothesized that, in contrast, aging rather improves the properties of red wine in several ways. A translational approach, including (I) in vitro experiments, (II) a randomized, blinded crossover trial of acute clinical effects, and (III) a standardized red wine blind tasting was used. Three monovarietal wines (Cabernet Sauvignon, Chianti, Shiraz) in three different vintages (range 2004–2016), each 5 years different, were assessed. Assessments were performed at a German university hospital (I, II) and on a garden terrace during a mild summer evening (III). Young wines induced cell stress and damage while significantly reducing cytoprotective proteins in HepG2 hepatoma cells. Sympathetic activity and multitasking skills were altered depending on wines’ ages. Hangovers tended to be aggravated by young red wine. Aged variants performed better in terms of aroma and overall quality but worse in optical appearance. We found no evidence for a red wine storage lesion. However, we plead for consensus-based guidelines for proper storage, as it is common in clinical medicine.

Pro-inflammatory T helper 17 directly harms oligodendrocytes in neuroinflammation

T helper (Th)17 cells are considered to contribute to inflammatory mechanisms in diseases such as multiple sclerosis (MS). However, the discussion persists regarding their true role in patients. Here, we visualized central nervous system (CNS) inflammatory processes in models of MS live in vivo and in MS brains and discovered that CNS-infiltrating Th17 cells form prolonged stable contact with oligodendrocytes. Strikingly, compared to Th2 cells, direct contact with Th17 worsened experimental demyelination, caused damage to human oligodendrocyte processes, and increased cell death. Importantly, we found that in comparison to Th2 cells, both human and murine Th17 cells express higher levels of the integrin CD29, which is linked to glutamate release pathways. Of note, contact of human Th17 cells with oligodendrocytes triggered release of glutamate, which induced cell stress and changes in biosynthesis of cholesterol and lipids, as revealed by single-cell RNA-sequencing analysis. Finally, exposure to glutamate decreased myelination, whereas blockade of CD29 preserved oligodendrocyte processes from Th17-mediated injury. Our data provide evidence for the direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for therapeutic opportunities in MS.

HO-1 nuclear accumulation and interaction with NPM1 protect against stress-induced endothelial senescence independent of its enzymatic activity

Heme oxygenase-1 (HO-1) has attracted accumulating attention for its antioxidant enzymatic activity. However, the exact regulatory role of its non-enzymatic activity in the cardiovascular system remains unaddressed. Here, we show that HO-1 was accumulated in the nuclei of stress-induced senescent endothelial cells, and conferred protection against endothelial senescence independent of its enzymatic activity. Overexpression of ΔHO-1, a truncated HO-1 without transmembrane segment (TMS), inhibited H2O2-induced endothelial senescence. Overexpression of ΔHO-1H25A, the catalytically inactive form of ΔHO-1, also exhibited anti-senescent effect. In addition, infection of recombinant adenovirus encoding ΔHO-1 with three nuclear localization sequences (NLS), alleviated endothelial senescence induced by knockdown of endogenous HO-1 by CRISPR/Cas9. Moreover, repression of HO-1 nuclear translocation by silencing of signal peptide peptidase (SPP), which is responsible for enzymatic cleavage of the TMS of HO-1, exacerbated endothelial senescence. Mechanistically, nuclear HO-1 interacted with NPM1 N-terminal portion, prevented NPM1 translocation from nucleolus to nucleoplasm, thus disrupted NPM1/p53/MDM2 interactions and inhibited p53 activation by NPM1, finally resisted endothelial senescence. This study provides a novel understanding of HO-1 as a promising therapeutic strategy for vascular senescence-related cardiovascular diseases.

