The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9.

Siglec-9 is a sialic-acid-binding lectin expressed predominantly on myeloid cells. Aberrant glycosylation occurs in essentially all types of cancers and results in increased sialylation. Thus, when the mucin MUC1 is expressed on cancer cells, it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). Here we found that this cancer-specific MUC1 glycoform, through engagement of Siglec-9, 'educated' myeloid cells to release factors associated with determination of the tumor microenvironment and disease progression. Moreover, MUC1-ST induced macrophages to display a tumor-associated macrophage (TAM)-like phenotype, with increased expression of the checkpoint ligand PD-L1. Binding of MUC1-ST to Siglec-9 did not activate the phosphatases SHP-1 or SHP-2 but, unexpectedly, induced calcium flux that led to activation of the kinases MEK-ERK. This work defines a critical role for aberrantly glycosylated MUC1 and identifies an activating pathway that follows engagement of Siglec-9.

Development of a perfusion process for continuous lentivirus production using stable suspension producer cell lines.

The large-scale production of clinical-grade lentiviral vectors (LVs) for gene therapy applications is a remaining challenge. The use of adherent cell lines and methods like transient transfection are cost-intensive and hamper process scalability as well as reproducibility. This study describes the use of two suspension-adapted stable packaging cell lines, called GPRGs and GPRTGs, for the development of a scalable and serum-free LV production process. Both stable packaging cell lines are based on an inducible Tet-off system, thus requiring doxycycline removal for initiation of the virus production. Therefore, we compared different methods for doxycycline removal and inoculated three independent 5 L bioreactors using a scalable induction method by dilution, an acoustic cell washer and manual centrifugation. The bioreactors were inoculated with a stable producer cell line encoding for a LV carrying a clinically relevant gene. LV production was performed in perfusion mode using a cell retention device based on acoustic wave separation. Comparable cell-specific productivities were obtained with all three methods and cumulative functional yields up to 6.36 × 1011 transducing units per bioreactor were generated in a 234-h long process, demonstrating the usability of stable Tet-off cell lines for an easily scalable suspension process. Remarkably, cell viabilities >90% were maintained at high cell densities without compromising productivity throughout the whole process, allowing to further extend the process time. Given its low effects of toxicity during virus production, the presented cell lines are excellent candidates to develop a fully continuous LV production process to overcome the existing bottlenecks in LV manufacturing.

Quantifying and mathematical modelling of the influence of soluble adenylate cyclase on cell cycle in human endothelial cells with Bayesian inference.

Adenosine-3', 5'-cyclic monophosphate (cAMP) produced by adenylate cyclases (ADCYs) is an established key regulator of cell homoeostasis. However, its role in cell cycle control is still controversially discussed. This study focussed on the impact of soluble HCO3 - -activated ADCY10 on cell cycle progression. Effects are quantified with Bayesian inference integrating a mathematical model and experimental data. The activity of ADCY10 in human umbilical vein endothelial cells (HUVECs) was either pharmacologically inhibited by KH7 or endogenously activated by HCO3 - . Cell numbers of individual cell cycle phases were assessed over time using flow cytometry. Based on these numbers, cell cycle dynamics were analysed using a mathematical model. This allowed precise quantification of cell cycle dynamics with model parameters that describe the durations of individual cell cycle phases. Endogenous inactivation of ADCY10 resulted in prolongation of mean cell cycle times (38.7 ± 8.3 h at 0 mM HCO3 - vs 30.3 ± 2.7 h at 24 mM HCO3 - ), while pharmacological inhibition resulted in functional arrest of cell cycle by increasing mean cell cycle time after G0 /G1 synchronization to 221.0 ± 96.3 h. All cell cycle phases progressed slower due to ADCY10 inactivation. In particular, the G1 -S transition was quantitatively the most influenced by ADCY10. In conclusion, the data of the present study show that ADCY10 is a key regulator in cell cycle progression linked specifically to the G1 -S transition.

Glycolysis is integral to histamine-induced endothelial hyperpermeability.

