A flow system for the study of shear forces upon cultured endothelial cells

A parallel plate chamber in a flow system has been designed to study the effects of fluid shear stresses on cells. The system was applied to the study of cultured endothelial cells grown on cover slips which were accommodated in recessed wells in the base plate. Dye injection studies in the chamber indicated laminar flow over the cells. Shear rates measured over the cover slips by an electrochemical technique were found to be linear with flow rate. Laser doppler anemometry showed parabolic profiles between the plates. Endothelial cells subjected to flow showed a correlation between the time required for orientation and the magnitude of the shear stress.     

Some observations on the use of cultured corneal endothelial cells as a model for intact corneal endothelium

Major differences have been identified between corneal endothelial cells in situ and those grown in culture. Cells in intact porcine corneal endothelium were studied and compared with primary cultures of the same cells either in suspension or in monolayers which had been grown on plastic (Nunc, Permonax). Differences were identified in the organization of the cytoskeleton (filamentous actin) between the cells in situ and in monolayer culture. The ability to withstand exposure to cryoprotective concentrations of Me(2)SO also varied substantially depending on whether the cells were in situ or in culture. These results underline the need for caution in the use of cells in culture as a model for studying the nature of injury to cells during the freezing of whole tissues.     

Laser Doppler velocimetry measurement in the model flow of a fish olfactory organ

The velocity field of the flow in the olfactory organ of a fish,exemplified by the swordtail Xiphophorus helleri, was investigatedby a three-component laser Doppler velocimetry (LDV) systemin a simulated stationary model flow at an enlarged scale, butconserving hydrodynamic similarity. Both the crossed beam andthe reference beam methods were applied to study the inflowphase. The complexity of the vector field is depicted in sectionalviews by isolines and arrow patterns. The power of the LDV techniqueand model simulation for such investigations is demonstrated.     

A model for shear stress-induced deformation of a flow sensor on the surface of vascular endothelial cells

Fluid mechanical shear stress elicits humoral, metabolic, and structural responses in vascular endothelial cells (ECs); however, the mechanisms involved in shear stress sensing and transduction remain incompletely understood. Beyond being responsive to shear stress, ECs distinguish among and respond differently to different types of shear stress. Recent observations suggest that endothelial shear stress sensing may occur through direct interaction of the flow with cell-surface structures that act as primary flow sensors. This paper presents a mathematical model for the shear stress-induced deformation of a flow sensor on the EC surface. The sensor is modeled as a cytoskeleton-coupled viscoelastic structure exhibiting standard linear solid behavior. Since ECs respond differently to different types of flow, the deformation and resulting velocity of the sensor in response to steady, non-reversing pulsatile, and oscillatory flow have been studied. Furthermore, the sensitivity of the results to changes in various model parameters including the magnitude of applied shear stress, the constants that characterize the viscoelastic behavior, and the pulsatile flow frequency (f) has been investigated. The results have demonstrated that in response to a suddenly applied shear stress, the sensor exhibits a level of instantaneous deformation followed by gradual creeping to the long-term response. The peak deformation increases linearly with the magnitude of the applied shear stress and decreases for viscoelastic constants that correspond to stiffer sensors. While the sensor deformation depends on f for low f values, the deformation becomes f -independent above a critical threshold frequency. Finally, the peak sensor deformation is considerably larger for steady and non-reversing pulsatile flow than for oscillatory flow. If the extent of sensor deformation correlates with the intensity of flow-mediated endothelial signaling, then our results suggest possible mechanisms by which ECs distinguish among steady, non-reversing pulsatile, and oscillatory shear stress.     

Computational mechanical model studies on the spontaneous emergent morphogenesis of the cultured endothelial cells

To address questions concerning why and how the morphology of endothelial cells (ECs) forms under shear stress loading, a computational fluid dynamics (CFD) three-dimensional (3D) model of ECs simulating cell shape was designed. A full 3D non-linear CFD simulation was conducted to estimate the wall shear stress (WSS) distribution. The model cell was capable of random rotation, deformation, migration, and proliferation. Flow was computed after each update of the cell shape with infinitesimal configuration changes. After a finite interval of the flow computation, only the infinitesimal configuration changes that reduced the WSS were allowed to accumulate. As a result of the very long free-run computation experiment, starting with a sub-confluent pattern of cells, the model cells became confluent and were elongated and aligned, with a shape index (SI) very close to that reported for cells in vivo. The average WSS converged to the lowest value at the same time.     

