Spin-Peierls Instability of Three-Dimensional Spin Liquids with Majorana Fermi Surfaces.

Three-dimensional (3D) variants of the Kitaev model can harbor gapless spin liquids with a Majorana Fermi surface on certain tricoordinated lattice structures such as the recently introduced hyperoctagon lattice. Here, we investigate Fermi surface instabilities arising from additional spin exchange terms (such as a Heisenberg coupling) which introduce interactions between the emergent Majorana fermion degrees of freedom. We show that independent of the sign and structure of the interactions, the Majorana surface is always unstable. Generically, the system spontaneously doubles its unit cell at exponentially small temperatures and forms a spin liquid with line nodes. Depending on the microscopics, further symmetries of the system can be broken at this transition. These spin-Peierls instabilities of a 3D spin liquid are closely related to BCS instabilities of fermions.

Interactions of silica nanoparticles with lung epithelial cells and the association to flotillins.

Amorphous silica nanoparticles (aSNPs) gain increasing popularity for industrial and therapeutic claims. The lung with its surface area of 100-140 m(2) displays an ideal target for therapeutic approaches, but it represents also a serious area of attack for harmful nanomaterials. The exact nature of the cytotoxic effects of NPs is still unknown. Furthermore, cellular pathways and the destiny of internalized NPs are still poorly understood. Therefore, we examined the cytotoxicity (MTS, LDH) and inflammatory responses (IL-8) for different-sized aSNPs (30, 70, 300 nm) on our lung epithelial cells line NCI H441 and endothelial cell line ISO-HAS-1. Additionally, colocalization studies have been conducted via immunofluorescence staining for flotillin-1- and flotillin-2-bearing endocytic vesicles. Subsequently, the relevance of flotillins concerning the viability of aSNP-exposed epithelial cells has been evaluated using flotillin-1/2 depleted cells (siRNA). This study reveals the relevance of the nanoparticle size regarding cytotoxicity (MTS, LDH) and inflammatory responses (IL-8), whereat the smaller the size of the nanoparticle is, the more harmful are the effects. All different aSNP sizes have been incorporated in flotillin-1- and flotillin-2-labelled vesicles in lung epithelial and endothelial cells, which display a marker for late endosomal or lysosomal structures and appear to exhibit a clathrin- or caveolae-independent mode of endocytosis. Flotillin-depleted H441 showed a clearly decreased uptake of aSNPs. Additionally, the viability of aSNP-exposed cells was reduced in these cells. These findings indicate a contribution of flotillins in as yet unknown (clathrin or caveolae-independent) endocytosis mechanisms and (or) endosomal storage.

Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures.

BACKGROUND: To date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures. METHODS: Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated. RESULTS: Regarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress. CONCLUSION: We present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

Self-Report Stress Measures to Assess Stress in Adults With Mild Intellectual Disabilities-A Scoping Review.

Stress has a major negative impact on the development of psychopathology and contributes to the onset of adverse physical conditions. Timely recognition and monitoring of stress-related problems are therefore important, especially in client populations that are more vulnerable to stress, such as people with mild intellectual disabilities (MID). Recent research on the use of physiological measures to assess stress levels emphasize that, in addition to these measures, self-report instruments are necessary to gain insight into the individual perception and impact of stress on daily life. However, there is no current overview of self-report stress measures that focus on the experience of stress in the present moment or in daily life. To provide an overview of the existing self-report stress measures for clinicians and researchers, a scoping review was conducted. In addition, to advise clinical professionals on the use of self-report measures of stress for people with MID, the results of an expert consultation were used to refine the preliminary findings. A systematic scoping literature search resulted in a total of 13 self-reported stress measures that met the final inclusion criteria, of which three were developed specifically for assessing stress in adults with MID (GAS-ID, LI, and SAS-ID). For each included self-report stress measure, the psychometric quality, assessment procedure, and suitability for adults with MID were reported. These were supplemented by the findings from the expert consultation. Implications for clinical practice on the use of self-report stress measures, particularly for people with MID, are discussed. Recommendations for future research and development are given.

Side-specific effects by cadmium exposure: apical and basolateral treatment in a coculture model of the blood-air barrier.

