Application Of Fluid Shear Stress Research Articles

PS-B07-1: BRAIN-TARGETED MECHANICAL INTERVENTION USING PASSIVE HEAD MOTION CAN BE ANTIHYPERTENSIVE

Objective: Reproduce the antihypertensive effect of physical exercise by applying mechanical intervention. Design and method: We conducted animal experiments in which we reproduced mechanical accelerations generated in the head during treadmill running at a moderate velocity. We performed in vitro experiments using cultured cells to determine what type of mechanical force was responsible for the antihypertensive effect of mechanical intervention. We carried out clinical studies to validate the clinical relevance of our findings. Results: Passive head motion in hypertensive rats, which reproduced the mechanical accelerations generated in their heads during treadmill running at a moderate velocity, decreased the expression of angiotensin II type 1 receptor (AT1R) in astrocytes in the rostral ventrolateral medulla (RVLM). This decrease resulted in lowering their blood pressure. Passive head motion generated interstitial fluid movement, which was estimated to exert shearing forces with an average magnitude of < 1 Pa on the cells in the rat medulla. Application of fluid shear stress of relevant magnitudes decreased the AT1R expression in cultured astrocytes, but not in neuronal cells. Furthermore, interference with interstitial fluid movement by hydrogel introduction in the RVLM of hypertensive rats eliminated the ability of passive head motion and treadmill running to decrease their blood pressure and AT1R expression in the RVLM astrocytes. Consistent with these results from animal experiments, vertically oscillating chair riding by hypertensive adult humans, which reproduced the mechanical accelerations generated in their heads during light jogging, lowered their blood pressure. Conclusion: Brain-targeted mechanical intervention can be antihypertensive by modulating the function of RVLM astrocytes through interstitial fluid shear stress.

Oxygen gradients dictate angiogenesis but not barriergenesis in a 3D brain microvascular model.

A variety of biophysical properties are known to regulate angiogenic sprouting, and in vitro systems can parse the individual effects of these factors in a controlled setting. Here, a three-dimensional brain microvascular model interrogates how variables including extracellular matrix composition, fluid shear stress, and radius of curvature affect angiogenic sprouting of cerebral endothelial cells. Tracking endothelial migration over several days reveals that application of fluid shear stress and enlarged vessel radius of curvature both attenuate sprouting. Computational modeling informed by oxygen consumption assays suggests that sprouting correlates to reduced oxygen concentration: both fluid shear stress and vessel geometry alter the local oxygen levels dictated by both ambient conditions and cellular respiration. Moreover, increasing cell density and consequently lowering the local oxygen levels yields significantly more sprouting. Further analysis reveals that the magnitude of oxygen concentration is not as important as its spatial concentration gradient: decreasing ambient oxygen concentration causes significantly less sprouting than applying an external oxygen gradient to the vessels. In contrast, barriergenesis is dictated by shear stress independent of local oxygen concentrations, suggesting that different mechanisms mediate angiogenesis and barrier formation and that angiogenic sprouting can occur without compromising the barrier. Overall, these results improve our understanding of how specific biophysical variables regulate the function and activation of cerebral vasculature, and identify spatial oxygen gradients as the driving factor of angiogenesis in the brain.

Foodborne compounds that alter plasma membrane architecture can modify the response of intestinal cells to shear stress in vitro

In order to ensure barrier function, intestinal cells need to respond promptly to biomechanical stimulation and to adapt constantly to physical cues. To this aim, cell membranes are essential and rely extensively on lipid metabolism and turnover. These can be tuned via nutrition, pharmacological treatment, or exposure to xenobiotics, however, knowledge on the impact of lifestyle and diet on intestinal cells' biomechanical compliance is relatively limited. Building on this, two intestinal cell models (non-transformed human colon epithelial cells HCEC-1CT and the colon adenocarcinoma cell line HT-29) were systematically compared in terms of cholesterol content, membrane fluidity, actin cytoskeletal organization, expression of mechano-gated PIEZO1 channels and caveolin-1. Biomechanical compliance was evaluated with the application of fluid shear stress (force response 0.75–1.5 dyn/cm2). As model substances the food contaminant mycotoxin alternariol (AOH, 0.01–10 μM) was chosen in virtue of its putative structural analogy with cholesterol. AOH was compared to the cholesterol lowering agent lovastatin (LOVA, 0.01–10 μM) and to water-soluble cholesterol (MβCD-CHOL, 0.01–10 μg/ml). Exposure to AOH, LOVA and MβCD-CHOL coherently modulated membrane cholesterol, expression of PIEZO1 and caveolin-1 as well as the formation of actin stress fibers. These effects were functionally relevant since they modified the force response profile to fluid shear stress (morphological adaption and [Ca2+]i). In sum, we could demonstrate a novel role for exogenous or endogenous molecules in shaping intestinal mechanotransduction via regulation of cholesterol homeostasis and plasma membrane architecture.