Cobalt ions induce metabolic stress in synovial fibroblasts and secretion of cytokines/chemokines that may be diagnostic markers for adverse local tissue reactions to hip implants

Adverse local tissue reactions (ALTRs) are a prominent cause of hip implant failure. ALTRs are characterized by aseptic necrosis and leukocyte infiltration of synovial tissue. The prevalence of ALTRs in hips with failing metal implants, with highest rates occurring in patients with metal-on-metal articulations, suggests a role for CoCrMo corrosion in ALTR formation. Although hypersensitivity reactions are the most accepted etiology, the precise cellular mechanism driving ALTR pathogenesis remains enigmatic. Here we show that cobalt ions released by failing hip implants induce mitochondrial stress and cytokine secretion by synovial fibroblasts: the presumptive initiators of ALTR pathogenesis. We found that in-vitro treatment of synovial fibroblasts with cobalt, but not chromium, generated gene expression changes indicative of hypoxia and mitophagy responses also observed in ALTRs biopsies. Inflammatory factors secreted by cobalt-exposed synovial fibroblasts were among those most concentrated in ALTR synovial fluid. Furthermore, both conditioned media from cobalt-exposed synovial fibroblasts, and synovial fluid from ALTRs patients, elicit endothelial activation and monocyte migration. Finally, we identify the IL16/CTACK ratio in synovial fluid as a possible diagnostic marker of ALTRs. Our results provide evidence suggesting that metal ions induce cell stress in synovial fibroblasts that promote an inflammatory response consistent with initiating ALTR formation. Statement of significanceWe demonstrate that the cytotoxic effects of cobalt ions on the synovial cells (fibroblast) is sufficient to trigger inflammation on hip joints with metal implants. Cobalt ions affect mitochondrial function, leading to the auto phagocytosis of mitochondria and trigger a hypoxic response. The cell's hypoxic response includes secretion of cytokines that are capable of trigger inflammation by activating blood vessels and enhancing leukocyte migration. Among the secreted cytokines is IL-16, which is highly concentrated in the synovial fluid of the patients with adverse local tissue reactions and could be use as diagnostic marker. In conclusion we define the cells of the hip joint as key players in triggering the adverse reactions to hip implants and providing biomarkers for early diagnosis of adverse reactions to hip implants.

Endothelial arginase 2 mediates retinal ischemia/reperfusion injury by inducing mitochondrial dysfunction

ObjectiveRetinal ischemic disease is a major cause of vision loss. Current treatment options are limited to late-stage diseases, and the molecular mechanisms of the initial insult are not fully understood. We have previously shown that the deletion of the mitochondrial arginase isoform, arginase 2 (A2), limits neurovascular injury in models of ischemic retinopathy. Here, we investigated the involvement of A2-mediated alterations in mitochondrial dynamics and function in the pathology. MethodsWe used wild-type (WT), global A2 knockout (A2KO-) mice, cell-specific A2 knockout mice subjected to retinal ischemia/reperfusion (I/R), and bovine retinal endothelial cells (BRECs) subjected to an oxygen-glucose deprivation/reperfusion (OGD/R) insult. We used western blotting to measure levels of cell stress and death markers and the mitochondrial fragmentation protein, dynamin related protein 1 (Drp1). We also used live cell mitochondrial labeling and Seahorse XF analysis to evaluate mitochondrial fragmentation and function, respectively. ResultsWe found that the global deletion of A2 limited the I/R-induced disruption of retinal layers, fundus abnormalities, and albumin extravasation. The specific deletion of A2 in endothelial cells was protective against I/R-induced neurodegeneration. The OGD/R insult in BRECs increased A2 expression and induced cell stress and cell death, along with decreased mitochondrial respiration, increased Drp1 expression, and mitochondrial fragmentation. The overexpression of A2 in BREC also decreased mitochondrial respiration, promoted increases in the expression of Drp1, mitochondrial fragmentation, and cell stress and resulted in decreased cell survival. In contrast, the overexpression of the cytosolic isoform, arginase 1 (A1), did not affect these parameters. ConclusionsThis study is the first to show that A2 in endothelial cells mediates retinal ischemic injury through a mechanism involving alterations in mitochondrial dynamics and function.