Cadmium (Cd(2+)) is a widespread environmental pollutant, which is associated with a wide variety of cytotoxic and metabolic effects. Recent studies showed that intoxication with the heavy metal most importantly targets the integrity of the epithelial barrier. In our study, the lung epithelial cell line, NCI H441, was cultured with the endothelial cell line, ISO-HAS-1, as a bilayer on a 24-well HTS-Transwell filter plate. This coculture model was exposed to various concentrations of CdCl(2). The transepithelial electrical resistance decreased on the apical side only after treatment with high Cd(2+) concentrations after 48 h. By contrast, a breakdown of TER to less than 5% of baseline could be observed much earlier (after 24 h) when Cd(2+) was administered from the basal side. Observations of cell layer fragmentation and widening of intercellular spaces confirmed the barrier breakdown only for the basolaterally treated samples. Furthermore, the cytotoxicity and release of proinflammatory markers was enhanced if samples were exposed to Cd(2+) from the basal side compared to treatment from the apical side. Moreover, we could demonstrate that a high concentration of Ca(2+) could prevent the barrier-disrupting effect of Cd(2+). In conclusion, the exposure of Cd(2+) to cocultures of lung cells caused a decrease in TER, major morphological changes, a reduction of cell viability and an increase of cytokine release, but the effects markedly differed between the two modes of exposure. Therefore, our results suggest that intact epithelial TJs may play a major role in protecting the air-blood barrier from inhaled Cd(2+).

Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung.

In order to delineate individual pathomechanisms in acute lung injury and pulmonary toxicology, we developed a primary coculture system to simulate the human alveolo-capillary barrier. Human pulmonary microvascular endothelial cells (HPMEC) were cocultivated with primary isolated human type II alveolar epithelial cells (HATII) on opposite sides of a permeable filter support, thereby constituting a bilayer. Within 7-11 days of coculture, the HATII cells partly transdifferentiated to type-I-like (HATI-like) cells, as demonstrated by morphological changes from a cuboidal to a flattened morphology, the loss of HATII-cell-specific organelles and the increase of HATI-cell-related markers (caveolin-1, aquaporin-5, receptor for advanced glycation end-products). Immunofluorescent analysis detected type-II-like and type-I-like alveolar epithelial cells mimicking the heterocellular composition of alveolar epithelium in vivo. The heterocellular epithelial monolayer showed a circumferential staining of tight-junctional (ZO-1, occludin) and adherens-junctional (E-cadherin, beta-catenin) proteins. HPMEC on the opposite side also developed tight and adherens junctions (VE-cadherin, beta-catenin). Under integral barrier properties, exposure to the proinflammatory cytokine tumour necrosis factor-alpha from either the endothelial (basolateral) or the epithelial (apical) side caused a largely compartmentalized release of the chemokines interleukin-8 and monocyte chemoattractant protein-1. Thus, the established coculture provides a suitable in vitro model to examine barrier function at the distal lung, including the interaction of microvascular endothelial cells with ATII-like and ATI-like epithelial cells. The compartmentalization of the barrier-forming bilayer also allows mechanisms of lung injury to be studied in both the epithelial (intra-alveolar) and the endothelial (intravascular) compartments.

Hypoxia-induced epithelial VEGF-C/VEGFR-3 upregulation in carcinoma cell lines.

