Shear Stress Exposure Research Articles

Erythrocyte deformability responses to intermittent and continuous subhemolytic shear stress.

Previous studies have demonstrated that red blood cells (RBC) either lyse or at least experience mechanical damage following prolonged exposure to high shear stress (≥100 Pa). Conversely, prolonged shear stress exposure within the physiological range (5-20 Pa, 300 s) was recently reported to improve RBC deformability. This study investigated the relationships between shear stress and RBC deformability to determine the breakpoint between beneficial vs. detrimental exposure to shear stress (i.e., "subhemolytic threshold"). A second aim of the study was to determine whether the frequency of intermittent application of shear stress influenced the subhemolytic threshold. RBC were exposed to various levels of shear stress (0-100 Pa) in a Couette type shearing system for 300 s. RBC deformability was then immediately measured via ektacytometry. Parallel experiments were conducted at the same shear stresses, except the application time differed while keeping constant the total exposure time: shear stress was applied either for 30 s and repeated 10 times (10×30 s) or applied for 15 s and repeated 20 times (20×15 s). For a range of donors, the subhemolytic threshold with constant shear stress application was between 30-40 Pa. When physiological shear stress was applied in an intermittent manner, more frequent applications tended to improve (i.e., increase) RBC deformability. However, when supra-physiological shear stress was applied, both continuous and intermittent protocols damaged RBC. Changes of RBC mechanical behavior occurred without increases of hemoglobin in the suspending media, thus attesting to the absence of hemolysis. Shear stress has a biphasic effect on the mechanical properties of RBC, with the duration and rate of exposure appearing to have minimal impact on the subhemolytic threshold when compared with the magnitude of applied shear stress.

The physical basis of renal fibrosis: effects of altered hydrodynamic forces on kidney homeostasis

Healthy kidneys are continuously exposed to an array of physical forces as they filter the blood: shear stress along the inner lumen of the tubules, distension of the tubular walls in response to changing fluid pressures, and bending moments along both the cilia and microvilli of individual epithelial cells that comprise the tubules. Dysregulation of kidney homeostasis via underlying medical conditions such as hypertension, diabetes, or glomerulonephritis fundamentally elevates the magnitudes of each principle force in the kidney and leads to fibrotic scarring and eventual loss of organ function. The purpose of this review is to summarize the progress made characterizing the response of kidney cells to pathological levels of mechanical stimuli. In particular, we examine important, mechanically responsive signaling cascades and explore fundamental changes in renal cell homeostasis after cyclic strain or fluid shear stress exposure. Elucidating the effects of these disease-related mechanical imbalances on endogenous signaling events in kidney cells presents a unique opportunity to better understand the fibrotic process.

Endothelial cell TIMP-1 is upregulated by shear stress via Sp-1 and the TGFβ1 signaling pathways

Laminar shear stress promotes vascular integrity by inhibiting proteolysis of the extracellular matrix (ECM) surrounding the microvasculature. We hypothesized that the matrix metalloproteinase inhibitor TIMP-1 would be upregulated in endothelial cells exposed to shear stress. Microvascular endothelial cells isolated from rat or mouse skeletal muscles were exposed to laminar shear stress for 2, 4, or 24 h. A biphasic increase in TIMP-1 protein was observed at 2 and 24 h of shear stress exposure. Sp-1 siRNA prevented the increase in TIMP-1 after 2, but not 24, hours of shear exposure. TGFβ production and Smad2/3 phosphorylation are increased by shear stress. Inhibition of TGFβ signaling, either by use of the TGFβ receptor 1 inhibitor SB-431542 or with Smad 2/3 siRNA, abrogated the shear stress-induced increase in TIMP-1 mRNA after 24 h of shear stress exposure. These results suggest that both acute and chronic elevated laminar shear stress act to maintain vessel integrity through increasing TIMP-1 production, but that the TGFβ signaling pathway is essential to maintain TIMP-1 expression during chronic shear stress.