205-OR: Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death through the AMPK-Dependent Signaling Pathway in Immortalized Adult Rat Schwann (IFRS1) Cells

Autophagy is a catabolic process that removes damaged intracellular organelles components by lysosomal degradation, but it is also activated by oxidative stress. We previously reported that the docosahexaenoic acid (DHA) suppressed oxidative stress-induced autophagy and cell death in immortalized adult rat Schwann (IFRS1) cells. However, the detailed mechanism has not been clarified yet. Therefore, we aimed to elucidate the intracellular signal transduction pathway by which DHA suppresses oxidative stress-induced autophagy in IFRS1 cells. Treatment with tert-butyl hydroperoxide (tBHP) for 3 hours decreased cell viability measured by MTT assay in IFRS1 cells, while pretreatment with 10 μM DHA for 12 hours significantly protected tBHP-induced cytotoxicity. The signal transduction of autophagy was assessed by immunoblotting of LC3, p62, AMP-activated protein kinase (AMPK) phosphorylation and UNC51-like kinase (ULK1) phosphorylation in IFRS1 cells. 50 μM tBHP increased the LC3-II/LC3-I ratio, and AMPK phosphorylation, whereas tBHP decreased the p62 protein expression, indicating that autophagy was induced by tBHP. Pretreatment with 10 μM DHA for 12 hours significantly decreased the LC3-II/LC3-I ratio and AMPK phosphorylation that were increased by tBHP, whereas DHA increased the p62 protein expression that was decreased by tBHP. p70S6Kinase and 4E-BP phosphorylation was not changed either by tBHP or by DHA. Autophagosome and autolysosome were induced by 50 μM tBHP in IFRS1 cells, as demonstrated by DAPRed and DALGreen staining. DHA pretreatment significantly reduced the production of tBHP-induced autophagosome, but suppression of autolysosome was partial. These results suggest that DHA may prevent against diabetic neuropathy by attenuating oxidative stress-induced autophagy and cell death through the AMPK-dependent signaling pathway in Schwann cells. Disclosure Y. Tatsumi: Research Support; Self; Daiichi Sankyo, Johnson & Johnson, Kyowa Kirin Co., Ltd., Lilly Diabetes, MSD K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Japan Tobacco Inc., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K. K., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. K. Sango: None. K. Kato: None. A. Kato: None. N. Niimi: None. H. Yako: None. T. Himeno: None. M. Kondo: None. S. Tsunekawa: None. Y. Kato: None. H. Kamiya: Speaker’s Bureau; Self; Eli Lilly Japan K. K., MDS Co., Ltd., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd. Funding Grant-in-Aid for Scientific Research (18K06763)

Therapeutic potential of astaxanthin and superoxide dismutase in Alzheimer's disease.

Oxidative stress, the imbalance of the antioxidant system, results in an accumulation of neurotoxic proteins in Alzheimer's disease (AD). The antioxidant system is composed of exogenous and endogenous antioxidants to maintain homeostasis. Superoxide dismutase (SOD) is an endogenous enzymatic antioxidant that converts superoxide ions to hydrogen peroxide in cells. SOD supplementation in mice prevented cognitive decline in stress-induced cells by reducing lipid peroxidation and maintaining neurogenesis in the hippocampus. Furthermore, SOD decreased expression of BACE1 while reducing plaque burden in the brain. Additionally, Astaxanthin (AST), a potent exogenous carotenoid, scavenges superoxide anion radicals. Mice treated with AST showed slower memory decline and decreased depositions of amyloid-beta (Aβ) and tau protein. Currently, the neuroprotective potential of these supplements has only been examined separately in studies. However, a single antioxidant cannot sufficiently resist oxidative damage to the brain, therefore, a combinatory approach is proposed as a relevant therapy for ameliorating pathological changes in AD.