Adaptation to hypoxia, a universal hallmark of carcinomas, is a critical step for tumor cell survival and growth. One of the principal regulators of hypoxia-responsive pathways is the transcription factor hypoxia-inducible factor-1 alpha (HIF-1 alpha). Currently, it is known that tumoral production of members of the vascular endothelial growth factor (VEGF)-family (VEGFs) may promote tumor growth and progression by acting on carcinoma cells that express the cognate receptors (VEGFRs). However, the influence of hypoxia in the formation of such a tumoral VEGF/VEGFR loop is not completely understood. In the present study we examined the potential existence of a HIF-1 alpha/VEGF/VEGFR autocrine loop on commonly occurring carcinomas. The experiments were performed on five colorectal carcinoma cell lines, one breast (MCF7) and one lung (A549) adenocarcinoma cell line under normoxic and oxygen stress conditions using HIF-1 alpha-EIA, VEGFs-ELISA as well as RT-PCR and immunofluorescence for VEGFRs. HIF-1 alpha overexpression was found already after 2 h of exposure to hypoxia in all above mentioned cell lines, thus documenting that activation of the transcription factor HIF-1 alpha is an early cellular event. Under hypoxic conditions a significant upregulation and activation of HIF-1 alpha accompanied by an increased production of VEGF in MCF7 and A549 was observed. The well-differentiated colorectal carcinoma cell lines were 'hypoxia-resistant' showing unchanged levels of HIF-1 alpha and VEGF under hypoxia. None of the cell lines used in this study expressed the VEGF receptors VEGFR-1 and VEGFR-2 under normoxia and hypoxia. Additionally, all colorectal carcinoma cell lines were negative for VEGFR-3 transcripts in both conditions. However, VEGFR-3 mRNA and protein were expressed and under hypoxia overexpressed in MCF7 and A549. Hypoxic cultures of both cell lines secreted in elevated levels the VEGFR-3 ligand VEGF-C but not VEGF-D. Our findings suggest that under hypoxic conditions an autocrine loop between VEGF-C/VEGFR-3 and HIF-1 alpha is possible in breast carcinoma and lung carcinoma but not in colorectal carcinoma cell lines.

Cell culture models of higher complexity in tissue engineering and regenerative medicine.

Cell culture techniques have tended to be used in biomaterial research as a screening method prior to embarking on specific in vivo experimentation. This presentation aims at showing that it is possible to develop more sophisticated in vitro systems using primary human cells in co-culture with other cell types and biomaterials in a three-dimensional setting. While the predictive value of such systems is still not proven these models can be employed to unravel the complexity of biological systems in order to understand molecular mechanisms of cell-cell and cell-material interactions. The brief overview is under the headings of basic principles of relevant culture systems, the study of inflammation and the healing response, scenarios for specific biomaterial applications and future directions. How human endothelial cells can be usefully incorporated into more complex cell culture models is presented as an example of how relevant questions in tissue engineering and regenerative medicine can be addressed. The central tenet of this paper is that it is possible to refine in vitro methodology using cells of human origin to establish relevant assay systems that more closely simulate the cellular and molecular microenvironment encountered in a specific situation of regeneration using biomaterials.

Assessment of alterations in barrier functionality and induction of proinflammatory and cytotoxic effects after sulfur mustard exposure of an in vitro coculture model of the human alveolo-capillary barrier.

Acute lung injury after sulfur mustard (SM) inhalation is characterized by massive, localized hemorrhage and alveolar edema, which implies severe disruption of the vascular and distal airway barrier. In this study, we tested a recently established in vitro coculture model of the alveolo-capillary barrier for its applicability to investigate acute toxic effects of SM at the human respiratory unit. The epithelial compartment of cocultures was exposed to varying concentrations of SM (0-1000 microM; t = 30 min). Following exposure, functional and structural barrier integrity of cocultures was monitored over a period of 24 h. A 50% reduction of transbilayer electrical resistance (TER) within 12-24 h after exposure to 300 microM SM and within 8 h after 1000 microM SM revealed a time- and concentration-dependent impairment of barrier functionality, which was associated with structural loss of both cell layers. Subsequent quantification of interleukin (IL)-6 and IL-8 in cell culture supernatants of exposed cocultures showed enhanced liberation of proinflammatory markers. Highest mediator levels were detected after 300 microM SM, with pronounced stimulation in the endothelial compartment. SM-related cytotoxicity was determined by assessing adenylate kinase (AK) release and by quantifying the fraction of DNA-fragmented nuclei using terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) and nuclear Hoechst staining. Both methods exposed a concentration-dependent increase of SM-mediated cytotoxic effects with high effects on endothelial cells. We conclude that the described in vitro model reflects important characteristics of SM-mediated acute lung injury in vivo and thus can be used to explore involved pathophysiological pathways.

A responsive human triple-culture model of the air-blood barrier: incorporation of different macrophage phenotypes.

