The RhoA guanine exchange factor ABR: a glucose-sensitive mediator of actin reorganization in feto-placental arterial endothelial cells altered by gestational diabetes mellitus.

In utero exposure to gestational diabetes mellitus (GDM) programs the fetus, increasing offspring risk for endothelial dysfunction and cardiovascular disease later in life. Hyperglycaemia is widely recognized as the driving force of diabetes-induced programming. We have previously shown that GDM exposure alters DNA methylation and gene expression associated with actin remodelling in primary feto-placental arterial endothelial cells (fpEC). Thus, we hypothesized that hyperglycaemic insults underlie programmed changes in fpEC morphology and actin organization by GDM. Therefore, arterial fpECs isolated after normal and GDM pregnancy, as well as normal fpECs that were exposed to hyperglycaemia in vitro, were analysed for the effect of GDM and hyperglycaemia on actin organization and network formation. Integration of gene expression and DNA methylation data identified the RhoA activator active BCR-related (ABR) as programmed by GDM and altered by in vitro hyperglycaemia. ABR silencing in GDM-exposed cells reduced RhoA activity by 34 ± 26% (P = 0.033) and restored normal fpEC phenotype. In fact, in vitro hyperglycaemia induced a similar fpEC phenotype as intrauterine exposure to GDM, i.e. round morphology and increased network formation on Matrigel by 34 ± 33% (P = 0.022) vs. 22 ± 20% for GDM (P = 0.004). Thus, we identified ABR as a novel glucose sensitive regulator of actin organization and cell shape, programmed by GDM and upregulated by hyperglycaemia. Identification of mechanisms induced by hyperglycaemia and affecting endothelial function in the long term will contribute to understanding GDM-induced programming of offspring endothelial dysfunction and cardiovascular disease. Future studies could focus on investigating the prevention or reversal of such malprogramming. KEY POINTS: In utero exposure to gestational diabetes mellitus (GDM) affects future health of the offspring, with an increased risk for endothelial dysfunction and cardiovascular disease in later life. GDM alters DNA methylation and expression of ABR in feto-placental arterial endothelial cells (fpEC), a model for endothelial cells exposed to the intrauterine environment of the fetus. GDM phenotype of fpECs is also induced by hyperglycaemia in vitro, and is characterized by altered actin organization and cell shape, which can be restored by ABR silencing. Revealing the cellular mechanisms induced by GDM and hyperglycaemia is important for understanding the mechanisms of how these conditions disturb endothelial function in the offspring.

Atraumatic access to human glioblastoma in a xenograft animal model by cerebral open flow microperfusion.

BACKGROUND: Orthotopic xenograft studies promote the development of targeted/personalized therapies to improve the still poor life expectancy of glioblastoma patients. NEW METHOD: We implemented an atraumatic access to glioblastoma with cerebral Open Flow Microperfusion (cOFM) by implantation of xenograft cells in rat brain with intact blood brain barrier (BBB) and subsequent development of a xenograft glioblastoma at the interface between the cOFM probe and surrounding brain tissue. Human glioma U87MG cells were implanted at a well-defined position into immunodeficient Rowett nude rat´s brain via cOFM (cOFM group) and syringe (control group). Characteristics of the mature tumors from both groups were assessed. RESULTS: For the first time xenograft cells were successfully introduced into rat brain with intact BBB using cOFM, and the tumor tissue developing around the cOFM probe was unaffected by the presence of the probe. Thereby an atraumatic access to the tumor was created. The success rate of glioblastoma development in the cOFM group was high (>70%). The mature cOFM-induced tumors (20-23 days after cell-implantation) resembled the syringe-induced ones and showed typical features of human glioblastoma. COMPARISON WITH EXISTING METHOD: Examining xenograft tumor microenvironment with currently available methods inevitably causes trauma that could affect the reliability of obtained data. CONCLUSION: This novel atraumatic access to human glioblastoma in rat brain provides the possibility to collect interstitial fluid from functional tumor tissue in vivo without trauma generation. Thereby, reliable data can be generated promoting drug research, biomarker identification, and enabling investigation of the BBB of an intact tumor.