Protective effects of Dioscorea birmanica extract against oxidative stress-induced damage in cultured normal hepatocytes and its phytochemical constituents

The rhizome ethanolic extract of Dioscorea birmanica Prain & Burkill (DBE) has long been used as Thai traditional remedies for treatments of chronic diseases whose development is linked to long-term oxidative stress. However, its protective effects against such stress have never been reported. This study evaluated antioxidant actions of DBE using multiple chemical assays and further assessed its protective effects in the normal hepatic BNL CL.2 cell line against sodium nitroprusside (SNP)-induced oxidative stress by measuring cell viability, oxidative damage to biomolecules, and the activities of antioxidant enzymes. Free radical levels in BNL CL.2 cells were determined to verify molecular mechanisms underlying the protective effects of DBE. The contents of phenolics and flavonoids were measured by colorimetric assays, and some of them were structurally elucidated by LC-ESI-QTOF-MS/MS. The results showed that DBE exerted potent scavenging activities against synthetic and natural radicals, including moderate metal chelating activity. Moreover, DBE improved the cell viability, attenuated oxidative damage to lipids, proteins, and DNA, as well as recovered the activities of antioxidant enzymes against SNP-induced oxidative stress with significant degrees in comparison to only stress-induced cells. In parallel with its cytoprotection, DBE exhibited significant reductions of excess levels of intracellular free radicals potentially through its potent antioxidant actions, thereby revealing the protective mechanism of DBE. For phytochemical analysis, high contents of phenolics and flavonoids were detected in DBE using colorimetric assays, and the structures of 14 potential antioxidants were elucidated using LC-ESI-QTOF-MS/MS. Therefore, DBE has the potential to develop into herbal dietary supplements for liver protection.

Elevation of gene expression of Btg2, Gadd 153, and antioxidant markers in RONS-induced PC12 cells

BackgroundFree radicals generated in the biological system bring about modifications in biological molecules causing damage to their structure and function. Identifying the damage caused by ROS and RNS is important to predict the pathway of apoptosis due to stress in PC12 cells. The first defense mechanisms against them are antioxidants which act in various pathways through important cellular organelles like the mitochondria and endoplasmic reticulum. Specific biomarkers like Gadd153 which is a marker for endoplasmic reticulum stress, Nrf2 which responds to the redox changes and translocates the antioxidant response elements, and Btg2 which is an antioxidant regulator have not been addressed in different stress conditions previously in PC12 cells. Therefore, the study was conducted to analyze the gene expression pattern (SOD, Catalase, Btg2, Gadd153, and Nrf2) and the protein expression pattern (iNOS and MnSOD) of the antioxidant stress markers in differential stress-induced PC12 cells. Peroxynitrite (1 μM), rotenone (1 μM), H2O2(100 mM), and high glucose (33 mM) were used to induce oxidative and nitrosative stress in PC12 cells.ResultsThe results obtained suggested that rotenone-induced PC12 cells showed a significant increase in the expression of catalase, Btg2, and Gadd153 compared to the control. Peroxynitrite-induced PC12 cells showed higher expression of Btg2 compared to the control. H2O2 and high glucose showed lesser expression compared to the control in all stress marker genes. In contrast, the Nrf2 gene expression is downregulated in all the stress-induced PC12 cells compared to the control. Further, MnSOD and iNOS protein expression studies suggest that PC12 cells exhibit a selective downregulation. Lower protein expression of MnSOD and iNOS may be resulted due to the mitochondrial dysfunction in peroxynitrite-, high glucose-, and H2O2-treated cells, whereas rotenone-induced cells showed lower expression, which could be the result of a dysfunction of the endoplasmic reticulum.ConclusionDifferent stress inducers like rotenone, peroxynitrite, H2O2, and high glucose increase the NO and ROS. Btg2 and Gadd153 genes were upregulated in the stress-induced cells, whereas the Nrf2 was significantly downregulated in differential stress-induced PC12 cells. Further, antioxidant marker genes were differentially expressed with different stress inducers.

Overexpression of MicroRNA-122 Resists Oxidative Stress-Induced Human Umbilical Vascular Endothelial Cell Injury by Inhibition of p53.

