An in vitro model of the tumor-lymphatic microenvironment with simultaneous transendothelial and luminal flows reveals mechanisms of flow enhanced invasion.

The most common cancers, including breast and skin, disseminate initially through the lymphatic system, yet the mechanisms by which tumor cells home towards, enter and interact with the lymphatic endothelium remain poorly understood. Transmural and luminal flows are important biophysical cues of the lymphatic microenvironment that can affect adhesion molecules, growth factors and chemokine expression as well as matrix remodeling, among others. Although microfluidic models are suitable for in vitro reconstruction of highly complex biological systems, the difficult assembly and operation of these systems often only allows a limited throughput. Here we present and characterize a novel flow chamber which recapitulates the lymphatic capillary microenvironment by coupling a standard Boyden chamber setup with a micro-channel and a controlled fluidic environment. The inclusion of luminal and transmural flow renders the model more biologically relevant, combining standard 3D culture techniques with advanced control of mechanical forces that are naturally present within the lymphatic microenvironment. The system can be monitored in real-time, allowing continuous quantification of different parameters of interest, such as cell intravasation and detachment from the endothelium, under varied biomechanical conditions. Moreover, the easy setup permits a medium-high throughput, thereby enabling downstream quantitative analyses. Using this model, we examined the kinetics of tumor cell (MDA-MB-231) invasion and transmigration dynamics across lymphatic endothelium under varying flow conditions. We found that luminal flow indirectly upregulates tumor cell transmigration rate via its effect on lymphatic endothelial cells. Moreover, we showed that the addition of transmural flow further increases intravasation, suggesting that distinct flow-mediated mechanisms regulate tumor cell invasion.

A computational model of nitric oxide production and transport in a parallel plate flow chamber.

We developed a mathematical model to investigate the production and transport of nitric oxide (NO) generated by a monolayer of cultured endothelial cells exposed to flow in a parallel plate flow chamber. The objectives were to provide a theoretical framework for interpreting experimental observations and to suggest a quantitative relationship between shear stress and NO production rate. NO production was described as a combination of a basal production rate term and a shear-dependent term. Our results show that the shear stress-dependence of the production of NO by the endothelium influences the nature of mass transport within the boundary layer. We found that the steady state NO concentration near the endothelial surface exhibits a biphasic dependence on shear stress, in which at low flow, NO concentration decreases owing to the enhanced removal by convective transport while only at higher shear stresses does the increased production cause an increase in NO concentration. The unsteady response to step changes in flow exhibits transient fluctuations in NO that can be explained by time-dependent changes in the diffusive and convective mass transport as the concentration profile evolves. Our results indicate that this model can be used to determine the relationship between shear stress and NO production rate from measurements of NO concentration.

Real time monitoring of the effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cell adhesion process using thickness shear mode (TSM) sensor.

The effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cellular interactions of the cell membrane with different substrates to determine the kinetics of cell adhesion was studied using thickness shear mode (TSM) sensor. The TSM sensor was operated at its first, third, fifth and seventh harmonics. Since the penetration depth of the shear wave decreases with increases in frequency, the multi-resonance operation of the TSM sensor was used to monitor the changes in the kinetics of the cell-substrate interaction at different distances from the sensor surface. During the sedimentation and the initial attachment of the cells on the sensor surface, the changes in the sensor resonant frequency and the magnitude response were monitored. First, HSPGs were partially digested with the enzyme Heparinase III to evaluate the effect of HSPG on the cell adhesion process. The results indicated that HSPG did not have any effect on the kinetics of the initial attachment, but it did reduce the strength of steady-state cell adhesion. Next, we investigated the effect of the electrostatic interactions of the cell membrane with the substrate on the cell adhesion. In this case, the sensor surface was coated with positively charged Poly-D-Lysine (PDL). It was observed that electrostatic interaction of the negatively charged cell membrane with the PDL surface promoted the initial cell adhesion but did not support long-term cell adhesion. The multi-resonant TSM technique was shown to be a very promising method for monitoring specific interfacial effects involving in cell adhesion process in real-time.

Heterogeneous response of microvascular endothelial cells to shear stress.

We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.

Heterogeneous cytoplasmic calcium response in microvascular endothelial cells.

We investigated changes in calcium concentration in response to the administration of ATP and the onset of shear stress with cultured rat adrenomedulary endothelial cells (RAMECs, microvascular). A substantial heterogeneity in time and space in the calcium response was observed. The onset of shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells The application of uniform exogenous ATP produced similar heterogeneous calcium transients. The size of the responding groups was dependent on ATP concentration. The propagation of calcium waves induced by either ATP or shear stress challenge was significantly suppressed by suramin, a non-specific purinergic receptor blocker. We investigated some of the mechanisms leading to the heterogeneity, and the results indicated that the main source of variation is the heterogeneous distribution of purinergic receptor. The application of ATP or shear stress stimulates cells to release ATP causing an increase of [Ca2+]ivia purinergic receptor in the cells that have high sensitivity. Subsequently, additional ATP is released and the elevation of ATP concentration in the vicinity of the initially responding cells mediates the calcium propagation. These data suggest a mechanism by which ATP acts as an autocrine and paracrine mediator to integrate individual cell responses that result in coordination of vascular functions in situ.

Selective modulation of endothelial cell [Ca2+]i response to flow by the onset rate of shear stress.

The response of endothelial cells (ECs) to their hemodynamic environment strongly influences normal vascular physiology and the pathogenesis of atherosclerosis. Unique responses to the complex flow patterns in lesion-prone regions imply that the temporal and spatial features of the mechanical stimuli modulate the cellular response to flow. We report the first systematic study of the effects of temporal gradients of shear stress on ECs. Flow was applied to cultured ECs using a novel cone-and-plate device allowing precise and independent control of the shear stress magnitude and the onset rate. Intracellular free calcium concentration ([Ca2+]i) increased rapidly following the onset of flow, and the characteristics of the transient were modulated by both the shear stress magnitude and onset rate. ECs were most sensitive to shear stress applied at physiological onset rates. Furthermore, the relative contribution of extracellular calcium and IP3-mediated release were dependent upon the specific flow regime.

In vitro cell shearing device to investigate the dynamic response of cells in a controlled hydrodynamic environment.

Mechanical stresses and strains play important roles in the normal growth and development of biological tissues, yet the cellular mechanisms of mechanotransduction have not been identified. A variety of in vitro systems for applying mechanical loads to cell populations have been developed to gain insight into these mechanisms. However, limitations in the ability to control precisely relevant aspects of the mechanical stimuli have obscured the physical relationships between mechanical loading and the biochemical signals that mediate the cellular response. We present a novel in vitro cell shearing device based on the principles of a cone and plate viscometer that utilizes microstepper motor technology to control independently the dynamic and steady components of a hydrodynamic shear-stress environment. Physical measurements of the cone velocity demonstrated faithful reproduction of user-defined input wave forms. Computational modeling of the fluid environment for the unsteady startup confirmed small inertial contributions and negligible secondary flows. Finally, we present experimental results demonstrating the onset rate dependence of functional and structural responses of endothelial cell cultures to dynamically applied shear stress. The controlled cell shearing device is a novel tool for elucidating mechanisms by which mechanical forces give rise to the biological signals that modulate cellular morphology and metabolism.

Spatial relationships in early signaling events of flow-mediated endothelial mechanotransduction.

Blood flow interactions with the vascular endothelium represent a specialized example of mechanical regulation of cell function that has important physiological and pathological cardiovascular consequences. The endothelial monolayer in vivo acts as a signal transduction interface for forces associated with flowing blood (hemodynamic forces) in the acute regulation of artery tone and chronic structural remodeling of arteries, including the pathology of atherosclerosis. Mechanisms related to spatial relationships at the cell surfaces and throughout the cell that influence flow-mediated endothelial mechanotransduction are discussed. In particular, flow-mediated ion channel activation and cytoskeletal dynamics are considered in relation to topographic analyses of the luminal and abluminal surfaces of living endothelial cells.

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro.

Exposure of endothelium to a nominally uniform flow field in vivo and in vitro frequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.

Deconvolution of gel filtration chromatographs of human plasma lipoproteins.

Gel filtration chromatographs of lipoproteins represent a superposition, or convolution, of the intrinsic polydispersity of the solute and the dispersion due to transport phenomena. We describe a deconvolution technique for improving the resolution of gel filtration chromatographs applicable to lipoproteins and other polydisperse solutes. A matrix of spreading functions, characterizing the dispersive properties of the column, was determined by fitting chromatographic data from a series of monodisperse standards with the solution to the transport equations and interpolating between the fit parameters. A successive approximation scheme was used in which a test distribution was incrementally corrected by an amount proportional to the error between the measured chromatograph and that derived from the test distribution. A nonlinear relaxing function was used to constrain the correction term such that the solution remained physically realizable (i.e., nonnegative absorbance) as it evolved. Deconvolved chromatographs of lipoproteins provided resolution of peaks that were obscured by spreading in the original data. The distribution of particle sizes within each fraction was calculated and verified experimentally by further separating the contents of fractions by gradient gel electrophoresis. Our technique, however, provided comparable resolution of the peaks without the additional experimental procedure.

Changes in surface topography in endothelial monolayers with time at confluence: influence on subcellular shear stress distribution due to flow.

It is well known that the morphology of endothelial cells in culture changes dramatically as they go from subconfluence to confluence. After reaching confluence, however, the morphology continues to change but much more subtly as cell density increases and they become more uniform in size and shape. Measurements of surface topography of confluent cells by atomic force microscopy (AFM) showed that cell heights became more uniform and that the root-mean-square amplitude of surface undulations decreased by 7% compared with monolayers that had just reached confluence. Computational fluid dynamics simulations of flow over the endothelial surface geometries measured by AFM showed that the change in topography with time after confluence altered the shear stress distribution, resulting in an increase in the stress concentrations experienced by the cells despite the reduced amplitude of the surface undulation. These data suggest that the starting point for in vitro experiments may influence the measured responses to shear stress, particularly transient responses that occur before structural adaptation takes place. In addition, changes in surface topography may reflect changes in cell tension, cytoskeletal structure, and adhesion to the substratum, all of which are associated with the regulation of growth in anchorage-dependent cells.

Subcellular distribution of shear stress at the surface of flow-aligned and nonaligned endothelial monolayers.

The stresses acting on the luminal surface of endothelial cells due to shear flow were determined on a subcellular scale. Atomic force microscopy was used to measure the surface topography of confluent endothelial monolayers cultured under no-flow conditions or exposed to steady shear stress (12 dyn/cm2 for 24 h). Flow over these surface geometries was simulated by computational fluid dynamics, and the distribution of shear stress on the cell surface was calculated. Flow perturbations due to the undulating surface produced cell-scale variations of shear stress magnitude and hence large shear stress gradients. Reorganization of the endothelial surface in response to prolonged exposure to steady flow resulted in significant reductions in the peak shear stresses and shear stress gradients. From the relationship between surface geometry and the resulting shear stress distribution, we have defined a hydrodynamic shape factor that characterizes the three-dimensional morphological response of endothelial cells to flow. The analysis provides a complete description of the spatial distribution of stresses on individual endothelial cells within a confluent monolayer on a scale relevant to the study of physical mechanisms of mechanotransduction.

Strain measurements in cultured vascular smooth muscle cells subjected to mechanical deformation.

Early work in the field of biomechanics employed rigorous application of the principles of mechanics to the study of the macroscopic structural response of tissues to applied loads. Interest in the functional response of tissues to mechanical stimulation has lead researchers to study the biochemical responses of cells to mechanical loading. Characterization of the experimental system (i.e., specimen geometry and boundary conditions) is no less important on the microscopic scale of the cell than it is for macroscopic tissue testing. We outline a method for appropriate characterization of cell deformation in a cell culture model; describe a system for applying a uniform, isotropic strain field to cells in culture; and demonstrate a dependence of cell deformation on morphology and distribution of adhesion sites. Cultured vascular smooth-muscle cells were mechanically deformed by applying an isotropic strain to the compliant substrate to which they were adhered. The state of strain in the cells was determined by measurement of the displacements of fluorescent microspheres attached to the cell surface. The magnitude and orientation of principal strains were found to vary spatially and temporally and to depend on cell morphology. These results show that cell strain can be highly variable and emphasize the need to characterize both the loading conditions and the actual cellular deformation in this type of experimental model.

Shear stress-induced reorganization of the surface topography of living endothelial cells imaged by atomic force microscopy.

We report the first topographical data of the surface of living endothelial cells at sub-light-microscopic resolution, measurements essential for a detailed understanding of force distribution in the endothelium subjected to flow. Atomic force microscopy was used to observe the surface topography of living endothelial cells in confluent monolayers maintained in static culture or subjected to unidirectional shear stress in laminar flow (12 dyne/cm2 for 24 hours). The surface of polygonal unsheared cells was smooth, with mean excursion of surface undulation between peak height (over the nucleus) and minima (at intercellular junctions) of 3.4 +/- 0.7 microns (mean +/- SD); the mean height to length ratio was 0.11 +/- 0.02. In cells that were aligned in the direction of flow after a 24-hour exposure to laminar shear stress, height differentials were significantly reduced (mean, 1.8 +/- 0.5 micron), and the mean height to length ratio was 0.045 +/- 0.009. Calculation of maximum shear stress and maximum gradient of shear stress (delta tau/delta x, where tau is shear stress at the cell surface) at constant flow velocity revealed substantial streamling of aligned cells that reduced delta tau/delta x by more than 50% at a nominal shear stress of 10 dyne/cm2. Aligned cells exhibited ridges extending in the direction of flow that represented imprints of submembranous F-actin stress-fiber bundles mechanically coupled to the cell membrane. The surface ridges, approximately 50 nm in height and 200 to 1000 nm in width, were particularly prominent in the periphery of the aligned cells.(ABSTRACT TRUNCATED AT 250 WORDS)