Modeling of shear stress experienced by endothelial cells cultured on microstructured polymer substrates in a parallel plate flow chamber

The application of physical stimuli to cell populations in tissue engineering and regenerative medicine may facilitate significant scientific and clinical advances. However, for the most part, these stimuli are evaluated in isolation, rather than in combination. This study was designed to combine two physical stimuli. The first being a microstructured tissue culture polystyrene substrate, known to produce changes in cell shape and orientation, and the second being laminar shear stress in a parallel plate flow chamber. The combined effects of these stimuli on endothelial cell monolayers cells were evaluated in a parallel plate flow chamber and using a computational fluid dynamics (CFD) model. The topography of the cell monolayers cultured on different microstructured surfaces was determined using confocal laser scanning microscopy (CLSM), and this topographic information was used to construct the CFD model. This research found that while the specific underlying structures were effectively planarized by the cell monolayer, significant differences in cell shape and orientation were observed on the different microstructured surfaces. Cells cultured on grooved substrates aligned in the direction of the grooves and showed higher retention after 1-h LSS conditioning than those cultured on pillars. The modeled shear stress distributions also showed differences. While minor differences in the magnitude of shear stress were noted, aligned cell monolayers experienced significantly lower spatial gradients of shear stress when compared with cells that were not pre-aligned by surface features. The results presented here provide an analysis of how one form of physical stimulus can be moderated by another and also provide a methodology by which the understanding of cell responses to topographic and mechanical stimuli can be further advanced.     

Heparinlike molecules with anticoagulant activity are synthesized by cultured endothelial cells

Cultured microvascular endothelial cells isolated from rat epididymal fat pads produce glycosaminoglycans that accelerate thrombin-antithrombin complex formation. The heparinlike nature of these macromolecules was established by complete destruction of their anticoagulant activity employing purified Flavobacterium heparinase. Only 15% of the biologic activity of these complex carbohydrates was expressed when the heparin binding domain on the protease inhibitor was chemically modified at the Trp 49 residue. The anticoagulantly active species contains disaccharides which constitute the unique antithrombin binding region of the mucopolysaccharide. Removal of the biologically active heparinlike components from endothelial cells with 0.05% trypsin suggests that these molecular species are present on the cell surface.     

Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.     

Flow field analysis in a compliant acinus replica model using particle image velocimetry (PIV)

Inhaled particles reaching the alveolar walls have the potential to cross the blood–gas barrier and enter the blood stream. Experimental evidence of pulmonary dosimetry, however, cannot be explained by current whole lung dosimetry models. Numerical and experimental studies shed some light on the mechanisms of particle transport, but realistic geometries have not been investigated. In this study, a three dimensional expanding model including two generations of respiratory bronchioles and five terminal alveolar sacs was created from a replica human lung cast. Flow visualization techniques were employed to quantify the fluid flow while utilizing streamlines to evaluate recirculation. Pathlines were plotted to track the fluid motion and estimate penetration depth of inhaled air. This study provides evidence that the two generations immediately proximal to the terminal alveolar sacs do not have recirculating eddies, even for intense breathing. Results of Peclet number calculations indicate that substantial convective motion is present in vivo for the case of deep breathing, which significantly increases particle penetration into the alveoli. However, particle diffusion remains the dominant mechanism of particle transport over convection, even for intense breathing because inhaled particles do not reach the alveolar wall in a single breath by convection alone. Examination of the velocity fields revealed significant uneven ventilation of the alveoli during a single breath, likely due to variations in size and location. This flow field data, obtained from replica model geometry with realistic breathing conditions, provides information to better understand fluid and particle behavior in the acinus region of the lung.     

Validity of a model of cultured myocardial cells for assessment of cardioplegia

Myocardial protection is usually studied in vitro on perfused heart preparations, but never directly on cultured cardiomyocytes. We evaluated a model of cultured newborn rat cardiomyocytes to study both the cytotoxicity and the protective effect against chemical hypoxia of three cardioplegic solutions (St Thomas' I, Bretschneider, St Thomas' II) under normothermic (37°C) and hypothermic (4°C) conditions. Cytotoxicity was evaluated in 50% and 100% concentrations of the cardioplegic solutions with incubation times from 90 to 360 min. Myocardial protection was studied in 50% cardioplegic solution with metabolic inhibitors. Immediate and late viabilities, after 24 h of recovery in the medium, were evaluated by simultaneous staining with fluorescein diacetate and propidium iodide.At 37°C, the 50% concentration of the three cardioplegic solutions did not modify cell viability. At 37°C, with 360 min of incubation, the 100% concentration of the St Thomas' I and Bretschneider solutions diminished immediate viability (mean ± SD: medium 87% ± 2%; St Thomas' I 58% ± 5%; Bretschneider 37% ± 8%; St Thomas' II 89% ± 3%) as well as late viability (medium 69% ± 2%; St Thomas' I 32% ± 3%; Bretschneider 24% ± 7%; St Thomas' II 65% ± 4%). At 4°C, immediate and late viabilities were unaffected by cardioplegic solutions.At 37°C, after 360 min incubation time, metabolic inhibitors diminished immediate viability to 29% ± 1% and late viability to zero. None of the three cardioplegic solutions used at 50% concentration prevented this effect.At 4°C, immediate viability was not significantly affected by metabolic inhibitors (73% ± 10%), but the use of Bretschneider cardioplegic solution seemed to be detrimental (53% ± 9%). On the other hand, recovery phase after pretreatment with metabolic inhibitors with or without cardioplegic solutions for 360 min significantly diminished late viability (medium 63% ± 7%; metabolic inhibitors 17% ± 8%; St Thomas' I 17% ± 6%; Bretschneider 8% ± 6%; St Thomas' II 15% ± 3%) and again cardioplegia was inefficient. In conclusion, in this in vitro model for the study of cardioplegic solutions, only pure concentrations of the St Thomas' I and Bretschneider solutions under normothermic conditions were cytotoxic. The well-known protective effects of hypothermia against ischemia and reperfusion injury were both reproduced. Therefore, and even though cardioplegia failed to have any protective effect, probably owing to a severe metabolic inhibition, this model may be useful for studying myocardial protection.     

