Angiotensinergic innervation of the kidney: Localization and relationship with catecholaminergic postganglionic and sensory nerve fibers

We describe an angiotensin (Ang) II-containing innervation of the kidney. Cryosections of rat, pig and human kidneys were investigated for the presence of Ang II-containing nerve fibers using a mouse monoclonal antibody against Ang II (4B3). Co-staining was performed with antibodies against synaptophysin, tyrosine 3-hydroxylase, and dopamine beta-hydroxylase to detect catecholaminergic efferent fibers and against calcitonin gene-related peptide to detect sensory fibers. Tagged secondary antibodies and confocal light or laser scanning microscopy were used for immunofluorescence detection. Ang II-containing nerve fibers were densely present in the renal pelvis, the subepithelial layer of the urothelium, the arterial nervous plexus, and the peritubular interstitium of the cortex and outer medulla. They were infrequent in central veins and the renal capsule and absent within glomeruli and the renal papilla. Ang II-positive fibers represented phenotypic subgroups of catecholaminergic postganglionic or sensory fibers with different morphology and intrarenal distribution compared to their Ang II-negative counterparts. The Ang II-positive postganglionic fibers were thicker, produced typically fusiform varicosities and preferentially innervated the outer medulla and periglomerular arterioles. Ang II-negative sensory fibers were highly varicose, prevailing in the pelvis and scarce in the renal periphery compared to the rarely varicose Ang II-positive fibers. Neurons within renal microganglia displayed angiotensinergic, catecholaminergic, or combined phenotypes. Our results suggest that autonomic fibers may be an independent source of intrarenal Ang II acting as a neuropeptide co-transmitter or neuromodulator. The angiotensinergic renal innervation may play a distinct role in the neuronal control of renal sodium reabsorption, vasomotion and renin secretion.

An optimized in vitro model of the respiratory tract wall to study particle cell interactions

As a part of the respiratory tissue barrier, lung epithelial cells play an important role against the penetration of the body by inhaled particulate foreign materials. In most cell culture models, which are designed to study particle-cell interactions, the cells are immersed in medium. This does not reflect the physiological condition of lung epithelial cells which are exposed to air, separated from it only by a very thin liquid lining layer with a surfactant film at the air-liquid interface. In this study, A549 epithelial cells were grown on microporous membranes in a two chamber system. After the formation of a confluent monolayer the cells were exposed to air. The morphology of the cells and the expression of tight junction proteins were studied with confocal laser scanning and transmission electron microscopy. Air-exposed cells maintained monolayer structure for 2 days, expressed tight junctions and developed transepithelial electrical resistance. Surfactant was produced and released at the apical side of the air-exposed epithelial cells. In order to study particle-cell interactions fluorescent 1 microm polystyrene particles were sprayed over the epithelial surface. After 4 h, 8.8% of particles were found inside the epithelium. This fraction increased to 38% after 24 h. During all observations, particles were always found in the cells but never between them. In this study, we present an in vitro model of the respiratory tract wall consisting of air-exposed lung epithelial cells covered by a liquid lining layer with a surfactant film to study particle-cell interactions.

Pyroelectric and piezoelectric scanning microscopy applied to reveal the bipolar state of 4-iodo-4′-nitrobiphenyl (INBP)

Two recent scanning probe techniques were applied to investigate the bipolar twin state of 4-iodo-4′-nitrobiphenyl (INBP) crystals. Solution grown crystals of INBP show typically a morphology which does not express that of a mono-domain polar structure (Fdd2, mm2). From previous X-ray diffraction a twinning volume ratio of [similar]70 : 30 is now explained by two unipolar domains (Flack parameter: 0.075(29)) of opposite orientation of the molecular dipoles, joined by a transition zone showing a width of [similar]140 μm. Scanning pyroelectric microscopy (SPEM) demonstrates a continuous transition of the polarization P from +P into −P across the zone. Application of piezoelectric force microscopy (PFM) confirms unipolar alignment of INBP molecules down to a resolution of [similar]20 nm. A previously proposed real structure for INBP crystals built from lamellae with antiparallel alignment is thus rejected. Anomalous X-ray scattering was used to determine the absolute molecular orientation in the two domains. End faces of the polar axis 2 are thus made up by NO2 groups. Using a previously determined negative pyroelectric coefficient pc leads to a confirmation also by a SPEM analysis. Calculated values for functional group interactions (DA), (AA), (DD) and the stochastic theory of polarity formation allow us to predict that NO2 groups should terminate corresponding faces. Following the present analysis, INBP may represent a first example undergoing dipole reversal upon growth to end up in a bipolar state.