Deep venous thrombosis (DVT) constitutes a great threat to health worldwide. Endothelial cell injury and dysfunction comprise the critical contributor for the development of DVT. However, the mechanism behind it remains poorly elucidated. The study is aimed at investigating the role of microRNA-122 (miR-122) and oxidative stress on DVT. The results showed that miR-122 overexpression dampened H2O2-evoked cytotoxic injury in human umbilical vein endothelial cells (HUVECs) by increasing cell viability, suppressing cell apoptosis and oxidative stress injury. Notably, miR-122 overexpression attenuated provasoconstriction factor endothelin-1 (ET-1) expression in HUVECs exposed to H2O2 but enhanced the productions of vasodilatation factor Prostaglandin F1α (PGF1α). Moreover, inhibition of miR-122 had the opposite results. miR-122 could inhibit the expression of p53. Low expression of p53 could enhance the protection of miR-122 on HUVEC injury. This study highlights that miR-122 overexpression may restore H2O2-induced HUVEC injury by regulating the expression of p53.

An evaluation of sorter induced cell stress (SICS) on peripheral blood mononuclear cells (PBMCs) after different sort conditions - Are your sorted cells getting SICS?

Flow cytometry and fluorescence-activated cell sorting have become invaluable tools to analyze and isolate specific cell populations in a wide range of biomedical research and clinical applications. In countless approaches worldwide, scientists are using single cell analyses to better understand the significance and variation within different cellular populations, and fluorescence-activated cell sorting has become a major technique for cell isolation in both basic and clinical research. However, majority of available cell sorters are pressurized, droplet-based systems, which apply significant environmental pressure and shear stress to cells during sorting. Recently, the flow cytometry community has become increasingly aware about the potential negative effects this could have on sorted cells and the term “sorter induced cell stress” (SICS) has been proposed. However, up to date only a limited number of studies have investigated the effects of cell sorting on cell viability and function. Therefore, solid data on the effects of sheath pressure and nozzle size on survival and function of sorted cells are surprisingly rare. With this in mind, we sorted “CD4+” T-cells and “live” cells from human peripheral blood mononuclear cells (PBMCs) at different sort conditions and analyzed their quality before and after sorting in a series of assays. Here we present our findings in reference to cell viability and cell proliferation following sorting on different instruments (BD FACSAria III SORP and BD FACSJazz), utilizing different nozzle sizes (70 to 100 μm) and sheath pressure settings (20 to 70 psi). The results show no significant differences in cell viability and proliferation after the different tested sort conditions, but rather differences between individual experiments. These findings are evaluated and their potential significance in cell sorting experiments is discussed.

Gb3-cSrc complex in glycosphingolipid-enriched microdomains contributes to the expression of p53 mutant protein and cancer drug resistance via β-catenin-activated RNA methylation.

Glucosylceramide synthase (GCS) is a key enzyme catalyzing ceramide glycosylation to generate glucosylceramide (GlcCer), which in turn serves as the precursor for cells to produce glycosphingolipids (GSLs). In cell membranes, GSLs serve as essential components of GSL‐enriched microdomains (GEMs) and mediate membrane functions and cell behaviors. Previous studies showed that ceramide glycosylation correlates with upregulated expression of p53 hotspot mutant R273H and cancer drug resistance. Yet, the underlying mechanisms remain elusive. We report herewith that globotriaosylceramide (Gb3) is associated with cSrc kinase in GEMs and plays a crucial role in modulating expression of p53 R273H mutant and drug resistance. Colon cancer cell lines, either WiDr homozygous for missense‐mutated TP53 (R273H+/+) or SW48/TP53‐Dox bearing heterozygous TP53 mutant (R273H/+), display drug resistance with increased ceramide glycosylation. Inhibition of GCS with Genz‐161 (GENZ 667161) resensitized cells to apoptosis in these p53 mutant‐carrying cancer cells. Genz‐161 effectively inhibited GCS activity, and substantially suppressed the elevated Gb3 levels seen in GEMs of p53‐mutant cells exposed to doxorubicin. Complex formation between Gb3 and cSrc in GEMs to activate β‐catenin was detected in both cultured cells and xenograft tumors. Suppression of ceramide glycosylation significantly decreased Gb3‐cSrc in GEMs, β‐catenin, and methyltransferase‐like 3 for m6A RNA methylation, thus altering pre‐mRNA splicing, resulting in upregulated expression of wild‐type p53 protein, but not mutants, in cells carrying p53 R273H. Altogether, increased Gb3‐cSrc complex in GEMs of membranes in response to anticancer drug induced cell stress promotes expression of p53 mutant proteins and accordant cancer drug resistance.