A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

Glycosaminoglycans (GAGs) presented on the surface of endothelial cells (ECs) are believed to influence leukocyte recruitment during inflammation, but their roles remain uncertain. Here we report an in vitro model of prolonged culture of human EC in which the contributions of heparan sulphate (HS) and hyaluronan (HA) to the process of neutrophil recruitment could be studied. Previously, we reported that increasing EC culture duration (up to 20 days) enhanced neutrophil recruitment in response to low dose (1 U/ml) but not high dose (100 U/ml) of tumour necrosis factor-α (TNF). Here we found that HS and HA were present at much higher levels on the surface of day 20 cultures than day 3 cultures. Neutrophil recruitment on both day 3 and day 20 ECs was mediated through CXCR chemokine receptors and interleukin-8 (IL-8). In addition, mRNA levels for TNF receptors, signalling pathway constituents, adhesion receptors, and chemokines involved in neutrophil recruitment were similar for day 3 and day 20 ECs. To test whether the enhanced neutrophil recruitment on day 20 EC was mediated by GAGs, they were removed enzymatically. Removal of HA (but not HS) inhibited neutrophil recruitment, as did antibody blockade of CD44, a counter-receptor for HA on neutrophils. Supernatants from hyaluronidase-treated day 20 ECs were more potent in activating neutrophils than supernatants from untreated EC. Thus, HA has a role in neutrophil recruitment that is revealed in long-term cultures where it increases potency of response to sub-optimal levels of TNF. This effect appears to occur through a dual mechanism involving chemokine presentation and interaction with CD44.     

A novel in vitro flow system for changing flow direction on endothelial cells

Atherosclerotic plaques localize to regions of flow disturbance, i.e. bifurcations, branch points and regions of high curvature. Shear stress in these regions can be multi-directional due to complex flow patterns such as time-varying vortices. However, commonly used in vitro flow models are incapable of changing flow orientation to any direction other than the reverse. We have developed a novel in vitro flow system to enable changes in flow direction to any angle. When cells were pre-aligned in laminar shear, and then rotated 90°, cells re-aligned over 24 h. Re-alignment involved actin remodeling by gradual rotation of actin stress fibers. This device will enable that analysis of how endothelial cells sense changes in flow direction as occur in vivo.     

A model of localised Rac1 activation in endothelial cells due to fluid flow

Endothelial cells respond to fluid flow by elongating in the direction of flow. Cytoskeletal changes and activation of signalling molecules have been extensively studied in this response, including: activation of receptors by mechano-transduction, actin filament alignment in the direction of flow, changes to cell-substratum adhesions, actin-driven lamellipodium extension, and localised activation of Rho GTPases. To study this process we model the force over a single cell and couple this to a model of the Rho GTPases, Rac and Rho, via a Kelvin-body model of mechano-transduction. It is demonstrated that a mechano-transducer can respond to the normal component of the force is likely to be a necessary component of the signalling network in order to establish polarity. Furthermore, the rate-limiting step of Rac1 activation is predicted to be conversion of Rac-GDP to Rac-GTP, rather than activation of upstream components. Modelling illustrates that the aligned endothelial cell morphology could attenuate the signalling network.     

Design and characterization of a system for exposure of cultured cells to extremely low frequency electric and magnetic fields over a wide range of field strengths

A system is described that is capable of producing extremely low frequency (ELF) magnetic fields for relatively short-term exposure of cultured mammalian cells. The system utilizes a ferromagnetic core to contain and direct the magnetic field of a 1,000 turn solenoidal coil and can produce a range of flux densities and induced electric fields much higher than those produced by Helmholtz coils. The system can generate magnetic fields from the microtesla (μT) range up to 0.14 T with induced electric field strengths on the order of 1.0 V/m. The induced electric field can be accurately varied by changing the sample chamber configuration without changing the exposure magnetic field. This gives the system the ability to separate the bioeffects of magnetic and induced electric fields. In the frequency range of 4–100 Hz and magnetic flux density range of 0.005–0.14 T, the maximum total harmonic distortion of the induced electric field is typically less than 1.0%. The temperature of the samples is held constant to within 0.4°C by constant perfusion of warmed culture medium through the sample chamber. © 1993 Wiley-Liss, Inc.     