Suitability of Common Collagen Scaffolds for Anterior Cruciate Ligament Repair

Introduction: Anterior cruciate ligament (ACL) injuries are very common; in Germany incidence of ACL ruptures is estimated at 32 per 100 000 in the general population and in the sports community this rate more than doubles. Current gold standard for anterior cruciate lig- ament repair is reconstruction using an autograft [1]. However, this approach has shown some limitations. A new method has been her- alded by the Knee Team at the Bern University Hospital (Inselspital) and the Sonnenhof clinic called Dynamic Intraligamentary Stabilization (DIS), which keeps ACL remnants in place in order to promote biologi- cal healing and makes use of a dynamic screw system [2]. The aim of this study was to investigate the cytocompatibility of collagen patches in combination with DIS to support regeneration of the ACL. The spe- cific hypothesis we tested was whether MSCs would differentiate towards TCs in co-culture. Materials and methods: Primary Tenocytes (TCs) and human bone marrow derived mesenchymal stem cells (MSCs) were harvested from ACL removed during knee prothesis or from bone marrow aspirations (Ethical Permit 187/10). Cells were seeded on two types of three dimensional carriers currently approved for cartilage repair, Novocart (NC, B. Brown) and Chondro-Gide (CG, Geistlich). These scaffolds comprise collagen structures with interconnecting pores originally developed for seeding of chondrocytes in the case of CG. ~40k cells were seeded on punched zylindrical cores of 8 mm in Ø and cultured on CG or NC patches for up to 7 days. The cells were either cultured as TC only, MSC only or co-cultured in a 1:1 mix on the scaffolds and on both sides of culture inserts (PET, high density pore Ø 0.4 mm, BD, Fal- con) with cell-cell contact. We monitored DNA content, GAG and HOP-content, tracked the cells using DIL and DIO fluorescent dyes (Molecular Probes, Life technologies) and confocal laser scanning and SEM microscopy as well as RT-PCR of tenocyte specific markers (i.e. col 1 and 3, TNC, TNMD, SCXA&B, and markers of dedifferentiation ACAN, col2, MMP3, MMP13). Finally, H&E stain was interpreted on cryosections and SEM images of cells on the scaffold were taken. Results: ThecLSMimagesshowedcellproliferationoverthe7dayson both matrices, however, on CG there were much fewer MSCs attached than on NC. SEM images showed a roundish chondrocyte-like pheno- type of cells on CG whereas on NC the phenotype was more teno- cyte-like (Fig. 1). Gene expression of both, MSC and TC seem to confirm a more favorable environment in 3D for both patches rather than monolayer control.

Autonomic angiotensinergic fibres in the human heart with an efferent sympathetic cophenotype

AIM The autonomic innervation of the heart consists of sympathetic and parasympathetic nerve fibres, and fibres of the intrinsic ganglionated plexus with noradrenaline and acytylcholine as principal neurotransmitters. The fibres co-release neuropeptides to modulate intracardiac neurotransmission by specific presynaptic and postsynaptic receptors. The coexpression of angiotensin II in sympathetic fibres of the human heart and its role are not known so far. METHODS Autopsy specimens of human hearts were studied (n=3; ventricles). Using immunocytological methods, cryostat sections were stained by a murine monoclonal antibody (4B3) directed against angiotensin II and co-stained by polyclonal antibodies against tyrosine hydroxylase, a catecholaminergic marker. Visualisation of the antibodies was by confocal light microscopy or laser scanning microscopy. RESULTS Angiotensin II-positive autonomic fibres with and without a catecholaminergic cophenotype (hydroxylase-positive) were found in all parts of the human ventricles. In the epicardium, the fibres were grouped in larger bundles of up to 100 and more fibres. They followed the preformed anatomic septa and epicardial vessels towards the myocardium and endocardium where the bundles dissolved and the individual fibres spread between myocytes and within the endocardium. Generally, angiotensinergic fibres showed no synaptic enlargements or only a few if they were also catecholaminergic. The exclusively catechalominergic fibres were characterised by multiple beaded synapses. CONCLUSION The autonomic innervation of the human heart contains angiotensinergic fibres with a sympathetic efferent phenotype and exclusively angiotensinergic fibers representing probably afferents. Angiotensinergic neurotransmission may modulate intracardiac sympathetic and parasympathetic activity and thereby influence cardiac and circulatory function.