Current pulmonary research underlines the relevance of the alveolar macrophage (AM) integrated in multicellular co-culture-systems of the respiratory tract to unravel, for example, the mechanisms of tissue regeneration. AMs demonstrate a specific functionality, as they inhabit a unique microenvironment with high oxygen levels and exposure to external hazards. Healthy AMs display an anti-inflammatory phenotype, prevent hypersensitivity to normally innocuous contaminants and maintain tissue homeostasis in the alveolus. To mirror the actual physiological function of the AM, we developed three different polarized [classically activated (M1) and alternatively activated (M2wh , wound-healing; M2reg , regulatory)] macrophage models using a mixture of differentiation mediators, as described in the current literature. To test their immunological impact, these distinct macrophage phenotypes were seeded on to the epithelial layer of an established in vitro air-blood barrier co-culture, consisting of alveolar epithelial cells A549 or H441 and microvascular endothelial cells ISO-HAS-1 on the opposite side of a Transwell filter-membrane. IL-8 and sICAM release were measured as functionality parameters after LPS challenge. The M1 model itself already provoked a severe inflammatory-like response of the air-blood barrier co-culture, thus demonstrating its potential as a useful in vitro model for inflammatory lung diseases. The two M2 models represent a 'non-inflammatory' phenotype but still showed the ability to trigger inflammation following LPS challenge. Hence, the latter could be used to establish a quiescent, physiological in vitro air-blood model. Thus, the more complex differentiation protocol developed in the present study provides a responsive in vitro triple-culture model of the air-blood-barrier that mimics AM features as they occur in vivo. © 2015 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

The role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro.

The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-ß) and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH)3), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-ß, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the Papp, the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells. In summary, this coculture model exhibits in vivo-like features, which cannot be observed in conventional monocultures, making the former more suitable to study interactions with external stimuli.

Pulmonary surfactant augments cytotoxicity of silica nanoparticles: Studies on an in vitro air-blood barrier model.

The air-blood barrier is a very thin membrane of about 2.2 µm thickness and therefore represents an ideal portal of entry for nanoparticles to be used therapeutically in a regenerative medicine strategy. Until now, numerous studies using cellular airway models have been conducted in vitro in order to investigate the potential hazard of NPs. However, in most in vitro studies a crucial alveolar component has been neglected. Before aspirated NPs encounter the cellular air-blood barrier, they impinge on the alveolar surfactant layer (10-20 nm in thickness) that lines the entire alveolar surface. Thus, a prior interaction of NPs with pulmonary surfactant components will occur. In the present study we explored the impact of pulmonary surfactant on the cytotoxic potential of amorphous silica nanoparticles (aSNPs) using in vitro mono- and complex coculture models of the air-blood barrier. Furthermore, different surface functionalisations (plain-unmodified, amino, carboxylate) of the aSNPs were compared in order to study the impact of chemical surface properties on aSNP cytotoxicity in combination with lung surfactant. The alveolar epithelial cell line A549 was used in mono- and in coculture with the microvascular cell line ISO-HAS-1 in the form of different cytotoxicity assays (viability, membrane integrity, inflammatory responses such as IL-8 release). At a distinct concentration (100 µg/mL) aSNP-plain displayed the highest cytotoxicity and IL-8 release in monocultures of A549. aSNP-NH2 caused a slight toxic effect, whereas aSNP-COOH did not exhibit any cytotoxicity. In combination with lung surfactant, aSNP-plain revealed an increased cytotoxicity in monocultures of A549, aSNP-NH2 caused a slightly augmented toxic effect, whereas aSNP-COOH did not show any toxic alterations. A549 in coculture did not show any decreased toxicity (membrane integrity) for aSNP-plain in combination with lung surfactant. However, a significant augmented IL-8 release was observed, but no alterations in combination with lung surfactant. The augmented aSNP toxicity with surfactant in monocultures appears to depend on the chemical surface properties of the aSNPs. Reactive silanol groups seem to play a crucial role for an augmented toxicity of aSNPs. The A549 cells in the coculture seem to be more robust towards aSNPs, which might be a result of a higher differentiation and polarization state due the longer culture period.

Flotillin-involved uptake of silica nanoparticles and responses of an alveolar-capillary barrier in vitro.

Drug and gene delivery via nanoparticles across biological barriers such as the alveolar-capillary barrier of the lung constitutes an interesting and increasingly relevant field in nanomedicine. Nevertheless, potential hazardous effects of nanoparticles (NPs) as well as their cellular and systemic fate should be thoroughly examined. Hence, this study was designed to evaluate the effects of amorphous silica NPs (Sicastar) and (poly)organosiloxane NPs (AmOrSil) on the viability and the inflammatory response as well as on the cellular uptake mechanisms and fate in cells of the alveolar barrier. For this purpose, the alveolar epithelial cell line (NCI H441) and microvascular endothelial cell line (ISO-HAS-1) were used in an experimental set up resembling the alveolar-capillary barrier of the lung. In terms of IL-8 and sICAM Sicastar resulted in harmful effects at higher concentrations (60 µg/ml) in conventional monocultures but not in the coculture, whereas AmOrSil showed no significant effects. Immunofluorescence counterstaining of endosomal structures in NP-incubated cells showed no evidence for a clathrin- or caveolae-mediated uptake mechanism. However, NPs were enclosed in flotillin-1 and -2 marked vesicles in both cell types. Flotillins appear to play a role in cellular uptake or trafficking mechanisms of NPs and are discussed as indicators for clathrin- or caveolae-independent uptake mechanisms. In addition, we examined the transport of NPs across this in vitro model of the alveolar-capillary barrier forming a tight barrier with a transepithelial electrical resistance of 560±8 Ω cm(2). H441 in coculture with endothelial cells took up much less NPs compared to monocultures. Moreover, coculturing prevented the transport of NP from the epithelial compartment to the endothelial layer on the bottom of the filter insert. This supports the relevance of coculture models, which favour a differentiated and polarised epithelial layer as in vitro test systems for nanoparticle uptake.

An impaired alveolar-capillary barrier in vitro: effect of proinflammatory cytokines and consequences on nanocarrier interaction.

The alveolar region of the lung is an important target for drug and gene delivery approaches. Treatment with drugs is often necessary under pathophysiological conditions, in which there is acute inflammation of the target organ. Therefore, in vitro models of the alveolar-capillary barrier, which mimic inflammatory conditions in the alveolar region, would be useful to analyse and predict effects of novel drugs on healthy or inflamed tissues. The epithelial cell line H441 was cultivated with primary isolated human pulmonary microvascular endothelial cells (HPMECs) or the endothelial cell line ISO-HAS-1 on opposite sides of a permeable filter support under physiological and inflammatory conditions. Both epithelial and endothelial cell types grew as polarized monolayers in bilayer coculture and were analysed in the presence and absence of the proinflammatory stimuli tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In addition, the nanocarrier polyethyleneimine (PEI) was chosen as a model compound to study cell uptake (Oregon Green (OG)-labelled PEI) and gene transfer (PEI-pDNA complex). Upon treatment with TNF-alpha and IFN-gamma, both cocultures exhibited comparable effects on the trans-bilayer electrical resistance, the transport of sodium fluorescein and the increase in secondary cytokine release. Basolateral (endothelial side) exposure to TNF-alpha or simultaneous exposure to TNF-alpha and IFN-gamma generated an alveolar-capillary barrier with inflammation-like characteristics, impaired barrier function and a local disruption of the continuous apical labelling of the tight junction plaque protein zonula occludens-1 (ZO-1). Although transfection rates of 8 per cent were obtained for H441 cells in non-polarized monocultures, apical-basolateral-differentiated (polarized) H441 in coculture could not be transfected. After basolateral cytokine exposure, uptake of fluorescently labelled PEI in polarized H441 was predominantly detected in those areas with a local disruption of ZO-1 expression. Accordingly, transfected cells were only sparsely found in coculture after basolateral costimulation with TNF-alpha and IFN-gamma. We designed a coculture model that mimics both the structural architecture of the alveolar-capillary barrier and inflammatory mechanisms with consequences on barrier characteristics, cytokine production and nanoparticle interaction. Our model will be suitable to systematically study adsorption, uptake and trafficking of newly synthesized nanosized carriers under different physiological conditions.

Retention of a differentiated endothelial phenotype by outgrowth endothelial cells isolated from human peripheral blood and expanded in long-term cultures.

Rapid adequate vascularization by autologous human endothelial cells remains a limiting step in the treatment of ischemic tissues and the generation of new tissues. We have expanded outgrowth endothelial cells (OEC) from human peripheral blood and investigated their phenotypic stability in long-term cultures. Our goal has been to obtain suitable numbers of autologous endothelial cells for pro-angiogenic cell therapies. Mononuclear cells were isolated from human peripheral blood. During culture, cells were characterized for several endothelial and stem cell markers in mono- or in co-culture with mature endothelial cells. In cultures from peripheral blood, we observed cells with a variable ability to assume a differentiated endothelial phenotype. Most of the cells showed markers reported for endothelial progenitor cells or hemangioblasts (CD31, KDR, VE-cadherin, CD34, CD117, CD45) but failed to develop a differentiated phenotype. Caveolin-1 was not detectable in these cells by reverse transcription/polymerase chain reaction (RT-PCR) or immunofluorescence. Another cell type arising from the same cultures expressed a differentiated phenotype and was designated as an OEC. This subset as an OEC was expanded in long-term cultures and analyzed by immunofluorescence, flow-cytometry, and RT-PCR for a stable endothelial phenotype. OEC showed several markers of a differentiated endothelium, such as high levels of caveolin-1 throughout all tested passages, and the ability to form angiogenic sprouts in vitro. Thus, OEC in long-term expansion cultures from blood mononuclear cells are phenotypically highly stable, a feature that is an important prerequisite for using OEC from peripheral blood for autologous endothelial cell therapies.

FVIII production by human lung microvascular endothelial cells.

While extrahepatic factor VIII (FVIII) synthesis suffices for hemostasis, the extrahepatic production sites are not well defined. We therefore investigated the ability of the human lungs to produce FVIII. Lungs from heart-beating donors who were declined for transplantation were perfused and ventilated in an isolated reperfusion model for 2 hours. A progressive accumulation of FVIII and von Willebrand factor (VWF) was recorded in the perfusion medium in 3 of 4 experiments. By contrast, factor V, fibrinogen, and immunoglobulin G (IgG) levels remained constant during the perfusion period, indicating that the accumulation of FVIII and VWF was not due to diffusion from the intercellular medium into the vascular system. Purified human lung microvascular endothelial cells produced FVIII during at least 2 passages in vitro. Altogether, these data identify the lung endothelial cells as a FVIII production site in humans.

Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro.

We have established a coculture system of human distal lung epithelial cells and human microvascular endothelial cells in order to study the cellular interactions of epithelium and endothelium at the alveolocapillary barrier in both pathogenesis and recovery from acute lung injury. The aim was to determine conditions for the development of functional cellular junctions and the formation of a tight epithelial barrier similar to that observed in vivo. The in vitro coculture system consisted of monolayers of human lung epithelial cell lines (A549 or NCI H441) and primary human pulmonary microvascular endothelial cells (HPMEC) on opposite sides of a permeable filter membrane. A549 failed to show sufficient differentiation with respect to formation of a tight epithelial barrier with intact cell-cell junctions. Stimulated with dexamethasone, the cocultures of NCI H441 and HPMEC established contact-inhibited differentiated monolayers, with NCI H441 showing a continuous, circumferential immunostaining of the tight junctional protein, ZO-1 and the adherens junction protein, E-cadherin. The generation of a polarized epithelial cell monolayer with typical junctional structures was confirmed by transmission electron microscopy. Dexamethasone treatment resulted in average transbilayer electrical resistance (TER) values of 500 Omega cm(2) after 10-12 days of cocultivation and correlated with a reduced flux of the hydrophilic permeability marker, sodium-fluorescein. In addition, basolateral distribution of the proinflammatory cytokine tumour necrosis factor-alpha caused a significant reduction of TER-values after 24 h exposure. This decrease in TER could be re-established to control level by removal of the cytokine within 24 h. Thus, the coculture system of the NCI H441 with HPMEC should be a suitable in vitro model system to examine epithelial and endothelial interactions in the pathogenesis of acute lung injury, infectious lung diseases and toxic lung injury. In addition, it could be used to improve techniques of lung drug delivery that also requires a functional barrier.