Roles for GP IIb/IIIa and αvβ3 integrins in MDA-MB-231 cell invasion and shear flow-induced cancer cell mechanotransduction

Adhesion of cancer cell to endothelial cells and the subsequent trans-endothelial migration are key steps in hematogenous metastasis. However, the molecular mechanisms of cancer cell/endothelial cell interaction under hemodynamic shear flow and how shear flow-induced cancer cell mechanotransduction are yet to be fully defined. In this study, we identified that the integrins of both platelet glycoprotein IIb/IIIa (GP IIb/IIIa) and αvβ3 were crucial for hematogenous metastasis of human breast carcinoma MDA-MB-231cells. The cell migration and invasion were studied by using Millicell cell culture insert system. The numbers of invaded MDA-MB-231 cells significantly increased by thrombin-activated platelets and reduced by eptifibatide, a platelet inhibitor. Meanwhile, RGDWE peptides, a specific inhibitor of αvβ3 integrin, also inhibited MDA-MB-231 cell invasion. We further used a parallel-plate flow chamber to investigate MDA-MB-231 cell adhesion under flow conditions. Alike in static condition, the adhesion capability of MDA-MB-231 cells to endothelial monolayer was also significantly affected by GP IIb/IIIa and αvβ3 integrins. The expression of matrix metalloproteinase-2 (MMP-2), MMP-9 and αvβ3 integrin in MDA-MB-231 cells were up-regulated after low shear stress exposure (1.84dynes/cm2, 2h). Moreover, we also demonstrated that low shear stress induced a sustained activation of p85 (a regulatory subunit of PI3K) and Akt. Pre-treating MDA-MB-231 cells with the specific PI3K inhibitor of LY294002 abolished the shear stress induced-Akt activation, and the expression of MMP-2, MMP-9, vascular endothelial growth factor (VEGF) and αvβ3 integrin were also down-regulated. Immunofluorescence assay showed that low shear stress also induced αvβ3 integrin clustering and nuclear factor-κB (NF-κB) activation. Interestingly, shear stress-induced activation of Akt and NF-κB was attenuated by LM609, a specific antibody of αvβ3 integrin. It suggests that αvβ3 integrin might be as a mechanosensor to trigger both PI3K/Akt and NF-κB signaling pathways. Taken together, these results establish that GP IIb/IIIa and αvβ3 integrins are essential mediators, and provide insight into how shear stress-induced αvβ3 integrin activation and the downstream pathways for contribution to MDA-MB-231 cell adhesion, migration and invasion.

Role of Hemolysis in Red Cell Adenosine Triphosphate Release in Simulated Exercise Conditions In Vitro

Specific adenosine triphosphate (ATP) release from red blood cells has been discussed as a possible mediator controlling microcirculation in states of decreased tissue oxygen. Because intravascular hemolysis might also contribute to plasma ATP, we tested in vitro which portion of ATP release is due to hemolysis in typical exercise-induced strains to the red blood cells (shear stress, deoxygenation, and lactic acidosis). Human erythrocytes were suspended in dextran-containing media (hematocrit 10%) and were exposed to shear stress in a rotating Couette viscometer at 37°C. Desaturation (oxygen saturation of hemoglobin ∼20%) was achieved by tonometry with N2 before shear stress exposure. Cells not exposed to shear stress were used as controls. Na lactate (15 mM), lactic acid (15 mM, pH 7.0), and HCl (pH 7.0) were added to simulate exercise-induced lactic acidosis. After incubation, extracellular hemoglobin was measured to quantify hemolysis. ATP was measured with the luciferase assay. Shear stress increased extracellular ATP in a stress-related and time-dependent manner. Hypoxia induced a ∼10-fold increase in extracellular ATP in nonsheared cells and shear stress-exposed cells. Lactic acid had no significant effect on ATP release and hemolysis. In normoxic cells, approximately 20%-50% of extracellular ATP was due to hemolysis. This proportion decreased to less than 10% in hypoxic cells. Our results indicate that when exposing red blood cells to typical strains they encounter when passing through capillaries of exercising skeletal muscle, ATP release from red blood cells is caused mainly by deoxygenation and shear stress, whereas lactic acidosis had only a minor effect. Hemolysis effects were decreased when hemoglobin was deoxygenated. Together, by specific release and hemolysis, extracellular ATP reaches values that have been shown to cause local vasodilatation.