HBP1 deficiency protects against stress-induced premature senescence of nucleus pulposus.

Senescence of nucleus pulposus (NP) cells is involved in the pathological process of intervertebral disc degeneration (IVDD). HMG-box transcription factor 1 (HBP1) is a transcriptional inhibitor that prevents proliferation and regulates premature senescence of cells. The aim of this study was to confirm whether HBP1 deficiency could protect stress-induced NP cells premature senescence. Firstly, HBP1 protein level in human degenerated intervertebral disc tissues was detected. Then, NP cells were isolated from disc samples and transfected with plasmid to upregulate HBP1expression. H2O2 and interleukin-1b (IL-1b) were used to induce NP cells premature senescence in a different manner. Thereafter, cell viability, proliferation, and apoptosis were measured, and the protein expressions of collagen II, HBP1, and p16, were determined by Western blot or immunofluorescence. Finally, the mRNA levels of aggrecan, collagen I, IL-6, Transforming Growth Factor-α (TNF-α), and matrix metalloproteinase-3 (MMP-3) were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The data indicated that HBP1 was upregulated in degenerated NP tissues. HBP1 gene overexpression increased p16 expression, affected NP cell proliferation, and caused cell apoptosis. In addition, HBP1 also decreased the collagen II and aggrecan expressions but increased collagen I, IL-6, TNF-α, and MMP-3 levels. Moreover, the silencing of HBP1 markedly reversed the H2O2 and IL-1b induced NP cell senescence by reducing p16 expression, apoptotic cell population, and inflammatory response and by promoting cell proliferation. In summary, HBP1 accumulation contributes to the senescence of NP cells, and HBP1 deficiency protects stress-induced NP cells premature senescence.

Cytotoxic effect of PEI-coated magnetic nanoparticles on the regulation of cellular focal adhesions and actin stress fibres

The biocompatibility of surface-coated magnetic nanoparticles (MNPs) is key to their successful use in biomedical applications. Polyethyleneimine-coated MNPs (MNP-PEIs) provide improved in vitro nucleic acid transfection efficiency and are safer compared to conventional chemicals. Commercial cell toxicity assays are useful for end-point and high-throughput screening, however, they only reports cells that have undergone an extreme toxic response leading to cell death. Cell toxicity is a complex process which can be expressed in many forms, through morphological, metabolic, and epigenetic changes. This study explores the effect of magnetic transfection with MNP-PEIs and an external magnetic field on cell toxicity, by studying particle internalization, changes in cellular morphology, and cell adhesion. We show that MNP-PEIs induce cell stress through a dose-dependent increase in cell adhesion via the overexpression of vinculin and formation of actin stress fibres. While the presence of PEI was the main contributor to increased cell stress, free PEI polyplexes induced higher toxicity compared to PEI bound to MNPs. MNPs without PEI coating however did not adversely affect cells, suggesting a chemical effect instead of a mechanical one. In addition, genes identified as being associated with actin fibre regulation and cell adhesion showed significant increases in expression from MNP-PEI internalization. From these results, we identify anomalous cell behaviour, morphology, and gene expression after interaction with MNP-PEIs, as well as a safe dosage to reduce acute cell toxicity.