Quantifying Lymphatic Endothelial Cell Morphological Changes in Response to Fluid Shear Stress, Cyclic Strain, or Combined Stress and Strain In Vitro

The lymphatic system performs vital transport of fluid, lipids, immune cells, and macromolecules. Lymphatic endothelial cells (LECs), which are highly mechanosensitive, are involved in modulating lymphatic contraction, permeability, etc. LECs are sensitive to the fluid shear stress (FSS) caused by lymph flow and the cyclic strain caused by wall contraction and relaxation. The effects of these mechanical forces on LECs are less understood compared to blood vascular endothelial cells, (BVEC), yet the relative changes in these forces on LECs are considerably greater in situ. To the best of our knowledge, no previous in vitro studies have applied simultaneous FSS and cyclic stretching to LECs. To address this information gap, we tested the hypothesis that both FSS and cyclic stretching will alter LEC structure and function. Toward this goal, we developed a novel bioreactor to apply simultaneous, independently-controlled FSS and cyclic uniaxial stretching to LECs on silicone membranes, and a second bioreactor for applying only uniaxial cyclic stretch. After subjecting LECs to FSS alone, stretch alone, or combined FSS and stretch in these systems, we imaged the cells using conventional or confocal fluorescence techniques and measured alterations in orientation and actin fiber alignment of the cultured LECs. Our results show statistically significant changes in LEC orientation compared to controls, aligning in the direction of low steady FSS (0.2 dynes/cm^2) after 12-24 hours. LECs subjected to the low steady flow combined with a 7.5% cyclic stretch, exhibited a more dramatic alignment that was parallel to flow and perpendicular to stretch after only 6 hours. These data indicate that, like BVEC, LEC morphological alignment is sensitive to both FSS and stretching, with a synergistic response when both are applied simultaneously. Similar levels of alignment were seen in LECs subjected to only cyclic stretch after 6 or 12 hours, but rather than aligning perpendicular to stretch, the distribution of cell angles was bimodal, with most cells aligning about +/- 15 degrees from perpendicular. This slightly off-axis alignment could be a function of applying stretch without fluid flow, or may be a response to the higher magnitude of stretch in those studies (40% compared to 7.5%). There was some visual indication of alignment of actin fibers in LEC subjected to combined FSS and stretching, but it was more difficult to determine clear trends due to spatial variation when imaging different regions of the membrane, and further study is therefore required. Taken together, our results show that application of FSS and strain differentially alter LEC alignment and morphology and provides an important platform to further investigate the mechanisms underlying the LEC response to complex physiologically relevant mechanical environments.

Cardiac and lung endothelial cells in response to fluid shear stress on physiological matrix stiffness and composition

Preconditioning of endothelial cells from different vascular beds has potential value for re-endothelialization and implantation of engineered tissues. Understanding how substrate stiffness and composition affects tissue-specific cell response to shear stress will aid in successful endothelialization of engineered tissues. We developed a platform to test biomechanical and biochemical stimuli. A novel polydimethylsiloxane-based parallel plate flow chamber enabled application of laminar fluid shear stress of 2dynes/cm2 for 12hours to microvascular cardiac and lung endothelial cells cultured on cardiac and lung-derived extracellular matrix. Optical imaging of cells was used to quantify cell changes in cell alignment. Analysis of integrin expression was performed using flow cytometry. Application of fluid shear stress caused the greatest cell alignment in cardiac endothelial cells seeded on polystyrene and lung endothelial cells on polydimethylsiloxane. This resulted in elongation of the lung endothelial cells. αv and β3 integrin expression decreased after application of shear stress in both cell types. Substrate stiffness plays an important role in regulating tissue-specific endothelial response to shear stress, which may be due to differences in their native microenvironments. Furthermore, cardiac and lung endothelial cell response to shear stress was significantly regulated by the type of coating used.