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.

Macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall

The human airway epithelium serves as structural and functional barrier against inhaled particulate antigen. Previously, we demonstrated in an in vitro epithelial barrier model that monocyte derived dendritic cells (MDDC) and monocyte derived macrophages (MDM) take up particulate antigen by building a trans-epithelial interacting network. Although the epithelial tight junction (TJ) belt was penetrated by processes of MDDC and MDM, the integrity of the epithelium was not affected. These results brought up two main questions: (1) Do MDM and MDDC exchange particles? (2) Are those cells expressing TJ proteins, which are believed to interact with the TJ belt of the epithelium to preserve the epithelial integrity? The expression of TJ and adherens junction (AJ) mRNA and proteins in MDM and MDDC monocultures was determined by RT-PCR, and immunofluorescence, respectively. Particle uptake and exchange was quantified by flow cytometry and laser scanning microscopy in co-cultures of MDM and MDDC exposed to polystyrene particles (1 μm in diameter). MDM and MDDC constantly expressed TJ and AJ mRNA and proteins. Flow cytometry analysis of MDM and MDDC co-cultures showed increased particle uptake in MDDC while MDM lost particles over time. Quantitative analysis revealed significantly higher particle uptake by MDDC in co-cultures of epithelial cells with MDM and MDDC present, compared to co-cultures containing only epithelial cells and MDDC. We conclude from these findings that MDM and MDDC express TJ and AJ proteins which could help to preserve the epithelial integrity during particle uptake and exchange across the lung epithelium.

Fate of inhaled particles after interaction with the lung surface

Inhaled particles may cause increased pulmonary and cardiovascular morbidity and mortality. The wall structures of airways and alveoli act as a series of structural and functional barriers against inhaled particles. Deposited particles are displaced and come into close association with epithelial cells, macrophages and dendritic cells. The cellular interplay after particle deposition in a triple cell co-culture model of the human airway wall was investigated by laser scanning microscopy. Furthermore, the cellular response was determined by measurement of TNF-alpha. Dendritic cells gained access to the apical side of the epithelium where they sampled particles and interacted with macrophages.

Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

Interaction of fine particles and nanoparticles with red blood cells visualized with advanced microscopic techniques

So far, little is known about the interaction of nanoparticles with lung cells, the entering of nanoparticles, and their transport through the blood stream to other organs. The entering and localization of different nanoparticles consisting of differing materials and of different charges were studied in human red blood cells. As these cells do not have any phagocytic receptors on their surface, and no actinmyosin system, we chose them as a model for nonphagocytic cells to study how nanoparticles penetrate cell membranes. We combined different microscopic techniques to visualize fine and nanoparticles in red blood cells: (I) fluorescent particles were analyzed by laser scanning microscopy combined with digital image restoration, (II) gold particles were analyzed by conventional transmission electron microscopy and energy filtering transmission electron microscopy, and (III) titanium dioxide particles were analyzed by energy filtering transmission electron microscopy. By using these differing microscopic techniques we were able to visualize and detect particles < or = 0.2 microm and nanoparticles in red blood cells. We found that the surface charge and the material of the particles did not influence their entering. These results suggest that particles may penetrate the red blood cell membrane by a still unknown mechanism different from phagocytosis and endocytosis.

Effects on hepatocellular carcinoma of doxorubicin-loaded immunoliposomes designed to target the VEGFR-2