Lysophosphatidic Acid Receptor 5 (LPA5) Knockout Ameliorates the Neuroinflammatory Response In Vivo and Modifies the Inflammatory and Metabolic Landscape of Primary Microglia In Vitro.

Systemic inflammation induces alterations in the finely tuned micromilieu of the brain that is continuously monitored by microglia. In the CNS, these changes include increased synthesis of the bioactive lipid lysophosphatidic acid (LPA), a ligand for the six members of the LPA receptor family (LPA1-6). In mouse and human microglia, LPA5 belongs to a set of receptors that cooperatively detect danger signals in the brain. Engagement of LPA5 by LPA polarizes microglia toward a pro-inflammatory phenotype. Therefore, we studied the consequences of global LPA5 knockout (-/-) on neuroinflammatory parameters in a mouse endotoxemia model and in primary microglia exposed to LPA in vitro. A single endotoxin injection (5 mg/kg body weight) resulted in lower circulating concentrations of TNFα and IL-1ß and significantly reduced gene expression of IL-6 and CXCL2 in the brain of LPS-injected LPA5-/- mice. LPA5 deficiency improved sickness behavior and energy deficits produced by low-dose (1.4 mg LPS/kg body weight) chronic LPS treatment. LPA5-/- microglia secreted lower concentrations of pro-inflammatory cyto-/chemokines in response to LPA and showed higher maximal mitochondrial respiration under basal and LPA-activated conditions, further accompanied by lower lactate release, decreased NADPH and GSH synthesis, and inhibited NO production. Collectively, our data suggest that LPA5 promotes neuroinflammation by transmiting pro-inflammatory signals during endotoxemia through microglial activation induced by LPA.

15d-PGJ2 Promotes ROS-Dependent Activation of MAPK-Induced Early Apoptosis in Osteosarcoma Cell In Vitro and in an Ex Ovo CAM Assay.

Osteosarcoma (OS) is the most common type of bone tumor, and has limited therapy options. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has striking anti-tumor effects in various tumors. Here, we investigated molecular mechanisms that mediate anti-tumor effects of 15d-PGJ2 in different OS cell lines. Human U2-OS and Saos-2 cells were treated with 15d-PGJ2 and cell survival was measured by MTT assay. Cell proliferation and motility were investigated by scratch assay, the tumorigenic capacity by colony forming assay. Intracellular ROS was estimated by H2DCFDA. Activation of MAPKs and cytoprotective proteins was detected by immunoblotting. Apoptosis was detected by immunoblotting and Annexin V/PI staining. The ex ovo CAM model was used to study growth capability of grafted 15d-PGJ2-treated OS cells, followed by immunohistochemistry with hematoxylin/eosin and Ki-67. 15d-PGJ2 substantially decreased cell viability, colony formation and wound closure capability of OS cells. Non-malignant human osteoblast was less affected by 15d-PGJ2. 15d-PGJ2 induced rapid intracellular ROS production and time-dependent activation of MAPKs (pERK1/2, pJNK and pp38). Tempol efficiently inhibited 15d-PGJ2-induced ERK1/2 activation, while N-acetylcystein and pyrrolidine dithiocarbamate were less effective. Early but weak activation of cytoprotective proteins was overrun by induction of apoptosis. A structural analogue, 9,10-dihydro-15d-PGJ2, did not show toxic effects in OS cells. In the CAM model, we grafted OS tumors with U2-OS, Saos-2 and MG-63 cells. 15d-PGJ2 treatment resulted in significant growth inhibition, diminished tumor tissue density, and reduced tumor cell proliferation for all cell lines. Our in vitro and CAM data suggest 15d-PGJ2 as a promising natural compound to interfere with OS tumor growth.

Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model.