A systematic investigation of the effect of the fluid shear stress on Caco-2 cells towards the optimization of epithelial organ-on-chip models

Epithelial cells experience constant mechanical forces, including fluid shear stress (FSS) on their apical surface. These forces alter both structure and function. While precise recapitulation of the complex mechanobiology of organs remains challenging, better understanding of the effect of mechanical stimuli is necessary towards the development of biorelevant in vitro models. This is especially relevant to organs-on-chip models which allow for fine control of the culture environment. In this study, the effects of the FSS on Caco-2 cell monolayers were systematically determined using a microfluidic device based on Hele-Shaw geometry. This approach allowed for a physiologically relevant range of FSS (from ∼0 to 0.03 dyn/cm2) to be applied to the cells within a single device. Exposure to microfluidic FSS induced significant phenotypical and functional changes in Caco-2 cell monolayers as compared to cells grown in static conditions. The application of FSS significantly altered the production of mucus, expression of tight junctions, vacuolization, organization of cytoskeleton, formation of microvilli, mitochondrial activity and expression of cytochrome P450. In the context of the intestinal epithelium, this detailed understanding of the effects of the FSS will enable the realization of in vitro organs-on-chip models with well-defined and tailored characteristics to a specific purpose, including for drug and nanoparticle absorption studies. The Hele-Shaw approach used in this study could be readily applied to other cell types and adapted for a wide range of physiologically relevant FSS.

Application of fluid shear stress to engineered vascular wall using microchannel

Application of fluid shear stress to engineered vascular wall using microchannel

Endothelial dysfunction in pulmonary arterial hypertension: loss of cilia length regulation upon cytokine stimulation.

Pulmonary arterial hypertension (PAH) is a syndrome characterized by progressive lung vascular remodelling, endothelial cell (EC) dysfunction, and excessive inflammation. The primary cilium is a sensory antenna that integrates signalling and fine tunes EC responses to various stimuli. Yet, cilia function in the context of deregulated immunity in PAH remains obscure. We hypothesized that cilia function is impaired in ECs from patients with PAH due to their inflammatory status and tested whether cilia length changes in response to cytokines. Primary human pulmonary and mouse embryonic EC were exposed to pro- (TNFα, IL1β, and IFNγ) and/or anti-inflammatory (IL-10) cytokines and cilia length was quantified. Chronic treatment with all tested inflammatory cytokines led to a significant elongation of cilia in both control human and mouse EC (by ∼1 µm, P < 0.001). This structural response was PKA/PKC dependent. Intriguingly, withdrawal of the inflammatory stimulus did not reduce cilia length. IL-10, on the other hand, blocked and reversed the pro-inflammatory cytokine-induced cilia elongation in healthy ECs, but did not influence basal length. Conversely, primary cilia of ECs from PAH patients were significantly longer under basal conditions compared to controls (1.86 ± 0.02 vs. 2.43 ± 0.08 µm, P = 0.002). These cilia did not elongate further upon pro-inflammatory stimulation and anti-inflammatory treatment did not impact cilia length. The missing length modulation was specific to cytokine stimulation, as application of fluid shear stress led to increased cilia length in the PAH endothelium. We identified loss of cilia length regulation upon cytokine stimulation as part of the endothelial dysfunction in PAH.

Fluid shear stress induces cancer stem cell-like phenotype in MCF7 breast cancer cell line without inducing epithelial to mesenchymal transition.

Metastasis is the leading cause of cancer deaths due to the spread of cancer cells through the blood vessels and the subsequent formation of secondary tumors. Metastasizing cancer cells in the human vasculature are called circulating tumor cells (CTCs) and are characterized to express the epithelial cell adhesion molecule (EpCAM). They are further known to survive physiological fluid shear stress (FSS) conditions. However, the effect of FSS on CTC molecular phenotype, such as the epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC) expression, has not been extensively studied. Here, CTCs in FSS are evaluated in an invitro model system. MCF7 and MDA-MB-231 breast cancer cell lines were grown in adherent and suspension culture media. The cell lines were tested for EMT and CSC genetic and protein markers using qRT-PCR and flow cytometry, respectively. Suspension cells showed a significantly increased EMT signature compared to adherent cells (p<0.05), suggesting that they model cells detaching from primary tumors invivo. Upon application of FSS, MCF7 and MDA-MB-231 cells did not show a significant change in EMT expression (p>0.05), but there was a statistically significant increase of the CSC population in MCF7 suspension cultures (p<0.05). These results with MCF7 suggest that CTCs can be modeled invitro as non-adherent cancer cells in FSS and that they show an increased CSC-like signature during circulation, providing new insights to the importance of CSC-targeting strategies when treating metastatic patients.

