Causes of shear sensitivity of the toxic dinoflagellate Protoceratium reticulatum

Dinoflagellates have proven extremely difficult to culture because they are inhibited by low‐level shear forces. Specific growth rate of the toxic dinoflagellate Protoceratium reticulatum was greatly decreased compared with static control culture by intermittent exposure to a turbulent hydrodynamic environment with a bulk average shear rate that was as low as 0.3 s^?1. Hydrodynamic forces appeared to induce the production of reactive oxygen species (ROS) within the cells and this caused peroxidation of cellular lipids and ultimately cell damage. Exposure to damaging levels of shear rate correlated with the elevated level of lipoperoxides in the cells, but ROS levels measured directly by flow cytometry did not correlate with shear induced cell damage. This was apparently because the measured level of ROS could not distinguish between the ROS that are normally generated by photosynthesis and the additional ROS produced as a consequence of hydrodynamic shear forces. Continuously subjecting the cells to a bulk average shear rate value of about 0.3 s^?1 for 24‐h caused an elevation in the levels of chlorophyll a, peridinin and dinoxanthin, as the cells apparently attempted to counter the damaging effects of shear fields by producing pigments that are potential antioxidants. In static culture, limitation of carbon dioxide produced a small but measureable increase in ROS. The addition of ascorbic acid (0.1 mM) to the culture medium resulted in a significant protective effect on lipid peroxidation, allowing cells to grow under damaging levels of shear rates. This confirmed the use of antioxidant additives as an efficient strategy to counter the damaging effects of turbulence in photobioreactors where shear sensitive dinoflagellates are cultivated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cells in 3D matrices under interstitial flow: Effects of extracellular matrix alignment on cell shear stress and drag forces

Interstitial flow is an important regulator of various cell behaviors both in vitro and in vivo, yet the forces that fluid flow imposes on cells embedded in a 3D extracellular matrix (ECM), and the effects of matrix architecture on those forces, are not well understood. Here, we demonstrate how fiber alignment can affect the shear and pressure forces on the cell and ECM. Using computational fluid dynamics simulations, we show that while the solutions of the Brinkman equation accurately estimate the average fluid shear stress and the drag forces on a cell within a 3D fibrous medium, the distribution of shear stress on the cellular surface as well as the peak shear stresses remain intimately related to the pericellular fiber architecture and cannot be estimated using bulk-averaged properties. We demonstrate that perpendicular fiber alignment of the ECM yields lower shear stress and pressure forces on the cells and higher stresses on the ECM, leading to decreased permeability, while parallel fiber alignment leads to higher stresses on cells and increased permeability, as compared to a cubic lattice arrangement. The Spielman–Goren permeability relationships for fibrous media agreed well with CFD simulations of flow with explicitly considered fibers. These results suggest that the experimentally observed active remodeling of ECM fibers by fibroblasts under interstitial flow to a perpendicular alignment could serve to decrease the shear and drag forces on the cell.     

DIFFERENTIATION OF THE GREEN ALGA CODIUM FRAGILE

Clonal cultures of Codium fragile were established from both swimming cells and vegetative filaments. In the laboratory axis primordia differentiate from heterotrichous juveniles only when cultures are agitated on a reciprocating shaker. The shear forces created by mechanical agitation are essential both for initiation and maintenance of primordia. Contact guidance of growing coenocytic filaments indicates mutual adhesion of filaments as the basis for the differentiation process.     

Shear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T-cell migration

The leukocyte-specific β₂-integrin LFA-1 and its ligand ICAM-1, expressed on endothelial cells (ECs), are involved in the arrest, adhesion, and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well established, the impact of forces on its major ligand ICAM-1 has received less attention. Using a parallel-plate flow chamber combined with confocal and super-resolution microscopy, we show that prolonged shear flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering before LFA-1 engagement. T cells adhered to mechanically prestimulated ECs or nanoclustered ICAM-1 substrates developed a promigratory phenotype, migrated faster, and exhibited shorter-lived interactions with ECs than when adhered to non mechanically stimulated ECs or to monomeric ICAM-1 substrates. Together, our results indicate that shear forces increase ICAM-1/LFA-1 bonds because of ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. Our data also underscore the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation.     

Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells

Summary Vascular endothelial cells modulate their structure and functions in response to changes in hemodynamic forces such as fluid shear stress. We have studied how endothelial cells perceive the shearing force generated by blood flow and the substance(s) that may mediate such a response. We identify cytoplasmic-free calcium ion (Ca⁺⁺), a major component of an internal signaling system, as a mediator of the cellular response to fluid shear stress. Cultured monolayers of bovine aortic endothelial cells loaded with the highly fluorescent Ca⁺⁺-sensitive dye Fura 2 were exposed to different levels of fluid shear stress in a specially designed flow chamber, and simultaneous changes in fluorescence intensity, reflecting the intracellular-free calcium concentration ([Ca⁺⁺] _i ), were monitored by photometric fluorescence microscopy. Application of shear stress to cells by fluid perfusion led to an immediate severalfold increase in fluorescence within 1 min, followed by a rapid decline for about 5 min, and finally a plateau somewhat higher than control levels during the entire period of the stress application. Repeated application of the stress induced similar peak and plateau levels of [Ca⁺⁺] _i but at reduced magnitudes of response. These responses were observed even in Ca⁺⁺-free medium. Thus, a shear stress transducer might exist in endothelial cells, which perceives the shearing force on the membrane as a stimulus and mediates the signal to increase cytosolic free Ca⁺⁺. This work was partly supported by a grant-in-aid, for Special Project Research no. 61132008, from the Japanese Ministry of Education, Science and Culture and a research fund from the Atherosclerosis Study Association.     

A Model for Sheath Formation Coupled to Motility in Leptothrix ochracea

Optical and atomic force microscopy (AFM) of naturally occurring Leptothrix ochracea was used to study the fine structure of sheaths and cells. Morphology of young sheaths suggests the scaffold chains have strong self-adhesion. Evidence from unencapsulated cells indicates fresh scaffold production through cell walls. Simple diffusion arguments are used to explain the morphology of the sheath structure. We propose a novel cell motility model based on previously published video data, our AFM images of naked cells, and simple flow calculations. The model indicates that motility results from differential shear forces resulting from extrusion of sheath material that passively pushes a filament of connected cells forward as the surrounding sheath material hardens behind the cell train.     

Indicating shear stress with FST-hemispheres - effects of stream-bottom topography and water depth

1. In order to quantify the effects of substratum roughness on shear stress and Fliesswasserstammtisch-(FST)-hemisphere movement, hemispheres were calibrated against shear stress in a laboratory flume with fully developed turbulent flow. In five different runs, substratum roughness, water depth and location of hemispheres in relation to the surrounding particles, were varied. 2. FST-hemisphere results were strongly influenced by bottom topography. In the case of hydraulically rough flow a linear relationship exists between shear stress (χ_c) and hemisphere density (ρ_h), whereas in the case of quasi-smooth flow a power law was obtained for the χ_c/ρ_h, relationship. Shear stress for a given hemisphere and relative roughness h/k > 4 (h= water depth; k= height of the roughness elements) deviated up to one order of magnitude between roughnesses. In water depths, where h/k 4, the χ_c/ρ_h relationships are dependent on the ratio h/k, due to water surface effects on hemisphere movement. In the case of k = d₉₀=r_h (d₉₀= characteristic diameter of the largest particles of the bottom substratum; r_h= radius of the hemispheres), the location of the hemispheres in respect to the roughness elements is of secondary importance. 3. In the case of hydraulically rough flow and wake interference between the roughness elements, the turbulent flow field close to the substratum is three-layered, each layer being characterized by its own velocity distribution laws (Dittrich & Hammann de Salazar, 1993). Depending on the height of the roughness elements, FST-hemispheres will be subjected mainly to flow forces of the near-bed layer (in rough substrata) or to flow forces distant from the near-bed zone (in fine substrata). The dominant flow forces acting on bottom particles, organisms, or FST-hemispheres are shear force and lift force. 4. The Local Shear Stress Model (Lamouroux et al., 1992) leads to a correct prediction of hemisphere distribution in a stream with a cobble size substratum, but to an overestimation of hemisphere numbers in a sandy-bottom stream. The substratum-dependent shear stress values therefore need to be entered into the model and a measure of substratum roughness included. 5. Macroinvertebrate abundance correlates well with the movement of FST-hemispheres. Samples from points with very high or very low roughness did not contribute to scatter in the data, indicating that the sum of the near-bed flow forces is relevant to macroinvertebrate distribution, not shear stress alone. We conclude, that FST-hemispheres are well suited to characterize near-bottom hydraulics and therefore the microhabitat of the benthos.     

Growth inhibition of dinoflagellate algae in shake flasks: Not due to shear this time!

Large scale algae cultures present interesting challenges in that they exhibit characteristics of typical bacterial and animal cell cultures. One current commercial food additive, docosahexaenoic acid (DHA), is produced using the dinoflagellate algae, Crypthecodinium cohnii. Like animal cell culture, the perceived sensitivity of algae culture to hydrodynamic forces has potentially limited the agitation and aeration applied to these systems. However, the high density cultivation of C. cohnii required for an economically feasible process inevitably results in high oxygen demand. In this study, we demonstrated what first appeared to be a problem with shear sensitivity in shake flasks is most probably a mass transfer limitation. We subsequently demonstrated the limit of chronic and rapid energy dissipation rate, EDR, that C. cohnii cells can experience. This limit was determined using a microfluidic device connected in a recirculation loop to a stirred tank bioreactor, which has been previously used to repeatedly expose animal cells to high levels of EDR. Inhibition of cell growth was observed when C. cohnii cells were subjected to an EDR of 5.9 × 10⁶ W/m³ with an average frequency of 0.2/min or more. This level of EDR is sufficiently high that C. cohnii can withstand typically encountered hydrodynamic forces in bioprocesses. This result suggests that at least one dinoflagellate algae, C. cohnii, is quite robust with respect to hydrodynamic forces and the scale‐up of process using this type of algae should be more concerned with providing sufficient gas transfer given the relatively high oxygen demand. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Adhesion Strength of Settled Spores of the Green Alga Enteromorpha

Strengths of attachment of spores of the green fouling alga Enteromorpha to glass have been measured using a modified water jet apparatus. Surface pressures of ～250 kPa were required to quantitatively remove attached spores after 4 h contact with a surface. The development of adhesive and cohesive strength is highly time-dependent; after 8 h in contact with a surface spores did not detach, even at pressures in excess of 250 kPa. Spores settled in groups are more resistant to detachment than single spores, which suggests that the adaptive value of gregarious settlement behaviour may lie in the greater resistance of groups to detachment forces in a naturally turbulent environment. The interfacial forces exerted as water impinges on the surface and the derivation of adhesion strength values in terms of wall shear stress are discussed and compared with those obtained by other methods. A surface pressure of 250 kPa approximates to 325 Pa wall shear stress. Calculation using the power-law formula predicts that detachment forces of this magnitude are unlikely to be realized at operating speeds for most vessels and that most Enteromorpha spores would not detach from untreated hulls.     

A finite element evaluation of the moment arm hypothesis for altered vertebral shear failure force

The mechanism of vertebral shear failure is likely a bending moment generated about the pars interarticularis by facet contact, and the moment arm length (MAL) between the centroid of facet contact and the location of pars interarticularis failure has been hypothesised to be an influential modulator of shear failure force. To quantitatively evaluate this hypothesis, anterior shear of C3 over C4 was simulated in a finite element model of the porcine C3–C4 vertebral joint with each combination of five compressive force magnitudes (0–60% of estimated compressive failure force) and three postures (flexed, neutral and extended). Bilateral locations of peak stress within C3's pars interarticularis were identified along with the centroids of contact force on the inferior facets. These measurements were used to calculate the MAL of facet contact force. Changes in MAL were also related to shear failure forces measured from similar in vitro tests. Flexed and extended vertebral postures respectively increased and decreased the MAL by 6.6% and 4.8%. The MAL decreased by only 2.6% from the smallest to the largest compressive force. Furthermore, altered MAL explained 70% of the variance in measured shear failure force from comparable in vitro testing with larger MALs being associated with lower shear failure forces. Our results confirmed that the MAL is indeed a significant modulator of vertebral shear failure force. Considering spine flexion is necessary when assessing low-back shear injury potential because of the association between altered facet articulation and lower vertebral shear failure tolerance.     

The effect of shear stress on protein conformation: Physical forces operating on biochemical systems: The case of von Willebrand factor

Macromolecules and cells exposed to blood flow in the circulatory tree experience hydrodynamic forces that affect their structure and function. After introducing the general theory of the effects of shear forces on protein conformation, selected examples are presented in this review for biological macromolecules sensitive to shear stress. In particular, the biochemical effects of shear stress in controlling the von Willebrand Factor (VWF) conformation are extensively described. This protein, together with blood platelets, is the main actor of the early steps of primary haemostasis. Under the effect of shear forces > 30 dyn/cm², VWF unfolding occurs and the protein exhibits an extended chain conformation oriented in the general direction of the shear stress field. The stretched VWF conformation favors also a process of self aggregation, responsible for the formation of a spider web network, particularly efficient in the trapping process of flowing platelets. Thus, the effect of shear stress on conformational changes in VWF shows a close structure-function relationship in VWF for platelet adhesion and thrombus formation in arterial circulation, where high shear stress is present. The investigation of biophysical effects of shear forces on VWF conformation contributes to unraveling the molecular interaction mechanisms involved in arterial thrombosis.     

Elongation of Confluent Endothelial Cells in Culture: The Importance of Fields of Force in the Associated Alterations of Their Cytoskeletal Structure

Studies using either animal models or in vitro flow systems have shown that the shape of large-vessel endothelial cells (ECs) was sensitive to the amplitude of the flow imposed on them. In order to better understand the morphological changes experienced by ECs when exposed to physical forces such as shear stress, the mechanical integrity of confluent bovine aortic ECs (BAECs) was anisotropically perturbed using the five following types of experiments: (i) slicing and partial scraping of BAEC monolayers; (ii) culture of BAECs on narrow strips of adhesive plastic; (iii) incubation of confluent BAECs with media containing low Ca²⁺ concentrations; (iv) culture of ECs on top of rectangular collagen gels; and (v) exposure of BAECs to laminar steady shear stress. In all five experimental systems, BAECs exhibited an elongated morphology and aligned their major axes in specific directions. In addition, a preferential alignment of actin microfilaments, vimentin intermediate filaments, and streaks of vinculin with the major axes of the cells often occurred concomitantly with BAEC elongation. In all five systems, the elongation of ECs was analyzed in terms of a mechanical deformation borne by the cytoskeleton, and possibly caused by anisotropic distribution of the forces experienced by the cell structure. In addition, the strain-stress and stiffness-stress relationships characterizing the elongation of BAECs exposed to steady flow were qualitatively similar to those computed for the uniaxial deformation of a spherical geodesic. Our findings suggest that the cytoskeleton of ECs plays an important role in the transduction of those forces which cause an elongation of ECs.     

Lubrication and load-bearing properties of human salivary pellicles adsorbed ex vivo on molecularly smooth substrata

In a series of Surface Force Balance experiments, material from human whole saliva was adsorbed to molecularly smooth mica substrata (to form an ‘adsorbed salivary film’). Measurements were taken of normal (load bearing, F _n) and shear (frictional, F _s*) forces between two interacting surfaces. One investigation involved a salivary film formed by overnight adsorption from undiluted, centrifuged saliva, with the adsorbed film rinsed with pure water before measurement. Measurements were taken under pure water and 70 mM NaNO₃. In a second investigation, a film was formed from and measured under a solution of 7% filtered saliva in 10 mM NaNO₃. F _n results for both systems showed purely repulsive layers, with an uncompressed thickness of 35–70 nm for the diluted saliva investigation and, prior to the application of shear, 11 nm for the rinsed system. F _s* was essentially proportional to F _n for all systems and independent of shear speed (in the range 100–2000 nm s^?1), with coefficients of friction  μ ～ 0.24 and μ ～ 0.46 for the unrinsed and rinsed systems, respectively. All properties of the rinsed system remained similar when the pure water measurement environment was changed to 70 mM NaNO₃. For all systems studied, shear gave rise to an approximately threefold increase in the range of normal forces, attributed to the ploughing up of adsorbed material during shear to form debris that stood proud of the adsorbed layer. The results provide a microscopic demonstration of the wear process for a salivary film under shear and may be of particular interest for understanding the implications for in vivo oral lubrication under conditions such as rinsing of the mouth cavity. The work is interpreted in light of earlier studies that showed a structural collapse and increase in friction for an adsorbed salivary film in an environment of low ionic strength.     

The Impact of Desiccation on the Adhesion of Barnacles Attached to Non-stick Coatings

Fouling-release coatings prevent fouling of ships' hulls through hydrodynamic forces generated as the ship moves through the water. The effectiveness of such coatings may be evaluated by measuring the adhesion strength of settled organisms, e.g. barnacles. The influence of desiccation of the barnacle adhesive on such measurements was investigated. Shear forces required to remove barnacles of the genus Balanus increased during the course of desiccation up to the point when the barnacles suddenly self-detached. The increase was thought to be due to the rising cohesive strength of the adhesive. Growing tensile forces within the weakly cross-linked adhesive, however, are suggested to have led to self-detachment. The shear forces required to remove barnacles of the genus Elminius were generally low and did not differ significantly during the course of desiccation. The different results may be attributed to specific base morphologies. It was concluded that measuring the adhesion strength of members of the Balanidae on non-stick surfaces in air could produce flawed results due to the influence of desiccation of the barnacle adhesive. The investigations have also provided new insights into the characteristics of barnacle adhesive.     

Micro-PTV Measurement of the Fluid Shear Stress Acting on Adherent Leukocytes In Vivo

Leukocyte adhesion is determined by the balance between molecular adhesive forces and convective dispersive forces. A key parameter influencing leukocyte adhesion is the shear stress acting on the leukocyte. This measure is indispensable for determining the molecular bond forces and estimating cell deformation. To experimentally determine this shear stress, we used microparticle tracking velocimetry analyzing more than 24,000 images of 0.5 μm fluorescent microbeads flowing within mildly inflamed postcapillary venules of the cremaster muscle in vivo. Green fluorescent protein, expressed under the lysozyme-M promoter, made leukocytes visible. After applying stringent quality criteria, 3 of 69 recordings were fully analyzed. We show that endothelial cells, but not leukocytes, are covered by a significant surface layer. The wall shear rate is nearly zero near the adherent arc of each leukocyte and reaches a maximum at the apex. This peak shear rate is 2-6-fold higher than the wall shear rate in the absence of a leukocyte. Microbead trajectories show a systematic deviation toward and away from the microvessel axis upstream and downstream from the leukocyte, respectively. The flow field around adherent leukocytes in vivo allows more accurate estimates of bond forces in rolling and adherent leukocytes and improved modeling studies.     

Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression

Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm² in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (^·OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H₂O₂), ^·OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H₂O₂ was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow. J. Cell. Physiol. 175:156–162, 1998. © 1998 Wiley-Liss, Inc.     

Force via integrins but not E-cadherin decreases Oct3/4 expression in embryonic stem cells

Increasing evidence suggests that mechanical factors play a critical role in fate decisions of stem cells. Recently we have demonstrated that a local force applied via Arg-Gly-Asp (RGD) peptides coated magnetic beads to mouse embryonic stem (ES) cells increases cell spreading and cell stiffness and decreases Oct3/4 (Pou5f1) gene expression. However, it is not clear whether the effects of the applied stress on these functions of ES cells can be extended to natural extracellular matrix proteins or cell–cell adhesion molecules. Here we show that a local cyclic shear force applied via fibronectin or laminin to integrin receptors increased cell spreading and stiffness, downregulated Oct3/4 gene expression, and decreased cell proliferation rate. In contrast, the same cyclic force applied via cell–cell adhesion molecule E-cadherin (Cdh1) had no effects on cell spreading, Oct3/4 gene expression, and the self-renewal of mouse ES cells, but induced significant cell stiffening. Our findings demonstrate that biological responses of ES cells to force applied via integrins are different from those to force via E-cadherin, suggesting that mechanical forces might play different roles in different force transduction pathways to shape early embryogenesis.     

Orientation of the photosynthetic flagellate,Peridinium gatunense,in hypergravity

The photosynthetic freshwater flagellate,Peridinium gatunense, uses both positive phototaxis and negative gravitaxis to move upwards in the water column. At higher fluence rates approaching those at the surface of their habitat, the cells tend to become unoriented and thus stop their upward movement. Orientation and motility ofPeridinium gatunense has been studied in the slow rotating centrifuge microscope (NIZEMI), which allows observation of swimming behavior during centrifugation acceleration between 1g and 5g. The movement vectors were analyzed by real time image analysis capable of tracking many cells simultaneously. At 1g the orientation was not very precise, but the degree of orientation increased significantly at higher acceleration forces up to about 3g. Most cells were capable of swimming even against an acceleration vector of 3.8g; at higher acceleration forces the cells were not able to cope with the centrifugal force. The linear velocity of cells swimming against 1g was about 20% lower than that of cells moving in other directions. The velocity decreased even more in cells swimming against higher acceleration forces.