Histamine-induced vascular leakage is a core process of allergic pathologies, including anaphylaxis. Here, we show that glycolysis is integral to histamine-induced endothelial barrier disruption and hyperpermeability. Histamine rapidly enhanced glycolysis in endothelial cells via a pathway that involved histamine receptor 1 and phospholipase C beta signaling. Consistently, partial inhibition of glycolysis with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) prevented histamine-induced hyperpermeability in human microvascular endothelial cells, by abolishing the histamine-induced actomyosin contraction, focal adherens junction formation, and endothelial barrier disruption. Pharmacologic blockade of glycolysis with 3PO in mice reduced histamine-induced vascular hyperpermeability, prevented vascular leakage in passive cutaneous anaphylaxis and protected from systemic anaphylaxis. In conclusion, we elucidated the role of glycolysis in histamine-induced disruption of endothelial barrier integrity. Our data thereby point to endothelial glycolysis as a novel therapeutic target for human pathologies related to excessive vascular leakage, such as systemic anaphylaxis.

Hyperosmolality in CHO cell culture: effects on the proteome.

Chinese hamster ovary (CHO) cells are the most commonly used host cell lines for therapeutic protein production. Exposure of these cells to highly concentrated feed solution during fed-batch cultivation can lead to a non-physiological increase in osmolality (> 300 mOsm/kg) that affects cell physiology, morphology, and proteome. As addressed in previous studies (and indeed, as recently addressed in our research), hyperosmolalities of up to 545 mOsm/kg force cells to abort proliferation and gradually increase their volume-almost tripling it. At the same time, CHO cells also show a significant hyperosmolality-dependent increase in mitochondrial activity. To gain deeper insight into the molecular mechanisms that are involved in these processes, as detailed in this paper, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed CHO cells compared with control cells. Our analysis revealed differentially expressed key proteins that mediate mitochondrial activation, oxidative stress amelioration, and cell cycle progression. Our studies also demonstrate a previously unknown effect: the strong regulation of proteins can alter both cell membrane stiffness and permeability. For example, we observed that three types of septins (filamentous proteins that form diffusion barriers in the cell) became strongly up-regulated in response to hyperosmolality in the experimental setup. Overall, these new observations correlate well with recent CHO-based fluxome and transcriptome studies, and reveal additional unknown proteins involved in the response to hyperosmotic pressure by over-concentrated feed in mammalian cells.Key points• First-time comparative proteome analysis of CHO cells exposed to over-concentrated feed.• Discovery of membrane barrier-forming proteins up-regulation under hyperosmolality.• Description of mitochondrial and protein chaperones activation in treated cells.

Hyperosmolality in CHO culture: Effects on cellular behavior and morphology.

Exposure of Chinese hamster ovary cells (CHO) to highly concentrated feed solution during fed-batch cultivation is known to result in an unphysiological osmolality increase (>300 mOsm/kg), affecting cell physiology and morphology. Extending previous observation on osmotic adaptation, the present study investigates for the first time potential effects of hyperosmolality on CHO cells on both population and single-cell level. We intentionally exposed CHO cells to hyperosmolality of up to 545 mOsm/kg during fed-batch cultivation. In concordance with existing research data, hyperosmolality-exposed CHO cells showed a nearly triplicated volume accompanied by ablation of proliferation. On the molecular level, we observed a strong hyperosmolality-dependent increase in mitochondrial activity in CHO cells compared to control. In contrast to mitochondrial activity, hyperosmolality-dependent proliferation arrest of CHO cells was not accompanied by DNA accumulation or caspase-3/7-mediated apoptosis. Notably, we demonstrate for the first time a formation of up to eight multiple, small nuclei in single hyperosmolality-stressed CHO cells. The here presented observations reveal previously unknown hyperosmolality-dependent morphological changes in CHO cells and support existing data on the osmotic response in mammalian cells.

Development and application of a cultivation platform for mammalian suspension cell lines with single-cell resolution.

In bioproduction processes, cellular heterogeneity can cause unpredictable process outcomes or even provoke process failure. Still, cellular heterogeneity is not examined systematically in bioprocess research and development. One reason for this shortcoming is the applied average bulk analyses, which are not able to detect cell-to-cell differences. In this study, we present a microfluidic tool for mammalian single-cell cultivation (MaSC) of suspension cells. The design of our platform allows cultivation in highly controllable environments. As a model system, Chinese hamster ovary cells (CHO-K1) were cultivated over 150 h. Growth behavior was analyzed on a single-cell level and resulted in growth rates between 0.85 and 1.16 day-1 . At the same time, heterogeneous growth and division behavior, for example, unequal division time, as well as rare cellular events like polynucleation or reversed mitosis were observed, which would have remained undetected in a standard population analysis based on average measurements. Therefore, MaSC will open the door for systematic single-cell analysis of mammalian suspension cells. Possible fields of application represent basic research topics like cell-to-cell heterogeneity, clonal stability, pharmaceutical drug screening, and stem cell research, as well as bioprocess related topics such as media development and novel scale-down approaches.