Mutations in COMP cause familial carpal tunnel syndrome

Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment syndrome, affecting a large proportion of the general population. Genetic susceptibility has been implicated in CTS, but the causative genes remain elusive. Here, we report the identification of two mutations in cartilage oligomeric matrix protein (COMP) that segregate with CTS in two large families with or without multiple epiphyseal dysplasia (MED). Both mutations impair the secretion of COMP by tenocytes, but the mutation associated with MED also perturbs its secretion in chondrocytes. Further functional characterization of the CTS-specific mutation reveals similar histological and molecular changes of tendons/ligaments in patients’ biopsies and the mouse models. The mutant COMP fails to oligomerize properly and is trapped in the ER, resulting in ER stress-induced unfolded protein response and cell death, leading to inflammation, progressive fibrosis and cell composition change in tendons/ligaments. The extracellular matrix (ECM) organization is also altered. Our studies uncover a previously unrecognized mechanism in CTS pathogenesis.

Microwaves can reverse the tumour phenotype of human papillomavirus type 16 (HPV16)-positive keratinocytes in 3D cell culture models: a novel therapy for HPV-associated disease?

High-risk human papillomavirus (HPV) is the causative agent of benign, precancerous and cancerous lesions, in both anogenital and oropharyngeal sites. Increased expression of the viral oncoproteins E6 and E7 are responsible for tumour progression. The treatment of these precancerous and cancerous lesions is invasive, painful and with long-term side effects. Localised microwaves have been used successfully in the clinic for the treatment of verrucas, which are caused by low-risk HPV genotypes (>75% success rate versus >33% for cryotherapy). Moreover, local hyperthermia is known to have anti-tumour effects. Ten-second microwave treatment of 3D in vitro-grown cervical tumour tissues (HPV16-positive SiHa cell) resulted in cell death in the treated zone while the tissue integrity was disrupted in the adjacent area. Microwaves induced apoptosis (induction of cleaved caspase 3) and autophagy (induction of LC3) and inhibited cell proliferation (loss of Ki67 and MCM2) in the entire tissue. Furthermore, HPV16 E6 and E7 expression was reduced in cells in the treated and transition zones, with subsequent induction of expression of the apoptosis-regulator, p53 over a 24 hour period following microwave treatment. Thermal stress, identified with the Heat Shock Protein 70 (HSP70) and translational stress identified by G3BP expression, was observed in the transition zone. In conclusion, we demonstrate that the microwave treatment induces cell stress pathways and inhibits HPV oncoprotein expression that causes tumour progression. Induction of apoptosis and reduced cell proliferation suggest a reversal of the cervical tumour phenotype in the 3D tissues.

50-OR: Beta Cell GDF15 Contributes to Diabetic Stress-Induced Beta-Cell Loss

Endoplasmic reticulum (ER) stress has been shown to play an important role in beta cell loss in diabetes. However, the factors involved in this process are still not fully understood. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor beta (TGFβ) family, is a stress response gene and is involved in various diseases. GDF15 is expressed in adult human beta cells; however, its role in beta cells and diabetes had not been elucidated. Interestingly, we now have identified GDF15 as a key factor involved in ER stress-induced beta cell loss. We found that GDF15 mRNA, protein levels and promoter activity were significantly induced by ER stress in INS-1 cells, Akita beta cells, and primary human islets and we identified the transcription factor involved. Moreover, we discovered that overexpression of GDF15 exacerbated, while knockdown of GDF15 inhibited ER stress-induced beta cell apoptosis, at least in part by regulating the pro-apoptotic factor CHOP. Most importantly, we found that GDF15 knockout mice were protected from STZ-induced diabetes, maintained insulin producing beta cells and exhibited improved serum insulin levels. Taken together, our findings suggest that GDF15 plays an important role in ER stress-induced beta cell death and that inhibition of GDF15 may represent a novel strategy to reduce beta cell loss and treat diabetes. Disclosure G. Xu: None. S. Jo: None. J. Chen: None. T. Grayson: None. A. Shalev: None.