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.

HEMOLYSIS ANALYSIS OF AXIAL BLOOD PUMPS WITH VARIOUS STRUCTURE IMPELLERS

Low hemolysis is an important factor for axial blood pumps that has been used in patients with heart failure. The structure of impellers plays a key role in the hemolytic properties of axial blood pumps. Axial blood pumps with various structure impellers exhibit different hemolytic characteristic. In the present study, we aimed to investigate the type of impellers structures in axial blood pumps that contain the best low hemolytic properties. Also, it is expensive and time-consuming to validate the axial blood pump's hemolytic property by in vivo experiments. Therefore, in the present study, the numerical method was applied to analyze the hemolytic property in a blood pump. Specifically, the hemolysis of the pump was calculated by using a forward Euler approach based on the changes in shear stress and related exposure times along the particle trace lines. The different vane structures and rotational speed that affect hemolysis were analyzed and compared. The results showed that long–short alternant vanes exhibited the best hemolytic property which could be utilized in the optimization design of axial blood pumps.

Mechanical activation of a multimeric adhesive protein through domain conformational change.

The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

Evaluation of Shear-Induced Platelet Activation Models Under Constant and Dynamic Shear Stress Loading Conditions Relevant to Devices

The advent of implantable blood-recirculating devices such as left ventricular assist devices and prosthetic heart valves provides a viable therapy for patients with end-stage heart failure and valvular disease. However, device-generated pathological flow patterns result in thromboembolic complications that require complex and lifelong anticoagulant therapy, which entails hemorrhagic risks and is not appropriate for certain patients. Optimizing the thrombogenic performance of such devices utilizing numerical simulations requires the development of predictive platelet activation models that account for variations in shear-loading rates characterizing blood flow through such devices. Platelets were exposed in vitro to both dynamic and constant shear stress conditions emulating those found in blood-recirculating devices in order to determine their shear-induced activation and sensitization response. Both these behaviors were found to be dependent on the shear loading rates, in addition to shear stress magnitude and exposure time. We then critically examined several current models and evaluated their predictive capabilities using these results. Shear loading rate terms were then included to account for dynamic aspects that are either ignored or partially considered by these models, and model parameters were optimized. Independent optimization for each of the two types of shear stress exposure conditions tested resulted in different sets of best-fit constants, indicating that universal optimization may not be possible. Inherent limitations of the current models require a paradigm shift from these integral-based discretized power law models to better address the dynamic conditions encountered in blood-recirculating devices.

Computational Fluid Dynamics Evaluation of Equivalency in Hemodynamic Alterations Between Driver, Integrity, and Similar Stents Implanted Into an Idealized Coronary Artery

We tested the hypothesis that a slight modification in fabrication from the Driver to the Integrity stent (Medtronic) results in nearly equivalent distributions of wall shear stress (WSS) and mean exposure time (MET), reflective of flow stagnation, and that these differences are considerably less than the Multi-Link Vision (Abbott Vascular) or BX Velocity (Cordis) bare metal stents when evaluated by computational fluid dynamics (CFD). Arteries were modeled as idealized straight rigid vessels without lesions. Two vessel diameters (2.25 and 3.0 mm) were studied for each stent and 2.75 mm diameter Integrity stents were also modeled to quantify the impact from best- and worst-case orientations of the stent struts relative to the primary blood flow direction. All stents were 18 mm in length and over-deployed by 10%. The results indicated that, regardless of diameter, the BX Velocity stents had the greatest percentage of the vessel exposed to adverse WSS followed by the Vision, Integrity, and Driver stents. In general, when strut thickness and stent:lumen ratio are similar, the orientation of struts is a determining factor for deleterious flow patterns. For a given stent, the number of struts was a larger determinant of adverse WSS and MET than strut orientation, suggesting that favorable blood flow patterns can be achieved by limiting struts to those providing adequate scaffolding. In conclusion, the Driver and Integrity stents both limit their number of linkages to those which provide adequate scaffolding while also maintaining similar strut thickness and stent:lumen ratios. The Integrity stent also imparts a slight helical velocity component. The modest difference in the fabrication approach between the Driver and Integrity stents is, therefore, not hemodynamically substantial in this idealized analysis, particularly relative to potentially adverse flow conditions introduced by the other stents modeled. This data was used in conjunction with associated regulatory filings and submitted to the FDA as part of the documents facilitating the recent approval for sale of the Resolute Integrity stent in the United States.