Making the cut: Innovative methods for optimizing perfusion-based migration assays.

Application of fluid shear stress to adherent cells dramatically influences their cytoskeletal makeup and differentially regulates their migratory phenotype. Because cytoskeletal rearrangements are necessary for cell motility and migration, preserving these adaptations under in vitro conditions and in the presence of fluid flow are physiologically essential. With this in mind, parallel plate flow chambers and microchannels are often used to conduct in vitro perfusion experiments. However, both of these systems currently lack capacity to accurately study cell migration in the same location where cells were perfused. The most common perfusion/migration assays involve cell perfusion followed by trypsinization which can compromise adaptive cytoskeletal geometry and lead to misleading phenotypic conclusions. The purpose of this study was to quantitatively highlight some limitations commonly found with currently used cell migration approaches and to introduce two new advances which use additive manufacturing (3D printing) or laser capture microdissection (LCM) technology. The residue-free 3D printed insert allows accurate cell seeding within defined areas, increases cell yield for downstream analyses, and more closely resembles the reported levels of fluid shear stress calculated with computational fluid dynamics as compared to other residue-free cell seeding techniques. The LCM approach uses an ultraviolet laser for "touchless technology" to rapidly and accurately introduce a custom-sized wound area in otherwise inaccessible perfusion microchannels. The wound area introduced by LCM elicits comparable migration characteristics compared to traditional pipette tip-induced injuries. When used in perfusion experiments, both of these newly characterized tools were effective in yielding similar results yet without the limitations of the traditional modalities. These innovative methods provide valuable tools for exploring mechanisms of clinically important aspects of cell migration fundamental to the pathogenesis of many flow-mediated disorders and are applicable to other perfusion-based models where migration is of central importance. © 2016 International Society for Advancement of Cytometry.

Shear stress and circumferential stretch by pulsatile flow direct vascular endothelial lineage commitment of mesenchymal stem cells in engineered blood vessels.

Understanding the response of mesenchymal stem cells (MSCs) in the dynamic biomechanical vascular environment is important for vascular regeneration. Native vessel biomechanical stimulation in vitro is thought to be the most important contributor to successful endothelial differentiation of MSCs. However, the appropriate biomechanical stimulation conditions for differentiating MSCs into ECs have not been fully investigated. To accomplish an in vivo-like loading environment, a loading system was designed to apply flow induced stress and induce hMSC differentiation in vascular cells. Culturing MSCs on tubular scaffolds under flow-induced shear stress (2.5 dyne/cm(2)) for 4 days results in increased mRNA levels of EC markers (vWF, CD31, VE-cadherin and E-selectin) after one day. Furthermore, we investigated the effects of 2.5 dyne/cm(2) shear stress followed by 3% circumferential stretch for 3 days, and an additional 5% circumferential stretch for 4 days on hMSC differentiation into ECs. EC marker protein levels showed a significant increase after applying 5% stretch, while SMC markers were not present at levels sufficient for detection. Our results demonstrate that the expression of several hMSC EC markers cultured on double-layered tubular scaffolds were upregulated at the mRNA and protein levels with the application of fluid shear stress and cyclic circumferential stretch.

A cell-based sensor of fluid shear stress for microfluidics.

Microsystems designed for cell-based studies or applications inherently require fluid handling. Flows within such systems inevitably generate fluid shear stress (FSS) that may adversely affect cell health. Simple assays of cell viability, morphology or growth are typically reported to indicate any gross disturbances to cell physiology. However, no straightforward metric exists to specifically evaluate physiological implications of FSS within microfluidic devices, or among competing microfluidic technologies. This paper presents the first genetically encoded cell sensors that fluoresce in a quantitative fashion upon FSS pathway activation. We picked a widely used cell line (NIH3T3s) and created a transcriptional cell-sensor where fluorescence turns on when transcription of a relevant FSS-induced protein is initiated. Specifically, we chose Early Growth Factor-1 (a mechanosensitive protein) upregulation as the node for FSS detection. We verified our sensor pathway specificity and functionality by noting induced fluorescence in response to chemical induction of the FSS pathway, seen both through microscopy and flow cytometry. Importantly, we found our cell sensors to be inducible by a range of FSS intensities and durations, with a limit of detection of 2 dynes cm(-2) when applied for 30 minutes. Additionally, our cell-sensors proved their versatility by showing induction sensitivity when made to flow through an inertial microfluidic device environment with typical flow conditions. We anticipate these cell sensors to have wide application in the microsystems community, allowing the device designer to engineer systems with acceptable FSS, and enabling the end-user to evaluate the impact of FSS upon their assay of interest.