A positive pressure workstation for semi-automated peptide purification of complex proteomic samples.

RATIONALE: High-throughput reliable data generation has become a substantial requirement in many "omics" investigations. In proteomics the sample preparation workflow consists of multiple steps adding more bias to the sample with each additional manual step. Especially for label-free quantification experiments, this drastically impedes reproducible quantification of proteins in replicates. Here, a positive pressure workstation was evaluated to increase automation of sample preparation and reduce workload as well as consumables. METHODS: Digested peptide samples were purified utilizing a new semi-automated sample preparation device, the Resolvex A200, followed by nanospray liquid chromatography/electrospray ionization (nLC/ESI) Orbitrap tandem mass spectrometry (MS/MS) measurements. In addition, the sorbents Maestro and WWP2 (available in conventional cartridge and dual-chamber narrow-bore extraction columns) were compared with Sep-Pak C18 cartridges. Raw data was analyzed by MaxQuant and Perseus software. RESULTS: The semi-automated workflow with the Resolvex A200 workstation and both new sorbents produced highly reproducible results within 10-300 µg of peptide starting material. The new workflow performed equally as well as the routinely conducted manual workflow with similar technical variability in MS/MS-based identifications of peptides and proteins. A first application of the system to a biological question contributed to highly reliable results, where time-resolved proteomic data was separated by principal component analysis (PCA) and hierarchical clustering. CONCLUSIONS: The new workstation was successfully established for proteolytic peptide purification in our proteomic workflow without any drawbacks. Highly reproducible results were obtained in decreased time per sample, which will facilitate further large-scale proteomic investigations.

Exploring the molecular content of CHO exosomes during bioprocessing.

In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade, in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. However, exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be a ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we provide a brief overview of different separation techniques and subsequently perform a proteome and regulatory, non-coding RNA analysis of exosomes, derived from lab-scale bioreactor cultivations of a CHO-K1 cell line, to lay out reference data for further research in the field. Applying bottom-up orbitrap shotgun proteomics and next-generation small RNA sequencing, we detected 1395 proteins, 144 micro RNA (miRNA), and 914 PIWI-interacting RNA (piRNA) species differentially across the phases of a batch cultivation process. The exosomal proteome and RNA data are compared with other extracellular fractions and cell lysate, yielding several significantly exosome-enriched species. Graphical Abstract KEY POINTS: • First-time comprehensive protein and miRNA characterization of CHO exosomes. • Isolation protocol and time point of bioprocess strongly affect quality of extracellular vesicles. • CHO-derived exosomes also contain numerous piRNA species of yet unknown function.

Identification and evaluation of cell- growth-inhibiting bDtBPP-analogue degradation products from phosphite antioxidants used in polyolefin bioprocessing materials.

The inhibiting effect of the secondary phosphite antioxidant degradation product bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) on cell growth is well-known. The present study describes structurally related compounds which are likely to be formed from similar widely used phosphite antioxidants used in materials for the manufacturing of single-use (SU) equipment. Two potential candidates of such compounds-3,3',5,5'-tetra-tert-butyl-2,2'-dihydroxybiphenylphosphate (TtBBP) and bis(p-nonylphenyl)phosphate (bNPP)-were identified by chromatography and mass spectrometry followed by synthesis and X-ray structure elucidation. Additionally, the formation of TtBBP was confirmed in an analytical degradation study and its migration from SU bioprocessing material was estimated. The cytotoxicity evaluation by means of cell culture spiking experiments and flow cytometry analysis revealed that' even if cell growth was inhibited by all the compounds to some extent, bDtBPP showed the most severe effect and stoods out from the other two degradants investigated. Graphical abstract.

Predictive Validity of the Static-99 and Static-99R in Switzerland.