1F07 せん断応力負荷時における肝星細胞の遊走とPDGF-BBの関連性(GS17-2:バイオヒート・マストランスポート(2))

1F07 せん断応力負荷時における肝星細胞の遊走とPDGF-BBの関連性(GS17-2:バイオヒート・マストランスポート(2))

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1. Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3. This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts. The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses. Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs. This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts. The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12).

Effect of Zinc and Nitric Oxide on Monocyte Adhesion to Endothelial Cells under Shear Stress

This study describes the effect of zinc on monocyte adhesion to endothelial cells under different shear stress regimens, which may trigger atherogenesis. Human umbilical vein endothelial cells were exposed to steady shear stress (15 dynes/cm(2) or 1 dyne/cm(2)) or reversing shear stress (time average 1 dyne/cm(2)) for 24 h. In all shear stress regimes, zinc deficiency enhanced THP-1 cell adhesion, while heparinase III reduced monocyte adhesion following reversing shear stress exposure. Unlike other shear stress regimes, reversing shear stress alone enhanced monocyte adhesion, which may be associated with increased H(2)O(2) and superoxide together with relatively low levels of nitric oxide (NO) production. L-N(G)-Nitroarginine methyl ester (L-NAME) treatment increased monocyte adhesion under 15 dynes/cm(2) and under reversing shear stress. After reversing shear stress, monocyte adhesion dramatically increased with heparinase III treatment followed by a zinc scavenger. Static culture experiments supported the reduction of monocyte adhesion by zinc following endothelial cell cytokine activation. These results suggest that endothelial cell zinc levels are important for the inhibition of monocyte adhesion to endothelial cells, and may be one of the key factors in the early stages of atherogenesis.

DEVELOPMENT OF A BLOOD ANALOG FOR THE HEMODYNAMIC EFFICIENCY EVALUATION OF CARDIOVASCULAR DEVICES

Accurate simulation of blood hemolytic potential (i.e. the destruction of red blood cells) would be a valuable tool to evaluate blood-interacting devices. Indeed, such a model would alleviate several practical problems with manipulating real blood (addition of anticoagulants and antibiotics, filtration, etc.). In the present study, we have developed a blood analog which is a suspension of micron-sized natural polymeric gel particles, each encapsulating a dye agent. These particles release the dye agent through shear-induced diffusion and fragmentation from exposure to high, non-physiological shear rates. A controlled shear field was applied to blood analog samples using a custom-made grooved parallel plate geometry device as previously reported in the literature. The device was designed to apply a shear stress and shear exposure duration in the range of 0.15 – 269.18 Pa and 156 - 1297 ms, respectively. From the results obtained, a correlation between the human index of hemolysis and the concentration of the released dye agent can be found. Therefore, the use of the proposed suspensions appears suitable as a blood analog for the in vitro evaluation of the destructive effects of cardiovascular devices.