Fluid shear stress stimulates osteogenic differentiation of human periodontal ligament cells via the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways.

Fluid shear stress (FSS) is a major type of mechanical stress that is loaded on human periodontal ligament cells (hPDLCs) during mastication and orthodontic tooth movement. This study aims to clarify the effect of FSS on the osteogenic differentiation of hPDLCs and to further verify the involvement of mitogen-activated protein kinase (MAPK) signaling in this process. After isolation and characterization, hPDLCs were subjected to 2-hour FSS at 12 dynes/cm(2), and cell viability, osteogenic gene mRNA expression, alkaline phosphatase (ALP) activity, secretion of Type I collagen (COL-I), and calcium deposition were assayed. The levels of phosphorylated p38 and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) in response to FSS were detected by Western blot, and the involvement of ERK1/2 and p38 MAPK signaling pathways in hPDLC osteogenesis under FSS was investigated using the specific MAPK inhibitors U0126 (2Z,3Z)-2,3-bis[amino(2-aminophenylthio)methylene]succinonitrile,ethanol) and SB203580 (4-[4-(4-fluorophenyl)-2-(4-[methylsulfinyl]phenyl)-1H-imidazol-5-yl]pyridine). The application of FSS on hPDLCs induced an early morphologic change and rearrangement of filamentous actin. ALP activity, messenger RNA (mRNA) levels of osteogenic genes, COL-I, and osteoid nodules were significantly increased by FSS. Moreover, ERK1/2 and p38 were activated in different ways after FSS exposure. U0126 and SB203580 completely blocked the FSS-induced increases in ALP activity and osteogenic gene mRNA expression and osteoid nodules formation. FSS is an effective approach for stimulating osteogenic differentiation of hPDLCs. The ERK1/2 and p38 MAPK signaling pathways are involved in this cellular process.

Fluid shear stress‐induced osteoarthritis: roles of cyclooxygenase‐2 and its metabolic products in inducing the expression of proinflammatory cytokines and matrix metalloproteinases

The mechanical overloading of cartilage is involved in the pathophysiology of osteoarthritis (OA) by both biochemical and mechanical pathways. The application of fluid shear stress to chondrocytes recapitulates the earmarks of OA, as evidenced by the release of proinflammatory cytokines (PICs), matrix metalloproteinases (MMPs), and apoptotic factors. Dysregulations or mutations in these genes might directly cause OA in addition to determining the stage at which OA becomes apparent, the joint sites involved, and the severity of the disease and how rapidly it progresses. However, the underlying mechanisms remain unknown. In this review, we propose that the dysregulation of cyclooxygenase-2 (COX-2) is associated with fluid shear stress-induced OA via its metabolic products at different stages of the disease. Indeed, high fluid shear stress rapidly induces the production of PICs and MMPs via COX-2-derived prostaglandin (PG)E2 at the early stage of OA. In contrast, prolonged shear exposure (>12 h) aggravates the condition by concurrently up-regulating the expression of proapoptotic genes and down-regulating the expression of antiapoptotic genes in a 15-deoxy-Δ (12,14)-prostaglandin J2 (15d-PGJ2)-dependent manner at the late stage of disease. These observations may help to resolve long-standing questions in OA progression and provide insight for development of strategies to treat and combat OA.