Increasing evidence suggests that systemic inflammation triggers a neuroinflammatory response that involves sustained microglia activation. This response has deleterious consequences on memory and learning capability in experimental animal models and in patients. However, the mechanisms connecting systemic inflammation and microglia activation remain poorly understood. Here, we identify the autotaxin (ATX)/lysophosphatidic acid (LPA)/LPA-receptor axis as a potential pharmacological target to modulate the LPS-mediated neuroinflammatory response in vitro (the murine BV-2 microglia cell line) and in vivo (C57BL/6J mice receiving a single i.p. LPS injection). In LPS-stimulated (20 ng/mL) BV-2 cells, we observed increased phosphorylation of transcription factors (STAT1, p65, and c-Jun) that are known to induce a proinflammatory microglia phenotype. LPS upregulated ATX, TLR4, and COX2 expression, amplified NO production, increased neurotoxicity of microglia conditioned medium, and augmented cyto-/chemokine concentrations in the cellular supernatants. PF8380 (a type I ATX inhibitor, used at 10 and 1 µM) and AS2717638 (an LPA5 antagonist, used at 1 and 0.1 µM) attenuated these proinflammatory responses, at non-toxic concentrations, in BV-2 cells. In vivo, we demonstrate accumulation of PF8380 in the mouse brain and an accompanying decrease in LPA concentrations. In vivo, co-injection of LPS (5 mg/kg body weight) and PF8380 (30 mg/kg body weight), or LPS/AS2717638 (10 mg/kg body weight), significantly attenuated LPS-induced iNOS, TNFα, IL-1ß, IL-6, and CXCL2 mRNA expression in the mouse brain. On the protein level, PF8380 and AS2717638 significantly reduced TLR4, Iba1, GFAP and COX2 expression, as compared to LPS-only injected animals. In terms of the communication between systemic inflammation and neuroinflammation, both inhibitors significantly attenuated LPS-mediated systemic TNFα and IL-6 synthesis, while IL-1ß was only reduced by PF8380. Inhibition of ATX and LPA5 may thus provide an opportunity to protect the brain from the toxic effects that are provoked by systemic endotoxemia.

Lysophosphatidic Acid Induces Aerobic Glycolysis, Lipogenesis, and Increased Amino Acid Uptake in BV-2 Microglia.

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.

Myeloperoxidase-Derived 2-Chlorohexadecanal Is Generated in Mouse Heart during Endotoxemia and Induces Modification of Distinct Cardiomyocyte Protein Subsets In Vitro.

Sepsis is a major cause of mortality in critically ill patients and associated with cardiac dysfunction, a complication linked to immunological and metabolic aberrations. Cardiac neutrophil infiltration and subsequent release of myeloperoxidase (MPO) leads to the formation of the oxidant hypochlorous acid (HOCl) that is able to chemically modify plasmalogens (ether-phospholipids) abundantly present in the heart. This reaction gives rise to the formation of reactive lipid species including aldehydes and chlorinated fatty acids. During the present study, we tested whether endotoxemia increases MPO-dependent lipid oxidation/modification in the mouse heart. In hearts of lipopolysaccharide-injected mice, we observed significantly higher infiltration of MPO-positive cells, increased fatty acid content, and formation of 2-chlorohexadecanal (2-ClHDA), an MPO-derived plasmalogen modification product. Using murine HL-1 cardiomyocytes as in vitro model, we show that exogenously added HOCl attacks the cellular plasmalogen pool and gives rise to the formation of 2-ClHDA. Addition of 2-ClHDA to HL-1 cardiomyocytes resulted in conversion to 2-chlorohexadecanoic acid and 2-chlorohexadecanol, indicating fatty aldehyde dehydrogenase-mediated redox metabolism. However, a recovery of only 40% indicated the formation of non-extractable (protein) adducts. To identify protein targets, we used a clickable alkynyl analog, 2-chlorohexadec-15-yn-1-al (2-ClHDyA). After Huisgen 1,3-dipolar cycloaddition of 5-tetramethylrhodamine azide (N3-TAMRA) and two dimensional-gel electrophoresis (2D-GE), we were able to identify 51 proteins that form adducts with 2-ClHDyA. Gene ontology enrichment analyses revealed an overrepresentation of heat shock and chaperone, energy metabolism, and cytoskeletal proteins as major targets. Our observations in a murine endotoxemia model demonstrate formation of HOCl-modified lipids in the heart, while pathway analysis in vitro revealed that the chlorinated aldehyde targets specific protein subsets, which are central to cardiac function.

Periplocin mediates TRAIL-induced apoptosis and cell cycle arrest in human myxofibrosarcoma cells via the ERK/p38/JNK pathway.

BACKGROUND: Periploca sepium is traditionally used in Chinese medicine to treat particularly rheumatic disorders and as a tonic. Periplocin was found as the most cytotoxic compound of its root bark and induced death receptor mediated apoptosis in liposarcoma cells. Sarcomas are a rare type of cancer with only a few treatment options. The five-year survival rate of advanced tumors is low. PURPOSE: In this study, we investigated the effects of periplocin in two myxofibrosarcoma (MFS)cell lines, MUG-Myx2a and MUG-Myx2b, which are subclones of the same tumor and reflect the tumor´s heterogeneity, and in T60 primary myxofibrosarcoma cells. METHODS: The xCELLigence system and the CellTiter 96® AQueous assay were used for studying cell viability. FACS and Western blot experiments were used to investigate the effects of periplocin on apoptosis induction, cell cycle distribution, and the expression of cleaved PARP, caspase 3, p53, phospho-histone γH2AX, ERK/phospho ERK, p38/phospho p38, and, finally, JNK/phospho JNK. Additionally, the expression of the apoptotic markers Bim, NOXA, Bak, Bcl-2, Bcl-xl, and the death receptors IGFR, FADD, TRADD, TNFR1A, TRAIL-R1, and TRAIL-R2 were evaluated using reversed real-time PCR. RESULTS: Periplocin decreased dose-dependently the viability of all MFS cell lines and was more effective than the standard chemotherapeutic doxorubicin. It arrested the cells in the G2/M phase and led to caspase activation. Moreover, periplocin increased the mRNA expression of NOXA, Bak, Bcl-2, and death receptors such as TRAIL-R1 and TRAIL-R2 and the protein expression of ERK/phospho ERK, p38/phospho p38, and JNK/phospho JNK. In all cases, differences in the effects in the different subclones were observed. CONCLUSION: Periplocin showed promising effects in MFS cells. The higher effectiveness compared to doxorubicin is an important aspect for further research with regard as a treatment option. The different effects of periplocin in the two subclones showed the great importance of intratumoral heterogeneity in MFS therapy.

MAPK signaling determines lysophosphatidic acid (LPA)-induced inflammation in microglia.

BACKGROUND: In the extracellular environment, lysophosphatidic acid (LPA) species are generated via autotaxin (ATX)-mediated hydrolysis of lysophospholipid precursors. Members of the LPA family are potent lipid mediators transmitting signals via six different G protein-coupled LPA receptors (LPAR1-6). The LPA signaling axis is indispensable for brain development and function of the nervous system; however, during damage of the central nervous system, LPA levels can increase and aberrant signaling events counteract brain function. Here, we investigated regulation of the ATX/LPA/LPAR axis in response to lipopolysaccharide-induced systemic inflammation in mice and potential neurotoxic polarization programs in LPA-activated primary murine microglia. METHODS: In vivo, LPAR1-6 expression was established by qPCR in whole murine brain homogenates and in FACS-sorted microglia. ELISAs were used to quantitate LPA concentrations in the brain and cyto-/chemokine secretion from primary microglia in vitro. Transcription factor phosphorylation was analyzed by immunoblotting, and plasma membrane markers were analyzed by flow cytometry. We used MAPK inhibitors to study signal integration by the JNK, p38, and ERK1/2 branches in response to LPA-mediated activation of primary microglia. RESULTS: Under acute and chronic inflammatory conditions, we observed a significant increase in LPA concentrations and differential regulation of LPAR, ATX (encoded by ENPP2), and cytosolic phospholipase A2 (encoded by PLA2G4A) gene expression in the brain and FACS-sorted microglia. During pathway analyses in vitro, the use of specific MAPK antagonists (SP600125, SB203580, and PD98059) revealed that JNK and p38 inhibition most efficiently attenuated LPA-induced phosphorylation of proinflammatory transcription factors (STAT1 and -3, p65, and c-Jun) and secretion of IL-6 and TNFα. All three inhibitors decreased LPA-mediated secretion of IL-1ß, CXCL10, CXCL2, and CCL5. The plasma membrane marker CD40 was solely inhibited by SP600125 while all three inhibitors affected expression of CD86 and CD206. All MAPK antagonists reduced intracellular COX-2 and Arg1 as well as ROS and NO formation, and neurotoxicity of microglia-conditioned media. CONCLUSION: In the present study, we show that systemic inflammation induces aberrant ATX/LPA/LPAR homeostasis in the murine brain. LPA-mediated polarization of primary microglia via MAPK-dependent pathways induces features reminiscent of a neurotoxic phenotype.

Myeloperoxidase and Septic Conditions Disrupt Sphingolipid Homeostasis in Murine Brain Capillaries In Vivo and Immortalized Human Brain Endothelial Cells In Vitro.

During inflammation, activated leukocytes release cytotoxic mediators that compromise blood-brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO-/- compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.

Circulating cord blood HDL-S1P complex preserves the integrity of the feto-placental vasculature.

Perinatal and long-term offspring morbidities are strongly dependent on the preservation of placental vascular homeostasis during pregnancy. In adults, the HDL-apoM-S1P complex protects the endothelium and maintains vascular integrity. However, the metabolism and biology of cord blood-derived HDLs (referred to as neonatal HDL, nHDL) strikingly differ from those in adults. Here, we investigate the role of neonatal HDLs in the regulation of placental vascular function. We show that nHDL is a major carrier of sphingosine-1-phosphate (S1P), which is anchored to the particle through apoM (rs = 0.90, p < 0.0001) in the fetal circulation. Furthermore, this complex interacts with S1P receptors on the feto-placental endothelium and activates specifically extracellular signal-regulated protein kinases 1 and 2 (ERK) and phospholipase C (PLC) downstream signaling, promotes endothelial cell proliferation and calcium flux. Notably, the nHDL-S1P complex triggers actin filaments reorganization, leading to an enhancement of placental endothelial barrier function. Additionally, nHDL induces vasorelaxation of isolated placental chorionic arteries. Taken together, these results suggest that circulating nHDL exerts vasoprotective effects on the feto-placental endothelial barrier mainly via S1P signaling.

Sexual dimorphism of miRNA signatures in feto-placental endothelial cells is associated with altered barrier function and actin organization.

Endothelial function and the risk for endothelial dysfunction differ between males and females. Besides the action of estrogen, sex chromosome gene expression and programming effects also provoke this sexual dimorphism. MicroRNAs (miRNAs) have emerged as regulators of endothelial cell function and dysfunction. We here hypothesized distinct miRNA expression patterns in male versus female human endothelial cells that contribute to the functional differences. We used our well-established model of fetal endothelial cells isolated from placenta (fpEC) and analyzed sexual dimorphic miRNA expression and potentially affected biological functions. Next-generation miRNA sequencing of fpEC isolated after pregnancies with male and female neonates identified sex-dependent miRNA expression patterns. Potential biological pathways regulated by the altered set of miRNAs were determined using mirPath and mirSystem softwares, and suggested differences in barrier function and actin organization. The identified pathways were further investigated by monolayer impedance measurements (ECIS) and analysis of F-actin organization (Phalloidin). Nine miRNAs were differentially expressed in fpEC of male versus female neonates. Functional pathways most significantly regulated by these miRNAs included 'Adherens junction', 'ECM receptor interaction' and 'Focal adhesion'. These pathways control monolayer barrier function and may be paralleled by altered cytoskeletal organization. In fact, monolayer impedance was higher in fpEC of male progeny, and F-actin staining revealed more pronounced peripheral stress fibers in male versus female fpEC. Our data highlight that endothelial cell function differs between males and females already in utero, and that altered miRNAs are associated with sex dependent differences in barrier function and actin organization.