Effects of physiologically relevant dynamic shear stress on platelet complement activation

Disturbed shear stress, commonly found in cardiovascular diseases, plays important roles in platelet activation and functions. It has been reported that when activated by elevated shear stress, platelets were able to support complement activation to completion. In this study, through a dynamic cone and plate shearing device, three physiologically relevant shear stresses were applied to platelets, mimicking the shear conditions when platelets pass through a normal left coronary artery (0.05–1 Pa), a 60% stenosis (elevated shear stress at 6.5 Pa for less than 0.1 s), and when platelets are trapped in a recirculation zone past a stenosis (<0.5 Pa). After shear exposure, platelet-surface complement activation (C1q, C4d, iC3b, and SC5b-9 depositions) was measured using a solid-phase ELISA approach and flow cytometry. Production of complement regulatory proteins – C1-inhibitor (C1-INH) and complement receptor 1 (CR1), was also measured. Results demonstrated that low-pulsatile shear stress (recirculation) was able to initiate platelet complement activation, by increasing C1q deposition significantly. Both pathological shear stresses triggered significant increases in C1 inhibitor generation and noticeable changes in CR1 production, effectively preventing complement activation from completion. These results suggested that for platelets, low-pulsatile shear stress may be more pro-atherogenic, compared to elevated shear stress, especially when the shear stress exposure time is short.

Shear Stress Reverses Dome Formation in Confluent Renal Tubular Cells

Background/Aims. It has been shown that MDCK cells, a cell line derived from canine renal tubules, develop cell domes due to fluid pumped under cell monolayer and focal detachment from the adhesion surface. In vitro studies have shown that primary cilia of kidney tubular epithelial cells act as mechanosensors, increasing intracellular calcium within seconds upon changes in fluid shear stress (SS) on cell membrane. We then studied the effect of prolonged SS exposure on cell dome formation in confluent MDCK cell monolayers. Methods. A parallel plate flow chamber was used to apply laminar SS at 2 dynes/cm² to confluent cell monolayers for 6 hours. Control MDCK cell monolayers were maintained in static condition. The effects of Ca²⁺ blockade and cell deciliation on SS exposure were also investigated. Results. Seven days after reaching confluence, static cultures developed liquid filled domes, elevating from culture plate. Exposure to SS induced almost complete disappearance of cell domes (0.4±0.8 vs. 11.4±2.8 domes/mm², p < 0.01, n=14). SS induced dome disappearance took place within minutes to hours, as shown by time-lapse videomicroscopy. Exposure to SS importantly affected cell cytoskeleton altering actin stress fibers expression and organization, and the distribution of tight junction protein ZO-1. Dome disappearance induced by flow was completely prevented in the presence of EGTA or after cell deciliation. Conclusions. These data indicate that kidney tubular cells are sensitive to apical flow and that these effects are mediated by primary cilia by regulation of Ca²⁺ entry in to the cell. SS induced Ca²⁺ entry provokes contraction of cortical actin ring that tenses cell-cell borders and decreases basal stress fibers. These processes may increase paracellular permeability and decrease basal adhesion making dome disappear. Elucidation of the effects of apical fluid flow on tubular cell function may open new insights on the pathophysiology of kidney diseases associated with cilia dysfunction.

Changes in NO, iNOS and eNOS Expression in MLO-Y4 Cells After Low-intensity Pulsed Ultrasound Treatment With or Without Shear Stress Exposure

Bone formation in vivo is stimulated by mechanical loads. The primary endogenous biomechanical stimulus at the cell level, which results from mechanical loading, is fluid shear stress. Pulsed ultrasound (US) also induces bone formation and accelerates fracture healing. To determine the combined effect of these mechanical stimuli, MLO-Y4 cells were exposed sequentially to US (1.5 MHz, 30 mW/cm2, 1 kHz pulse rate, 20% duty cycle) and fluid shear stress (SS) (19 dynes/cm2) for a range of duration periods. Nitrite levels were measured which indirectly show the nitric oxide (NO) production from the cells. The amount of nitric oxide (NO) produced by cells exposed to combined US and SS treatments that consisted of equivalent exposure periods of 2.5, 5 and 10 min per treatment type, were respectively 0.7, 1.2 and 2.2 fold greater than the sum of the amounts of NO produced by cells exposed to lone US and shear stress treatments for the corresponding time periods (2.5, 5 and 10 min). Alone and in conjunction with mechanical stimuli, serum increases the NO production in the cells. 1400 W, a selective iNOS inhibitor, decreased by 33% the amount of NO produced by cells exposed sequentially to 10 min US and 10 min shear stress treatments. L-NAME, a non-selective inhibitor, completely inhibited NO production. Intermediate and long term experiments, where cells were exposed to US for 20 min and then to shear stress for 3 and 24 h, were also performed. For both durations nitrite levels were higher in the media of sequentially exposed cells than in the media of cells exposed to only one mechanical stimulus. In the long term, the rate of NO production decreased markedly. However, NO production remained significantly higher in the mechanically stimulated cells as compared to controls. In both the intermediate and long term experiments, US combined with SS to have a synergistic effect on eNOS mRNA levels and an additive effect on iNOS levels. These studies demonstrate that US and SS synergistically increase NO production in osteocyte-like cells and they suggest that it may be beneficial to use both exogenous US and endogenously derived SS to increase the formation of bone tissue.