Design of a side-view particle imaging velocimetry flow system for cell-substrate adhesion studies

Experimental models that mimic the flow conditions in microcapillaries have suggested that the local shear stresses and shear rates can mediate tumor cell and leukocyte arrest on the endothelium and subsequent sustained adhesion. However, further investigation has been limited by the lack of experimental models that allow quantitative measurement of the hydrodynamic environment over adherent cells. The purpose of this study was to develop a system capable of acquiring quantitative flow profiles over adherent cells. By combining the techniques of side-view imaging and particle image velocimetry (PIV), an in vitro model was constructed that is capable of obtaining quantitative flow data over cells adhering to the endothelium. The velocity over an adherent leukocyte was measured and the shear rate was calculated under low and high upstream wall shear. The microcapillary channel was modeled using computational fluid dynamics (CFD) and the calculated velocity profiles over cells under the low and high shear rates were compared to experimental results. The drag force applied to each cell by the fluid was then computed. This system provides a means for future study of the forces underlying adhesion by permitting characterization of the local hydrodynamic conditions over adherent cells.     

Interactions of cultured endothelial cells with TGF-beta, bFGF, PDGF and IGF-I

Endothelial cells in culture synthesize the growth factors transforming growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) and, perhaps, insulin like growth factor I (IGF-I). We have previously demonstrated that IGF-I and PDGF have both high affinity receptors and stimulate glucose and AIB uptake in the microvessel cells under study and that IGF-I, but not PDGF, has similar high affinity receptors in cultured large vessel endothelial cells. In the present study, cultured bovine endothelial cells were exposed to these four growth factors to determine a) their effects on the acute metabolic processes of neutral amino acid (AIB) and glucose uptake and b) their interactions at the endothelial cell surface. In microvessel endothelial cells, each growth factor stimulated AIB and glucose uptake 2-4 fold whereas in large vessel endothelial cells only bFGF stimulated glucose uptake. Each growth factor had specific high affinity binding to the microvessel cells that was not influenced by the presence of the other growth factors. In large vessel endothelial cells, similar high affinity binding was present only for IGF-I and to a lesser degree TGF-beta. When cells were exposed to a given growth factor for 18 hours, homologous receptor downregulation was observed, with a maximal 60-95% decrease in surface binding. These findings suggest several potential levels of interaction of the growth factors TGF-beta, bFGF, PDGF and IGF-I in cultured vascular endothelial cells.     

Modulation of Ca2+ transients in cultured endothelial cells in response to fluid flow through alphav integrin

In order to determine whether integrin dynamics is associated with intracellular Ca(2+) concentration ([Ca(2+)](i)) mobilization in ECs in response to hemodynamic forces, changes in [Ca(2+)](i) in fluo-4-loaded cultured bovine aortic endothelial cells (BAECs) under fluid flow conditions were visualized employing laser scanning confocal microscopy. Following the onset of flow stimulus, transient increases in [Ca(2+)](i) occurred several times in individual BAECs during the 30-min observation period. The frequency of these [Ca(2+)](i) transients was clearly reduced by the application of an integrin antagonist (GRGDSP peptide). Furthermore, treatment of cells with an integrin activator (Mn(2+)) resulted in reduction of peak [Ca(2+)](i) levels and elevated frequency, which was markedly rescued upon GRGDSP administration. In contrast, an actin de-polymerizing agent (cytochalasin D) exerted no inhibitory effects; rather, cytochalasin D more likely facilitated [Ca(2+)](i) transients. Moreover, [Ca(2+)](i) transients, which were suppressed by short interference RNA-induced silencing of alphav integrin, exhibited greater frequently in cells cultured on vitronectin substratum in comparison with those cultured on fibronectin or collagen substratum. Either removal of extracellular Ca(2+), application of an inhibitor of endoplasmic reticulum Ca(2+)-ATPase (thapsigargin) or non-selective cation channel blocker (La(3+)) inhibited the [Ca(2+)](i) transients. Additionally, [Ca(2+)](i) transients were attenuated by extracellular signal-regulated kinase (ERK) kinase inhibitor (U0126); in contrast, [Ca(2+)](i) transients were unaffected by tyrosine kinase inhibitor (genistein) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002). Therefore, our findings revealed that alphav integrin dynamics modulates the frequency of flow-induced [Ca(2+)](i) transients in BAECs in an ERK-dependent fashion.