This study evaluated the validity of the Static-99 and Static-99R in assessing sexual recidivism in Switzerland, based on a sample of 142 male sex offenders. Both tools showed predictive validity, but the Static-99R had better discrimination (OR = 1.82, AUC = .81) and calibration (Brier = .078, P/E = 0.96) than the Static-99. A cut score of four on the Static-99R maximized sensitivity (92.9%) and specificity (60.2%). However, although most offenders (98.7%) with a score < 4 did not commit sexual offenses in the 5-year follow-up period, only one in five (20.3%) offenders with a score ≥ 4 actually recidivated. Furthermore, the predicted number of recidivists in the well above average risk category (Static-99R ≥ 6) was 24% higher than expected in routine samples. The results suggest that the Static-99R may be a useful screening tool to identify low-risk individuals but offenders with scores ≥ 4 should be subjected to a more thorough assessment.

Clonal variations in CHO IGF signaling investigated by SILAC-based phosphoproteomics and LFQ-MS.

Chinese hamster ovary (CHO) cells are commonly used for the production of monoclonal antibodies. Omics technologies have been used to elucidate cellular switch points which result in higher monoclonal antibody (mAb) productivity and process yields in CHO and other biopharmaceutical production cell lines such as human or mouse. Currently, investigations of the phosphoproteome in CHO cell lines are rare yet could provide further insights into cellular mechanisms related to target product expression. Therefore, we investigated CHO IGF-signaling events using a comparative expression and phosphoproteomic approach in recombinant mAb-producing XL99 cell lines and corresponding parental strain. Differences were found on the level of protein expression between producer and parental cells in the exponential growth phase, mainly in proteins related to the lysosome, oligosaccharide metabolic processes, stress response, and cellular homeostasis. Within a stable isotope labeling by amino acids in cell culture (SILAC)-based phosphoproteomic investigation of IGF signaling, expected general regulation of phosphorylation sites and cell line-specific responses were observed. Detected early phosphorylation events can be associated to observed effects of IGF on cellular growth, metabolism, and cell cycle distribution. Producer cell line-specific signaling exhibited differences to parental cells in intracellular trafficking and transcriptional processes, along with an overall lower amount of observable cross talk to other signaling pathways. By combining label-free and SILAC-based expression for phosphoproteomic analyses, cellular differences in the highly interactive levels of signaling and protein expression were detected, indicating alterations in metabolism and growth following treatment with an exogenous growth factor. The characterization of cell lines and effects of IGF addition resulted in identification of metabolic switch points. With this data, it will be possible to modulate pathways towards increased CHO process yield by targeted application of small-molecule inhibitors.

Assessment of mixture toxicity of (tri)azoles and their hepatotoxic effects in vitro by means of omics technologies.

Consumers are constantly exposed to chemical mixtures such as multiple residues of different pesticides via the diet. This raises questions concerning potential combination effects, especially because these substances are tested for regulatory purposes on an individual basis. With approximately 500 active substances approved as pesticides, there are too many possible combinations to be tested in standard animal experiments generally requested for regulatory purposes. Therefore, the development of in vitro tools and alternative testing strategies for the assessment of mixture effects is extremely important. As a first step in the development of such in vitro tools, we used (tri)azoles as model substances in a set of different cell lines derived from the primary target organ of these substances, the liver (human: HepaRG, rat: H4IIE). Concentrations were reconciled with measured tissue concentrations obtained from in vivo experiments to ensure comparable effect levels. The effects of the substances were subsequently analyzed by transcriptomics and metabolomics techniques and compared to data from corresponding in vivo studies. The results show that similar toxicity pathways are affected by substances and combinations, thus indicating a similar mode of action and additive effects. Two biomarkers obtained by the approach, CAR and Cyp1A1, were used for mixture toxicity modeling and confirmed the concentration-additive effects, thus supporting the selected testing strategy and raising hope for the development of in vitro methods suitable to detect combination effects and prioritize mixtures of concern for further testing.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)-the effect of biomolecular ligands to balance cell adhesion and stimulated detachment.

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Impact of extracellular RNA on endothelial barrier function.

Different types of high and low molecular weight extracellular RNA (eRNA) are liberated from cells upon conditions of tissue damage or vascular diseases and have been demonstrated in vivo and in vitro to influence the integrity and barrier function of the vascular endothelium. Among the types of self eRNA studied in this respect, ribosomal RNA appears to engage cytokines to promote hyperpermeability, while counteracting RNase1 serves as a potent vessel-protective factor. Different microRNAs may change the expression program of endothelial cells with consequences for cellular contacts and stability. Non-self viral RNAs are recognized by Toll-like receptors that transmit intracellular inflammation signals to disturb the vascular barrier function, largely in connection with infectious diseases. Although derived from the same nucleotide building blocks, the various forms of eRNA exhibit a multitude of molecular interactions with the endothelium that may drastically change its phenotypical characteristics. The impact of eRNA on vascular integrity in health and disease is summarized in this concise review.