Hemodynamic shear stress regulates the apelin/APJ system in human endothelial cells

Introduction: The apelin/APJ system is being considered to play a crucial role in cardiovascular function. Namely, the isoforms apelin-12 and apelin-13 seem to be most active in the cardio vasculature. Application of fluid shear stress to endothelial cells elicits the formation of NO via PI3K/AKT signaling and phosphorylation of the endothelial NO synthase (eNOS). We suppose that the apelin/APJ system is involved in the transduction of mechanical forces into intracellular signaling. Methods: We applied hemodynamic shear stress (1.5 dyne/cm2 for venous, 20 dyne/cm2 for arterial conditions) on HUVEC, HCAEC, and HCASMC using a parallel plate flow chamber. Cells were stimulated with apelin-12 and apelin-13. Static controls were kept under similar conditions. After shear stress exposure cells were harvested and prepared for real-time qPCR and Western Blot analysis or fixed and stained for anti-APJ (IF). The supernatant was collected for nitrite measurements. Additionally, HUVEC were transfected with APJ siRNA. Results: After application of shear stress, apelin gene and protein expression were regulated in ECs only. In HUVEC, expression of apelin was increased by 40% under low shear stress, whereas under high shear stress conditions apelin expression decreased by 50% (p<0.01). Interestingly, HCAEC showed an opposite behaviour (p<0.001). Furthermore, in ECs APJ gene and protein expression increased after exposure to shear stress (p<0.05), which could be confirmed by IF. Incubation of HUVEC with apelin-12 and -13 revealed a dose-dependent increase of NO production only after incubation with apelin-12 (P<0.01). Western Blot Analysis underlined apelin isoform dependent cellular signaling. Incubation with apelin-12 for up to 15 min lead to a significant increase in PI3 kinase and Akt phosphorylation under static and dynamic conditions (p=0.02), whereas incubation with apelin-13 had no significant influence on PI3k/Akt signalling and lead to phosphorylation of Erk 1/2 instead. In order to demonstrate shear stress dependency of the apelin/APJ system, we transfected HUVEC with APJ siRNA and exposed them to our standard flow conditions. Transfection with APJ siRNA lead to a loss of shear stress dependent eNOS up-regulation and adhesion. Conclusion: Our results show for the first time a shear stress regulated adjustment of the Apelin/APJ system in human endothelial cells depending on arterial or venous flow conditions. This is confirmed by an enhanced eNOS expression and NO production. Interestingly, apelin-12 and apelin-13 activate different signaling pathways in HUVEC and give rise for various biological responses.

In Silico Modeling of Shear-Stress-Induced Nitric Oxide Production in Endothelial Cells through Systems Biology

Nitric oxide (NO) produced by vascular endothelial cells is a potent vasodilator and an antiinflammatory mediator. Regulating production of endothelial-derived NO is a complex undertaking, involving multiple signaling and genetic pathways that are activated by diverse humoral and biomechanical stimuli. To gain a thorough understanding of the rich diversity of responses observed experimentally, it is necessary to account for an ensemble of these pathways acting simultaneously. In this article, we have assembled four quantitative molecular pathways previously proposed for shear-stress-induced NO production. In these pathways, endothelial NO synthase is activated 1), via calcium release, 2), via phosphorylation reactions, and 3), via enhanced protein expression. To these activation pathways, we have added a fourth, a pathway describing actual NO production from endothelial NO synthase and its various protein partners. These pathways were combined and simulated using CytoSolve, a computational environment for combining independent pathway calculations. The integrated model is able to describe the experimentally observed change in NO production with time after the application of fluid shear stress. This model can also be used to predict the specific effects on the system after interventional pharmacological or genetic changes. Importantly, this model reflects the up-to-date understanding of the NO system, providing a platform upon which information can be aggregated in an additive way.

Fluid shear stress sensitizes cancer cells to receptor-mediated apoptosis via trimeric death receptors

Cancer metastasis, the process of cancer cell migration from a primary to distal location, typically leads to a poor patient prognosis. Hematogenous metastasis is initiated by intravasation of circulating tumor cells (CTCs) into the bloodstream, which are then believed to adhere to the luminal surface of the endothelium and extravasate into distal locations. Apoptotic agents such as tumor necrosis factor apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer cells, have shown promise in treating CTCs to reduce the probability of metastasis. The role of hemodynamic shear forces in altering the cancer cell response to apoptotic agents has not been previously investigated. Here, we report that human colon cancer COLO 205 and prostate cancer PC-3 cells exposed to a uniform fluid shear stress in a cone-and-plate viscometer become sensitized to TRAIL-induced apoptosis. Shear-induced sensitization directly correlates with the application of fluid shear stress, and TRAIL-induced apoptosis increases in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis is not affected by the application fluid shear stress. Interestingly, fluid shear stress does not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments reveal that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear forces can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents.