Small-Molecule Lysophosphatidic Acid Receptor 5 (LPAR5) Antagonists: Versatile Pharmacological Tools to Regulate Inflammatory Signaling in BV-2 Microglia Cells.

Lysophosphatidic acid (LPA) species in the extracellular environment induce downstream signaling via six different G protein-coupled receptors (LPAR1-6). These signaling cascades are essential for normal brain development and function of the nervous system. However, in response to acute or chronic central nervous system (CNS) damage, LPA levels increase and aberrant signaling events can counteract brain function. Under neuro-inflammatory conditions signaling along the LPA/LPAR5 axis induces a potentially neurotoxic microglia phenotype indicating the need for new pharmacological intervention strategies. Therefore, we compared the effects of two novel small-molecule LPAR5 antagonists on LPA-induced polarization parameters of the BV-2 microglia cell line. AS2717638 is a selective piperidine-based LPAR5 antagonist (IC50 0.038 µM) while compound 3 is a diphenylpyrazole derivative with an IC50 concentration of 0.7 µM in BV-2 cells. Both antagonists compromised cell viability, however, at concentrations above their IC50 concentrations. Both inhibitors blunted LPA-induced phosphorylation of STAT1 and STAT3, p65, and c-Jun and consequently reduced the secretion of pro-inflammatory cyto-/chemokines (IL-6, TNFα, IL-1ß, CXCL10, CXCL2, and CCL5) at non-toxic concentrations. Both compounds modulated the expression of intracellular (COX-2 and Arg1) and plasma membrane-located (CD40, CD86, and CD206) polarization markers yet only AS2717638 attenuated the neurotoxic potential of LPA-activated BV-2 cell-conditioned medium towards CATH.a neurons. Our findings from the present in vitro study suggest that the two LPAR5 antagonists represent valuable pharmacological tools to interfere with LPA-induced pro-inflammatory signaling cascades in microglia.

MICU1 controls cristae junction and spatially anchors mitochondrial Ca2+ uniporter complex.

Recently identified core proteins (MICU1, MCU, EMRE) forming the mitochondrial Ca2+ uniporter complex propelled investigations into its physiological workings. Here, we apply structured illumination microscopy to visualize and localize these proteins in living cells. Our data show that MICU1 localizes at the inner boundary membrane (IBM) due to electrostatic interaction of its polybasic domain. Moreover, this exclusive localization of MICU1 is important for the stability of cristae junctions (CJ), cytochrome c release and mitochondrial membrane potential. In contrast to MICU1, MCU and EMRE are homogeneously distributed at the inner mitochondrial membrane under resting conditions. However, upon Ca2+ elevation MCU and EMRE dynamically accumulate at the IBM in a MICU1-dependent manner. Eventually, our findings unveil an essential function of MICU1 in CJ stabilization and provide mechanistic insights of how sophistically MICU1 controls the MCU-Complex while maintaining the structural mitochondrial membrane framework.

Pharmacological Inhibition of Serine Palmitoyl Transferase and Sphingosine Kinase-1/-2 Inhibits Merkel Cell Carcinoma Cell Proliferation.

The majority of Merkel cell carcinoma, a highly aggressive neuroendocrine cancer of the skin, is associated with Merkel cell polyomavirus infection. Polyomavirus binding, internalization, and infection are mediated by glycosphingolipids. Besides receptor function, bioactive sphingolipids are increasingly recognized as potent regulators of several hallmarks of cancer. Merkel cell polyomavirus+ and Merkel cell polyomavirus- cells express serine palmitoyl transferase subunits and sphingosine kinase (SK) 1/2 mRNA. Induced expression of Merkel cell polyomavirus-large tumor antigen in human lung fibroblasts resulted in upregulation of SPTLC1-3 and SK 1/2 expression. Therefore, we exploited pharmacological inhibition of sphingolipid metabolism as an option to interfere with proliferation of Merkel cell polyomavirus+ Merkel cell carcinoma cell lines. We used myriocin (a serine palmitoyl transferase antagonist) and two SK inhibitors (SKI-II and ABC294640). In MKL-1 and WaGa cells myriocin decreased cellular ceramide, sphingomyelin, and sphingosine-1-phosphate content. SKI-II increased ceramide species but decreased sphingomyelin and sphingosine-1-phosphate concentrations. Aberrant sphingolipid homeostasis was associated with reduced cell viability, increased necrosis, procaspase-3 and PARP processing, caspase-3 activity, and decreased AKTS473 phosphorylation. Myriocin and SKI-II decreased tumor size and Ki-67 staining of xenografted MKL-1 and WaGa tumors on the chorioallantoic membrane. Our data suggest that pharmacological inhibition of sphingolipid synthesis could represent a potential therapeutic approach in Merkel cell carcinoma.