To maintain a tumour vasculature in proportion of the tumour growth, the endothelial cells proliferate and up-regulate the expression of the VEGF receptor 2 (VEGFR-2), whose expression is restricted to this cell type. This specificity implies that one therapeutically target the tumour endothelium. We investigated the use of immunoliposomes (IL), containing conjugated Fab' fragments of the monoclonal rat anti-VEGFR-2 antibody DC101 (DC101-IL) to cargo doxorubicin to the tumour endothelium. In vitro, fluorescein-labelled IL displayed a 7 fold better binding to VEGFR-2-positive 293T cells in comparison to unspecific liposomes. Balb/C mice were injected subcutaneously with syngeneic hepatocellular carcinoma cells. One set of animals was treated with DC101-IL filled with doxorubicin when the tumours were bigger than 400 mm3. A specific delivery of doxorubicin to endothelial cells of the tumour vessels could be demonstrated by the red fluorescence of doxorubicin with laser scanning microscopy, but neither a delay of tumour growth nor a shrinking of the tumour mass was observed. Yet necrosis in the tumours treated with doxorubicin containing vehicles was larger than in the tumours of the control groups. A second set of animals was treated with DC101-IL filled with doxorubicin when the tumours were smaller than 1 mm3. DC101-IL filled with doxorubicin led to a significant delay in tumour growth up to 7 weeks compared to empty DC101-IL, free doxorubicin, and HEPES/Glucose (HEPES/Glucose vs. DOX-DC101-IL, p = 0.001; unpaired, two-tailed Student's t-test) and to a higher amount of necrotic areas in the tumours (p = 0.053; 1 way ANOVA with 4 groups). These findings suggest that IL designed to bind specifically to VEGFR-2 can be used to deliver doxorubicin to the tumour endothelium and may impair the "angiogenic switch" of the tumours.

Translocation of particles and inflammatory responses after exposure to fine particles and nanoparticles in an epithelial airway model

ABSTRACT: BACKGROUND: Experimental studies provide evidence that inhaled nanoparticles may translocate over the airspace epithelium and cause increased cellular inflammation. Little is known, however, about the dependence of particle size or material on translocation characteristics, inflammatory response and intracellular localization. RESULTS: Using a triple cell co-culture model of the human airway wall composed of epithelial cells, macrophages and dendritic cells we quantified the entering of fine (1 mum) and nano-sized (0.078 mum) polystyrene particles by laser scanning microscopy. The number distribution of particles within the cell types was significantly different between fine and nano-sized particles suggesting different translocation characteristics. Analysis of the intracellular localization of gold (0.025 mum) and titanium dioxide (0.02-0.03 mum) nanoparticles by energy filtering transmission electron microscopy showed differences in intracellular localization depending on particle composition. Titanium dioxide nanoparticles were detected as single particles without membranes as well as in membrane-bound agglomerations. Gold nanoparticles were found inside the cells as free particles only. The potential of the different particle types (different sizes and different materials) to induce a cellular response was determined by measurements of the tumour necrosis factor-alpha in the supernatants. We measured a 2-3 fold increase of tumour necrosis factor-alpha in the supernatants after applying 1 mum polystyrene particles, gold nanoparticles, but not with polystyrene and titanium dioxide nanoparticles. CONCLUSION: Quantitative laser scanning microscopy provided evidence that the translocation and entering characteristics of particles are size-dependent. Energy filtering transmission electron microscopy showed that the intracellular localization of nanoparticles depends on the particle material. Both particle size and material affect the cellular responses to particle exposure as measured by the generation of tumour necrosis factor-alpha.

Diesel exhaust particles modulate the tight junction protein occludin in lung cells in vitro

ABSTRACT: BACKGROUND: Using an in vitro triple cell co-culture model consisting of human epithelial cells (16HBE14o-), monocyte-derived macrophages and dendritic cells, it was recently demonstrated that macrophages and dendritic cells create a transepithelial network between the epithelial cells to capture antigens without disrupting the epithelial tightness. The expression of the different tight junction proteins in macrophages and dendritic cells, and the formation of tight junction-like structures with epithelial cells has been demonstrated. Immunofluorescent methods combined with laser scanning microscopy and quantitative real-time polymerase chain reaction were used to investigate if exposure to diesel exhaust particles (DEP) (0.5, 5, 50, 125 mug/ml), for 24 h, can modulate the expression of the tight junction mRNA/protein of occludin, in all three cell types. RESULTS: Only the highest dose of DEP (125 mug/ml) seemed to reduce the occludin mRNA in the cells of the defence system however not in epithelial cells, although the occludin arrangement in the latter cell type was disrupted. The transepithelial electrical resistance was reduced in epithelial cell mono-cultures but not in the triple cell co-cultures, following exposure to high DEP concentration. Cytotoxicity was not found, in either epithelial mono-cultures nor in triple cell co-cultures, after exposure to the different DEP concentrations. CONCLUSION: We concluded that high concentrations of DEP (125 mug/ml) can modulate the tight junction occludin mRNA in the cells of the defence system and that those cells play an important role maintaining the epithelial integrity following exposure to particulate antigens in lung cells.

Toxic effects of brake wear particles on epithelial lung cells in vitro

ABSTRACT: BACKGROUND: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. RESULTS: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. CONCLUSION: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress.