Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.

Turning placenta into brain: placental mesenchymal stem cells differentiate into neurons and oligodendrocytes

We aimed to induce neural stem (NSC) and progenitor cells (NPC) from human placental tissues.

Hierarchical clustering of flow cytometry data for the study of conventional central chondrosarcoma

We have investigated the use of hierarchical clustering of flow cytometry data to classify samples of conventional central chondrosarcoma, a malignant cartilage forming tumor of uncertain cellular origin, according to similarities with surface marker profiles of several known cell types. Human primary chondrosarcoma cells, articular chondrocytes, mesenchymal stem cells, fibroblasts, and a panel of tumor cell lines from chondrocytic or epithelial origin were clustered based on the expression profile of eleven surface markers. For clustering, eight hierarchical clustering algorithms, three distance metrics, as well as several approaches for data preprocessing, including multivariate outlier detection, logarithmic transformation, and z-score normalization, were systematically evaluated. By selecting clustering approaches shown to give reproducible results for cluster recovery of known cell types, primary conventional central chondrosacoma cells could be grouped in two main clusters with distinctive marker expression signatures: one group clustering together with mesenchymal stem cells (CD49b-high/CD10-low/CD221-high) and a second group clustering close to fibroblasts (CD49b-low/CD10-high/CD221-low). Hierarchical clustering also revealed substantial differences between primary conventional central chondrosarcoma cells and established chondrosarcoma cell lines, with the latter not only segregating apart from primary tumor cells and normal tissue cells, but clustering together with cell lines from epithelial lineage. Our study provides a foundation for the use of hierarchical clustering applied to flow cytometry data as a powerful tool to classify samples according to marker expression patterns, which could lead to uncover new cancer subtypes.

VHL-gene deletion in single renal tubular epithelial cells and renal tubular cysts: further evidence for a cyst-dependent progression pathway of clear cell renal carcinoma in von Hippel-Lindau disease

Inheritance of a mutant allele of the von Hippel-Lindau tumor suppressor gene predisposes affected individuals to develop renal cysts and clear cell renal cell carcinoma. Von Hippel-Lindau gene inactivation in single renal tubular cells has indirectly been showed by immunohistochemical staining for the hypoxia-inducible factor alpha target gene product carbonic anhydrase IX. In this study we were able to show von Hippel-Lindau gene deletion in carbonic anhydrase IX positive nonneoplastic renal tubular cells, in epithelial cells lining renal cysts and in a clear cell renal cell carcinoma of a von Hippel-Lindau patient. This was carried out by means of laser confocal microscopy and immunohistochemistry in combination with fluorescence in situ hybridization. Carbonic anhydrase IX negative normal renal tubular cells carried no von Hippel-Lindau gene deletion. Furthermore, recent studies have indicated that the von Hippel-Lindau gene product is necessary for the maintenance of primary cilia stability in renal epithelial cells and that disruption of the cilia structure by von Hippel-Lindau gene inactivation induces renal cyst formation. In our study, we show a significant shortening of primary cilia in epithelial cells lining renal cysts, whereas, single tubular cells with a von Hippel-Lindau gene deletion display to a far lesser extent signs of cilia shortening. Our in vivo results support a model in which renal cysts represent precursor lesions for clear cell renal cell carcinoma and arise from single renal tubular epithelial cells owing to von Hippel-Lindau gene deletion.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Laser scanning microscopy combined with image restoration to analyse a 3D model of the human epithelial airway barrier

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.

An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

MET inhibition in tumor cells by PHA665752 impairs homologous recombination repair of DNA double strand breaks

Abnormal activation of cellular DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems has broad implications for both cancer biology and treatment. Recent studies suggest a potential link between DNA repair and aberrant activation of the hepatocyte growth factor receptor Mesenchymal-Epithelial Transition (MET), an oncogene that is overexpressed in numerous types of human tumors and considered a prime target in clinical oncology. Using the homologous recombination (HR) direct-repeat direct-repeat green fluorescent protein ((DR)-GFP) system, we show that MET inhibition in tumor cells with deregulated MET activity by the small molecule PHA665752 significantly impairs in a dose-dependent manner HR. Using cells that express MET-mutated variants that respond differentially to PHA665752, we confirm that the observed HR inhibition is indeed MET-dependent. Furthermore, our data also suggest that decline in HR-dependent DNA repair activity is not a secondary effect due to cell cycle alterations caused by PHA665752. Mechanistically, we show that MET inhibition affects the formation of the RAD51-BRCA2 complex, which is crucial for error-free HR repair of double strand DNA lesions, presumably via downregulation and impaired translocation of RAD51 into the nucleus. Taken together, these findings assist to further support the role of MET in the cellular DNA damage response and highlight the potential future benefit of MET inhibitors for the sensitization of tumor cells to DNA damaging agents.

Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.