Establishment of a CpG island microarray for analyses of genome-wide DNA methylation in Chinese hamster ovary cells.

Optimizing productivity and growth rates of recombinant Chinese hamster ovary (CHO) cells requires insight into the regulation of cellular processes. In this regard, the elucidation of the epigenetic process of DNA methylation, known to influence transcription by a differential occurrence in CpG islands in promoter regions, is increasingly gaining importance. However, DNA methylation has not yet been investigated on a genomic scale in CHO cells and suitable tools have not existed until now. Based on the genomic and transcriptomic CHO data currently available, we developed a customized oligonucleotide microarray covering 19598 CpG islands (89 % of total bioinformatically identified CpG islands) in the CHO genome. We applied our CHO-specific CpG island microarray to investigate the effect of butyrate treatment on differential DNA methylation in CHO cultures in a time-dependent approach. Supplementation of butyrate is known to enhance cell specific productivities in CHO cells and leads to alterations of epigenetic silencing events. Gene ontology clusters regarding, e.g., chromatin modification or DNA repair, were significantly overrepresented 24 h after butyrate addition. Functional classifications furthermore indicated that several major signaling systems such as the Wnt/ß-catenin pathway were affected by butyrate treatment. Our novel CHO-specific CpG island microarray will provide valuable information in future studies of cellular processes associated with productivity and product characteristics.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Hypoxia-reoxygenation-induced endothelial barrier failure: role of RhoA, Rac1 and myosin light chain kinase.

Hypoxia-reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho-GTPases under hypoxia-reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia-reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia-reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE-cadherin from cellular junctions. Inhibition of contractile activation with ML-7 partially protected against hypoxia-reoxygenation-induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia-reoxygenation-induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8-CPT-O-Me-cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE-cadherin at cellular junctions and abrogated reoxygenation-induced hyperpermeability. Similar results were reproduced in isolated saline-perfused rat hearts. These data show that activation of Rac1 but not the inhibition of RhoA preserves endothelial integrity against reoxygenation-induced loss of barrier function.

Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells.

Cytosolic free calcium concentration ([Ca(2+)]i) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca(2+)]i overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca(2+)]i overload can be prevented by lithium treatment. [Ca(2+)]i and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-d-glucose (5mM; 2-DG) plus sodium cyanide (5mM; NaCN) caused a significant decrease in cellular ATP content (14±1 nmol/mg protein vs. 18±1 nmol/mg protein in the control, n=6 culture dishes, P<0.05), an increase in [Ca(2+)]i (278±24 nM vs. 71±2 nM in the control, n=60 cells, P<0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10mM 2-DG led to a similar decrease in ATP content (14±2 nmol/mg vs. 18±1 nmol/mg in the control, P<0.05) with a delay of 5 min. The [Ca(2+)]i response of EC was biphasic with a peak after 1 min (183±6 nM vs. 71±1 nM, n=60 cells, P<0.05) followed by a sustained increase in [Ca(2+)]i. A 24-h pre-treatment with 10mM of lithium chloride before the inhibition of ATP synthesis abolished both phases of the 2-DG-induced [Ca(2+)]i increase. This effect was not observed when lithium chloride was added simultaneously with 2-DG. We conclude that lithium chloride abolishes the injurious [Ca(2+)]i overload in EC and that this most likely occurs by preventing inositol 3-phosphate-sensitive Ca(2+)-release from the endoplasmic reticulum. Though further research is needed, these findings provide a novel option for therapeutic strategies to protect the endothelium against imminent barrier failure.

Opposing effects of ATP and adenosine on barrier function of rat coronary microvasculature.

ATP can differentially affect the micro- and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular endothelial cells and microvessels, in vivo. We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular endothelial monolayers (RCEC) in vitro, as well as in rat mesentery vessels, and in rat hearts in vivo. ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracellular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5'-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A(1), and A(2) but not by A(3) receptor antagonists. Likewise, immunofluorescence microscopy of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo. In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.