Influence of eukaryotic translation initiation factor 6 on non-small cell lung cancer development and progression.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Dysregulation of protein synthesis plays a major role in carcinogenesis, a process regulated at multiple levels, including translation of mRNA into proteins. Ribosome assembly requires correct association of ribosome subunits, which is ensured by eukaryotic translation initiation factors (eIFs). eIFs have become targets in cancer therapy studies, and promising data on eIF6 in various cancer entities have been reported. Therefore, we hypothesised that eIF6 represents a crossroad for pulmonary carcinogenesis. High levels of eIF6 are associated with shorter patient overall survival in adenocarcinoma (ADC), but not in squamous cell carcinoma (SQC) of the lung. We demonstrate significantly higher protein expression of eIF6 in ADC and SQC than in healthy lung tissue based on immunohistochemical data from tissue microarrays (TMAs) and on fresh frozen lung tissue. Depletion of eIF6 in ADC and SQC lung cancer cell lines inhibited cell proliferation and induced apoptosis. Knockdown of eIF6 led to pre-rRNA processing and ribosomal 60S maturation defects. Our data indicate that eIF6 is upregulated in NSCLC, suggesting an important contribution of eIF6 to the development and progression of NSCLC and a potential for new treatment strategies against NSCLC.

A short-term in vivo model for Merkel Cell Carcinoma.

In vivo tumor models are essential for studying the biology of cancer, identifying tumor targets and evaluating antitumor drugs. Considering the request for the minimisation of animal experiments and following the "3R"-rule ("replacement," "refinement," "reduction"), it has become crucial to develop alternative experimental models in cancer biology. Several studies have already described the avian chorioallantoic membrane (CAM) model as an alternative to rodents, suitable to investigate growth, progression and metastasis of various types of cancer. In the present work, we grafted three Merkel cell carcinoma (MCC) cell lines onto the avian CAM and monitored tumor growth and development of solid tumor nodules. Morphology of xenograft was characterised histologically and immunohistochemically. Our results demonstrate CAM assay as a useful tool to study MCC pathophysiology.

2-Chlorohexadecanoic acid induces ER stress and mitochondrial dysfunction in brain microvascular endothelial cells.

Peripheral leukocytes induce blood-brain barrier (BBB) dysfunction through the release of cytotoxic mediators. These include hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system of activated phagocytes. HOCl targets the endogenous pool of ether phospholipids (plasmalogens) generating chlorinated inflammatory mediators like e.g. 2-chlorohexadecanal and its conversion product 2-chlorohexadecanoic acid (2-ClHA). In the cerebrovasculature these compounds inflict damage to brain microvascular endothelial cells (BMVEC) that form the morphological basis of the BBB. To follow subcellular trafficking of 2-ClHA we synthesized a 'clickable' alkyne derivative (2-ClHyA) that phenocopied the biological activity of the parent compound. Confocal and superresolution structured illumination microscopy revealed accumulation of 2-ClHyA in the endoplasmic reticulum (ER) and mitochondria of human BMVEC (hCMEC/D3 cell line). 2-ClHA and its alkyne analogue interfered with protein palmitoylation, induced ER-stress markers, reduced the ER ATP content, and activated transcription and secretion of interleukin (IL)-6 as well as IL-8. 2-ClHA disrupted the mitochondrial membrane potential and induced procaspase-3 and PARP cleavage. The protein kinase R-like ER kinase (PERK) inhibitor GSK2606414 suppressed 2-ClHA-mediated activating transcription factor 4 synthesis and IL-6/8 secretion, but showed no effect on endothelial barrier dysfunction and cleavage of procaspase-3. Our data indicate that 2-ClHA induces potent lipotoxic responses in brain endothelial cells and could have implications in inflammation-induced BBB dysfunction.