Pattern of P450 expression at the human blood–brain barrier: Roles of epileptic condition and laminar flow

P450 enzymes (CYPs) play a major role in hepatic drug metabolism. It is unclear whether these enzymes are functionally expressed by the diseased human blood-brain barrier (BBB) and are involved in local drug metabolism or response. We have evaluated the cerebrovascular CYP expression and function, hypothesizing possible implication in drug-resistant epilepsy. CYP P450 transcript levels were assessed by cDNA microarray in primary endothelial cultures established from a cohort of brain resections (n = 12, drug-resistant epilepsy EPI-EC and aneurism domes ANE-EC). A human brain endothelial cell line (HBMEC) and non-brain endothelial cell line (HUVEC) were used as controls. The effect of exposure to shear stress on CYP expression was evaluated. Results were confirmed by Western blot and immunohistochemistry on brain specimens. Endothelial drug metabolism was assessed by high performance liquid chromatography (HPLC-UV). cDNA microarray showed the presence of CYP enzymes in isolated human primary brain endothelial cells. Using EPI-EC and HBMEC we found that CYP mRNA levels were significantly affected by exposure to shear stress. CYP3A4 protein was overexpressed in EPI-EC (290 ± 30%) compared to HBMEC and further upregulated by shear stress exposure. CYP3A4 was increased in the vascular compartment at regions of reactive gliosis in the drug-resistant epileptic brain. Metabolism of carbamazepine was significantly elevated in EPI-EC compared to HBMEC. These results support the hypothesis of local drug metabolism at the diseased BBB. The direct association between BBB CYP enzymes and the drug-resistant phenotype needs to be further investigated.

Effect of Nitric Oxide Synthase and growth conditions on hydrogen peroxide production in cultured endothelial cells during shear stress

Endothelial cells release nitric oxide (NO) and reactive oxygen species (ROS) during acute shear stress (SS). We hypothesized that chronic SS attenuates the production of H2O2 during acute SS.Human microvascular endothelial cells (HMVEC) were exposed to one hour of laminar SS, either with or without a preceding 24 hour SS exposure (chronic SS). Cells were studied either in full serum media (5%) or after serum‐starving (to increase ROS) overnight (0.5% serum). 2′,7′‐dichlorofluorescein (DCF) assessed H2O2 formation with flow cytometry (N=3–7).Acute SS increased H2O2 release compare to static conditions (4.4±0.5 fold increase (F.I.) for 0.5% serum and 1.7±0.2 F.I for 5% serum (p&lt;0.05)). Presence of L‐Nitro‐Arginine Methyl Ester (L‐NAME; nitric oxide synthase inhibitor) during acute shear reduced production of H2O2 in serum starved cells (2.7±0.2 F.I. vs. 4.4±0.5 F.I. (p&lt;0.05)), but not in full serum. Acute SS after chronic SS reduced H2O2 in cells grown in low (1.9±0.8 F.I.) and full serum (0.9±0.1 F.I.). This reduction in H2O2 was reversed by L‐NAME in full serum (2.2±0.5 F.I. vs. 0.9±0.1 F.I. (p&lt;0.05)), but not in low serum.Preconditioning with 24 hrs of SS inhibits generation of H2O2 during acute SS. We conclude that NOS modulates H2O2 production differentially in high and low serum conditions.