Shear-Induced Resistance to Neutrophil Activation via the Formyl Peptide Receptor

The application of fluid shear stress on leukocytes is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. The formyl peptide receptor (FPR) on neutrophils, which binds to formyl-methionyl-leucyl-phenylalanine (fMLP) and plays a role in neutrophil chemotaxis, has been implicated as a fluid shear stress sensor that controls pseudopod formation. The role of shear forces on earlier indicators of neutrophil activation, such as L-selectin shedding and αMβ2 integrin activation, remains unclear. Here, human neutrophils exposed to uniform shear stress (0.1–4.0 dyn/cm2) in a cone-and-plate viscometer for 1–120 min showed a significant reduction in both αMβ2 integrin activation and L-selectin shedding after stimulation with 0.5 nM of fMLP. Neutrophil resistance to activation was directly linked to fluid shear stress, as the response increased in a shear stress force- and time-dependent manner. Significant shear-induced loss of FPR surface expression on neutrophils was observed, and high-resolution confocal microscopy revealed FPR internalized within neutrophils. These results suggest that physiological shear forces alter neutrophil activation via FPR by reducing L-selectin shedding and αMβ2 integrin activation in the presence of soluble ligand.

Conditional deletion ofPkd1in osteocytes disrupts skeletal mechanosensing in mice

We investigated whether polycystin-1 is a bone mechanosensor. We conditionally deleted Pkd1 in mature osteoblasts/osteocytes by crossing Dmp1-Cre with Pkd1(flox/m1Bei) mice, in which the m1Bei allele is nonfunctional. We assessed in wild-type and Pkd1-deficient mice the response to mechanical loading in vivo by ulna loading and ex vivo by measuring the response of isolated osteoblasts to fluid shear stress. We found that conditional Pkd1 heterozygotes (Dmp1-Cre;Pkd1(flox/+)) and null mice (Pkd1(Dmp1-cKO)) exhibited a ∼ 40 and ∼ 90% decrease, respectively, in functional Pkd1 transcripts in bone. Femoral bone mineral density (12 vs. 27%), trabecular bone volume (32 vs. 48%), and cortical thickness (6 vs. 17%) were reduced proportionate to the reduction of Pkd1 gene dose, as were mineral apposition rate (MAR) and expression of Runx2-II, Osteocalcin, Dmp1, and Phex. Anabolic load-induced periosteal lamellar MAR (0.58 ± 0.14; Pkd1(Dmp1-cKO) vs. 1.68 ± 0.34 μm/d; control) and increases in Cox-2, c-Jun, Wnt10b, Axin2, and Runx2-II gene expression were significantly attenuated in Pkd1(Dmp1-cKO) mice compared with controls. Application of fluid shear stress to immortalized osteoblasts from Pkd1(null/null) and Pkd1(m1Bei/m1Bei)-derived osteoblasts failed to elicit the increments in cytosolic calcium observed in wild-type controls. These data indicate that polycystin-1 is essential for the anabolic response to skeletal loading in osteoblasts/osteocytes.

Fluid shear stress changes cell morphology and regulates the expression of ATP6V1A and TCIRG1 mRNA in rat osteoclasts

Fluid shear stress (FSS) is a potent physical cell signal in the regulation of bone remodeling. Although the effects of FSS on bone cells of the osteoblastic lineage have been studied extensively, less is known about the direct effect of FSS on osteoclasts. In this study, we investigated the direct influence of FSS on rat osteoclasts isolated by a classic mechanical anatomical technique. Osteoclasts were exposed to an FSS of 2.9 dynes/cm2 for a given period, or subjected to various magnitudes of FSS for 30 min. Cell morphology under FSS was observed and the expression of the mRNA of ATP6V1A and TCIRG1, two crucial subunits of the vacuolar H+-ATPase gene, was analyzed by quantitative real-time PCR. Changes in osteoclast morphology were apparent after the application of FSS, including increased cell volume, strengthened refraction and decreased transmittance. Time-dependent and dose-dependent increases in ATP6V1A and TCIRG1 mRNA expression were seen in response to FSS within 30 min. However, a duration of FSS exceeding 30 min induced a significant decrease in ATP6V1A and TCIRG1 mRNA expression. FSS therefore appears to be a potent stimulus that is sensed by rat osteoclasts and results in significant changes in cell morphology and the gene regulation of ATP6V1A and TCIRG1. These effects might be strictly dependent on the duration of FSS.