Ethyl pyruvate inhibits oxidation of LDL in vitro and attenuates oxLDL toxicity in EA.hy926 cells.

BACKGROUND: Ethyl pyruvate (EP) exerts anti-inflammatory and anti-oxidative properties. The aim of our study was to investigate whether EP is capable of inhibiting the oxidation of LDL, a crucial step in atherogenesis. Additionally, we examined whether EP attenuates the cytotoxic effects of highly oxidized LDL in the human vascular endothelial cell line EA.hy926. METHODS: Native LDL (nLDL) was oxidized using Cu2+ ions in the presence of increasing amounts of EP. The degree of LDL oxidation was quantified by measuring lipid hydroperoxide (LPO) and malondialdehyde (MDA) concentrations, relative electrophoretic mobilities (REMs), and oxidation-specific immune epitopes. The cytotoxicity of these oxLDLs on EA.hy926 cells was assessed by measuring cell viability and superoxide levels. Furthermore, the cytotoxicity of highly oxidized LDL on EA.hy926 cells under increasing concentrations of EP in the media was assessed including measurements of high energy phosphates (ATP). RESULTS: Oxidation of nLDL using Cu2+ ions was remarkably inhibited by EP in a concentration-dependent manner, reflected by decreased levels of LPO, MDA, REM, oxidation-specific epitopes, and diminished cytotoxicity of the obtained oxLDLs in EA.hy926 cells. Furthermore, the cytotoxicity of highly oxidized LDL on EA.hy926 cells was remarkably attenuated by EP added to the media in a concentration-dependent manner reflected by a decrease in superoxide and an increase in viability and ATP levels. CONCLUSIONS: EP has the potential for an anti-atherosclerotic drug by attenuating both, the oxidation of LDL and the cytotoxic effect of (already formed) oxLDL in EA.hy926 cells. Chronic administration of EP might be beneficial to impede the development of atherosclerotic lesions.

Histone deacetylase inhibitors vorinostat and panobinostat induce G1 cell cycle arrest and apoptosis in multidrug resistant sarcoma cell lines.

Synovial sarcoma and high grade chondrosarcoma are characterized by their lack of response to conventional cytotoxic chemotherapy, the tendency to develop lung metastases, and low survival rates. Research within the field prioritizes the development and expansion of new treatment options for dealing with unresectable or metastatic diseases. Numerous clinical trials using histone deacetylases inhibitors (HDACi) have shown specific efficacy as an active antitumor agent for treating a variety of solid tumors. However, as of yet the effect of different HDACi on synovial- and chondrosarcoma cells has not been investigated. In this study, vorinostat (SAHA), panobinostat (LBH-589), and belinostat (PXD101) decreased cell viability of synovial sarcoma (SW-982) and chondrosarcoma (SW-1353) cells in a time- and dose dependent manner and arrested SW-982 cells in the G1/S phase. Western blot analysis determined the responsible cell cycle regulator proteins. In addition, we found apoptotic induction by caspase 3/7 activity, caspase 3 cleavage, and PARP cleavage. In SW-1353 cells only SAHA showed comparable effects. Noteworthy, all HDACi tested had synergistic effects with the topoisomerase II inhibitor doxorubicin in SW-1353 chondrosarcoma cells making the cells more sensitive to the chemotherapeutic drug. Our results show for the first time that SAHA and LBH-589 reduced viability of sarcoma cells and arrested them at the G1/S checkpoint, while also inducing apoptosis and enhancing chemotherapeutic sensitivity, especially in chondrosarcoma cells. These data demonstrate the exciting potential of HDACi for use in sarcoma treatment.