Laminin interactions with head and neck cancer cells under low fluid shear conditions lead to integrin activation and binding

Lymphatic metastasis of cancer cells involves movement from the primary tumor site to the lymph node, where the cells must be able to productively lodge and grow. It is there that tumor cells encounter cellular and non-cellular constituent elements that make up the lymph node parenchyma. Our work shows that head and neck squamous cell carcinoma (HNSCC) cell lines are able to bind to laminin, fibronectin, vitronectin, and hyaluronic acid, which are extracellular matrix elements within the lymph node parenchyma. HNSCC cell lines bound to laminin under lymphodynamic low shear stress (0.07 dynes/cm(2)), consistent with lymph flow via β1 integrins, including α2β1, α3β1, and α6β1. Binding occurred in the presence of shear stress and not in the absence of flow. Additionally, tumor cell binding to laminin under flow did result in calcium signaling. Our data indicate a novel role for β1 integrin-mediated binding of HNSCC cells to laminin under conditions of lymphodynamic flow that results in intracellular calcium signaling within the cancer cell.     

Morphological analysis of tumor cell/endothelial cell interactions under shear flow

In the process of hematogenous cancer metastasis, tumor cells (TCs) must shed into the blood stream, survive in the blood circulation, migrate through the vascular endothelium (extravasation) and proliferate in the target organs. However, the precise mechanisms by which TCs penetrate the endothelial cell (EC) junctions remain one of the least understood aspects of TC extravasation. This question has generally been addressed under static conditions, despite the important role of flow induced mechanical stress on the circulating cell-endothelium interactions. Moreover, flow studies were generally focused on transient or firm adhesion steps of TC-EC interactions and did not consider TCs spreading or extravasation. In this paper, we used a parallel-plate flow chamber to investigate TC-EC interactions under flow conditions. An EC monolayer was cultured on the lower plate of the flow chamber to model the endothelial barrier. Circulating TCs were introduced into the flow channel under a well-defined flow field and TC cell shape changes on the EC monolayer were followed in vitro with live phase contrast and fluorescence microscopy. Two spreading patterns were observed: radial spreading which corresponds to TC extravasation, and axial spreading where TCs formed a mosaic TC-EC monolayer. By investigating the changes in area and minor/major aspect ratio, we have established a simple quantitative basis for comparing spreading modes under various shear stresses. Contrary to radial spreading, the extent of axial spreading was increased by shear stress.     

Investigation of human platelet adhesion under low shear conditions in a rotational flow chamber

Even though blood pumps have come into clinical usage, thrombo-embolic complications still pose a major problem, and they have not yet been clarified and quantified. However, it is known that the basis of thrombus formation is platelet adhesion, which is thought to be closely associated with the shear rate. Therefore, our current interest focuses on the effect of shear conditions on platelet adhesion. We have designed and carried out an experimental setup allowing fluorescent microscopy of whole blood within a rotational viscometer under controllable shear conditions. A small area of the bottom plate was coated with type I collagen, which provided a model of the injured vessel as a target for platelet adhesion. Using this setup, the time course of platelet adhesion under several different shear rates, ranging from 127 to 723 s^?1, was studied. Platelet adhesion increased along with shear rates up to 283 s^?1, followed by a gradual decrease when the shear rate exceeded 346 s^?1. The adhesion amounts were statistically significant between 283 and 173 s^?1 (p = 0.02), 173 and 127 s^?1 (p = 0.035), and 283 and 503 s^?1 (p = 0.03), respectively. This result suggests that there is an optimal shear condition around 300 s^?1 for platelet adhesion to type I collagen.     

Electrochemical analysis of blood cell/substrate interactions under flow conditions

Interactions of blood cells (RBCs) with a microelectrode of 50 (im diameter have been examined under flow conditions using impedance measurements at high frequencies. At such frequencies, the electrolyte resistance (R_e,) is assimilated to the real part of impedance, and interactions are associated with transient fluctuations of R_e. Sedimentation experiments suggest that one erythrocyte contributes to a 1.1% R_e, increase. Effects of wall shear rate (from 25 to 140 s¹) and RBC concentration (from 8.4 × 10⁵ to 2.7 × 10⁶ cells/ml) have been investigated; the number of interactions rapidly decreases with wall shear rate. Event frequency is proportional to RBC concentration ranging from 3.1 × 10⁶ cells/ml to 1.3 × 10⁷ cells/ml. At high concentrations of RBCs, some transient events overlap. Videotaped images help to determine how many RBCs interact with the microelectrode at the same time on separate surface areas. Under flow conditions, the contribution of one RBC on the R_e increase is similar to the mathematical value obtained by sedimentation and decreases slightly with wall shear rate.     

A low shear stress modular bioreactor for connected cell culture under high flow rates

A generic “system on a plate” modular multicompartmental bioreactor array which enables microwell protocols to be transferred directly to the bioreactor modules, without redesign of cell culture experiments or protocols is described. The modular bioreactors are simple to assemble and use and can be easily compared with standard controls since cell numbers and medium volumes are quite similar. Starting from fluid dynamic and mass transport considerations, a modular bioreactor chamber was first modeled and then fabricated using “milli‐molding,” a technique adapted from soft lithography. After confirming that the shear stress was extremely low in the system in the range of useful flow rates, the bioreactor chambers were tested using hepatocytes. The results show that the bioreactor chambers can increase or maintain cell viability and function when the flow rates are below 500 µL/min, corresponding to wall shear stresses of 10^?5 Pa or less at the cell culture surface. Biotechnol. Bioeng. 2010; 106: 127–137. © 2010 Wiley Periodicals, Inc.     

Modeling cell interactions under flow

In this review, we summarize the current state of understanding of the processes by which leukocytes, and other cells, such as tumor cells interact with the endothelium under various blood flow conditions. It is shown that the interactions are influenced by cell-cell adhesion properties, shear stresses due to the flow field and can also be modified by the cells microrheological properties. Different adhesion proteins are known to be involved leading to particular mechanisms by which interactions take place during inflammation or metastasis. Cell rolling, spreading, migration are discussed, as well as the effect of flow conditions on these mechanisms, including microfluidic effects. Several mathematical models proposed in recent years capturing the essential features of such interaction mechanisms are reviewed. Finally, we present a recent model in which the adhesion is given by a kinetics theory based model and the cell itself is modeled as a viscoelastic drop. Qualitative agreement is found between the predictions of this model and in vitro experiments.     

Response of osteoblasts to low fluid shear stress is time dependent

The process of mechanotransduction of bone, the conversion of a mechanical stimulus into a biochemical response, is known to occur in osteoblasts in response to fluid shear stress. In order to understand the reaction of osteoblasts to various times of flow perfusion, osteoblasts were seeded on three-dimensional scaffolds, and cultured in the following conditions: continuous flow perfusion, intermittent flow perfusion, and static condition. We collected samples on day 4, 8 and 12 for analysis. Osteoblast proliferation was demonstrated by cell proliferation and scanning electron microscopy assay. Additionally, the expression of known markers of differentiation, including alkaline phosphatase and osteocalcin, were tested by qRT-PCR and alkaline phosphatase activity assay, and the deposition of calcium was used as an indicator of mineralization demonstrated by calcium content assay. The results supported that low fluid shear stress plays an important role in the activation of osteoblasts: enhance cell proliferation, increase calcium deposition, and promote the expression of osteoblastic markers. Furthermore, the continuous flow perfusion is a more favorable environment for the initiation of osteoblast activity compared with intermittent flow perfusion. Therefore, the force and time of fluid shear stress are important parameters for osteoblast activation.     

Renal tubular fluid shear stress promotes endothelial cell activation

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. In this study, we hypothesized that changes in urinary FSS represent a tubular aggression that contributes to the development of inflammation, a key event in progression of nephropathies. Human renal tubular cells (HK-2) were exposed to FSS for 30 min at 0.01 Pa. Treatment of human endothelial cells (HMEC-1) with the resulting conditioned medium (FSS-CM) increased C-C chemokine ligand 2 (CCL2) and tumor necrosis factor (TNF)-α protein secretion, increased endothelial vascular adhesion molecule-1 (VCAM-1) mRNA expression and stimulated adhesion of human (THP-1) monocytes to the endothelial monolayer. These effects were TNF-α dependent as they were abolished by neutralization of TNF-α. Interestingly, the origin of TNF-α was not epithelial, but resulted from autocrine endothelial production. However, in contrast to short term FSS, long term FSS (5 h) induced the release of the key inflammatory proteins CCL2 and TNF-α directly from tubular cells. In conclusion, these results suggest for the first time that urinary FSS can contribute to the inflammatory state involved in initiation/perpetuation of renal diseases.     

低切应力作用下体外培养动脉内皮细胞内皮素的表达

研究了切应力对完整血管的生物学作用以及应力引起血管重建过程中内皮素(ET)的变化．采用血管体外应力培养系统,将一段完整的猪颈总动脉在体外进行培养,设切应力分别为2Pa(％组)和0．5Pa(S5组),设置2、4、6．8．10．12、14．16和18h共9个时相观察点,非平衡法放射免疫检测灌流液中的ET含量．通过Logistic曲线方程拟合,分析切应力作用下完整动脉ET表达变化规律。结果显示：S20组ET总体变化不明显;S5组分泌速率在前12．37h内明显上升,而后又逐渐下降趋于稳定,且始终高于S20组。说明低切应力作用下ET的表达及分泌增高．结果提示,在低切应力引起的血管重建中,ET可能起着重要作用。     

Migration and differentiation of osteoclast precursors under gradient fluid shear stress

Biomechanics and Modeling in Mechanobiology - The skeleton can adapt to mechanical loading through bone remodeling, and osteoclasts close to microdamages are believed to initiate bone resorption....     

Bongkrekic acid facilitates glycolysis in cultured cells and induces cell death under low glucose conditions

Bongkrekic acid (BKA) inhibits adenine nucleotide translocator (ANT) and suppresses ADP/ATP exchange in the mitochondrial inner membrane. Previously, we demonstrated that BKA exhibited cytotoxic effects on 4T1 tumor cells, depending on the cell number in the culture, but not on NIH3T3 cells. However, the cause of this differential sensitivity was unelucidated. Here we demonstrate that BKA reduced the O₂ consumption in both cell lines and increased the mitochondrial membrane potential, thereby facilitating glucose consumption. BKA reduced cellular ATP in 4T1 cells in a dose-dependent manner but not in NIH3T3 cells. The cellular ATP of 4T1 cells was decreased with a reduced glucose concentration in the media, but that of NIH3T3 cells remained constant. We also demonstrated that BKA-induced cell death in both cell lines in low glucose media; however, the susceptibility to the reduced glucose concentration was slightly higher in 4T1 cells, which may be attributed to the difference in the dependency on glycolysis as their energy source. These results indicate that 4T1 tumor cells rely heavily on glucose for energy production. Our data demonstrate that BKA disturbs ATP production in mitochondria and increases the susceptibility to a low glucose condition.     

Modelling interactions between phytoplankton and bacteria under nutrient-regenerating conditions

Two of the algal parameter values in Table II are incorrect.They should read: _a,o minimun algal N:C ratio 0.037 mol N/mol C _a,m maximum algal N:C ratio 0.133 mol N/mol C     

Modelling interactions between phytoplankton and bacteria under nutrient-regenerating conditions

The interactions of phytoplankton and bacteria in a nitrogen-limitedsteady-state system with an organic nitrogen compound or ammoniumas the sole nitrogen source were modelled. The effects of variousalgal excretion rates and two different mathematical representationsof excretion were examined. The model predicted that higherexcretion elevated the bacterial steady-state biomass, and loweredthe algal biomass. Bacterial respiration, which directly determinednitrogen regeneration, had an important effect on the system.The bacterial growth yield in the model was mainly a functionof the growth rate, and not of the nitrogen:carbon ratio ofthe substrate. In one version of the model, where the excretionof organic carbon increased with decreasing growth rate, themodel started to oscillate when the multiplication product ofmaximum specific excretion of excreted organic carbon (EOC)and the bacterial yield on EOC exceeded the dilution rate, irrespectiveof the form of nitrogen (ammonium or dissolved organic nitrogen)in the medium. The model results were compared with chemostatexperiments with the alga Emiliania huxleyi and a bacterialisolate in pure and mixed culture at two different dilutionrates. The carbon and nitrogen biomass of the bacteria was {smalltilde}1.5 times higher in mixed culture than in pure culture.In the experiments with low dilution rate, the recovery of nitrogenin the form of biomass, ammonium or amino acids was low, suggestingthe excretion by the algae of a refractory nitrogen-containingproduct which the bacteria could not use.     

Self-assembly of protein superstructures by physical interactions under cytoplasm-like conditions

The structure-driven assembly of multimeric protein complexes and the formation of intracellular phase-like protein condensates have been the subject of intense research. However, the assembly of larger superstructures comprising cellular components, such as protein nanoparticles driven by general physical rather than specific biochemical interactions, remains relatively uncharacterized. Here, we use gas vesicles (GVs)—genetically encoded protein nanoparticles that form ordered intracellular clusters—as a model system to study the forces driving multiparticle assembly under cytoplasm-like conditions. Our calculations and experimental results show that the ordered assembly of GVs can be achieved by screening their mutual electrostatic repulsion with electrolytes and creating a crowding force with dissolved macromolecules. The precise balance of these forces results in different packing configurations. Biomacromolecules such as polylysine and DNA are capable of driving GV clustering. These results provide basic insights into how physically driven interactions affect the formation of protein superstructures, offer guidance for manipulating nanoparticle assembly in cellular environments through synthetic biology methods, and inform research on the biotechnology applications of GVs.     

CRP promotes monocyte-endothelial cell adhesion via Fcgamma receptors in human aortic endothelial cells under static and shear flow conditions

Monocyte-endothelial cell adhesion is a key early event in atherogenesis. C-reactive protein (CRP), a cardiovascular risk marker, is known to stimulate ICAM and VCAM in human aortic endothelial cells (HAEC) and induces monocyte-endothelial cell adhesion. In this study, we examined the mechanisms by which native CRP promotes monocyte-endothelial cell adhesion under static conditions and tested the effect of CRP on adhesion under shear flow. Incubation of HAEC with CRP (>25 microg/ml) upregulated NF-kappaB activity, and this resulted in a significant increase in ICAM (54% increase, P<0.001), VCAM (41% increase, P<0.01), and monocyte-endothelial cell adhesion (44% increase, P<0.02) compared with those of control. Preincubation with antibodies to CD32 and CD64 but not CD16 effectively inhibited this activation. Blocking NF-kappaB activity with inhibitors or a dominant negative inhibitory kappaB significantly decreased ICAM, VCAM upregulation, and subsequent monocyte-endothelial cell adhesion. Preincubation with antibodies to CD32 and CD64 or transient transfection with small interference RNA to CD32 attenuated CRP-induced NF-kappaB activity, ICAM, VCAM, and monocyte-endothelial cell adhesion under static conditions. Also, the Syk kinase inhibitor piceatannol and MG-132, a proteasome degradation inhibitor, produced similar attenuation in NF-kappaB activity, ICAM, VCAM, and adhesion. Furthermore, CRP-activated endothelial cells supported monocyte rolling, arrest, and transmigration in shear flow (2 dyn/cm2), and this was also inhibited by preincubation with antibodies to CD32 and CD64. Thus, in HAEC, CRP upregulates monocyte-endothelial adhesion by activation of NF-kappaB through engaging the Fcgamma receptors CD32 and CD64.