Flow Chamber System Research Articles

Radiation sensitization of tumor cells induced by shear stress: The roles of integrins and FAK

Recent studies revealed that the interstitial fluid flow in and around tumor tissue not only played an important role in delivering anticancer agents, but also affected the microenvironment, mostly hypoxia, in modulating tumor radio-sensitivity. The current study investigated the hypoxia-independent mechanisms of flow-induced shear stress in sensitizing tumors to radiation.Colon cancer cells were seeded onto glass slides pre-coated with fibronectin. A parallel-plate flow chamber system was used to impose fluid shear stress. Cell proliferation, apoptosis and colony assays were measured after shear stress and/or radiation. Cell cycle analysis and immunoblots of cell adhesion signal molecules were evaluated. The effect of shear stress was reversed by modulating integrin β1 or FAK.Shear stress of 12dyne/cm2 for 24h, but not 3h, enhanced the radiation induced cytotoxicity to colon cancer cells. Protein expression of FAK was significantly down-regulated but not transcriptionally suppressed. By modulating integrin β1 and FAK expression, we demonstrated that shear stress enhanced tumor radiosensitivity by regulating integrin β1/FAK/Akt as well as integrin β1/FAK/cortactin pathways. Shear stress in combination with radiation might regulate integrins signaling by recruiting and activating caspases 3/8 for FAK cleavage followed by ubiquitin-mediated proteasomal degradation.Shear stress enhanced the radiation toxicity to colon cancer cells through suppression of integrin signaling and protein degradation of FAK. The results of our study provide a strong rationale for cancer treatment that combines between radiation and strategy in modulating tumor interstitial fluid flow.

Effect of pulsatile and continuous flow on yes-associated protein.

Yes-associated protein (YAP) is a mechanosignaling protein that relays mechanical information to the nucleus by changing its level of phosphorylation. We hypothesize that different flow patterns show differential effect on phosphorylated YAP (pYAP) (S127) and total YAP and could be responsible for flow dependent localization of atherosclerosis. Confluent human umbilical vein endothelial cells (HUVECs) seeded on fibronectin-coated glass slides were exposed to continuous forward flow (CFF) and pulsatile forward flow (PFF) using a parallel plate flow chamber system for 30 minutes. Cell lysates were prepared and immunoblotted to detect the levels of phosphorylated YAP and total YAP. HUVECs exposed to both PFF and CFF showed a mild decrease in the levels of both pYAP (S127) and total YAP. While the levels of pYAP (S127) decreased to 87.85 and 85.21% of static control with PFF and CFF, respectively, the levels of total YAP significantly decreased to 91.31 and 92.27% of static control. No significant difference was seen between CFF and PFF on their effect on pYAP (S127), but both conditions resulted in a significant decrease in total YAP at 30 minutes. The results of this experiment show that the possible effect of different types of flow on YAP is not induced before 30 minutes. Experiments exposing endothelial cells to various types of flow for longer duration of time could help to elucidate the role of YAP in the pathogenesis of atherosclerosis.

In vitro method to observe E-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells.

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche. The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines. Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions. Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs. Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions. However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly. CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs. Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques. In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system. This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

MicroRNA-34a targets Forkhead box j2 to modulate differentiation of endothelial progenitor cells in response to shear stress

Flow shear stress plays important roles in modulating differentiation of endothelial progenitor cells (EPCs). MicroRNAs are crucial for diverse cellular processes, but the expressions and functions of microRNAs in EPCs responding to mechanical stimuli remain unclear. We sought to determine the effects of microRNA-34a (miR-34a) and a novel target Forkhead box j2 (Foxj2) on shear stress-induced EPC differentiation. Human umbilical cord blood-derived EPCs were exposed to laminar shear stress of 15dyn/cm2 with parallel plate flow chamber system. Real time RT-PCR showed that shear stress significantly increased miR-34a expression, which was accompanied by the endothelial differentiation of EPCs. Whereas Foxj2, a putative target of miR-34a predicted by multiple algorithms, was suppressed in this process. Dual luciferase reporter assays, as well as miR-34a mimics and inhibitor treatment were used to confirm the interplay between miR-34a and Foxj2. Our results revealed an inverse correlation of miR-34a and Foxj2 expressions implicated in the endothelial differentiation of EPCs. MiR-34a contributed to this process by up-regulating the expressions of endothelial cell markers, and down-regulating smooth muscular cell markers. In addition, Foxj2 overexpression attenuated endothelial differentiation of EPCs, while Foxj2 siRNA had the opposite effect. These data suggested a unique mechanism that shear stress induces the expression of miR-34a, which targets to Foxj2 and promotes endothelial differentiation of EPCs. The results provide new insights into miR-34a/Foxj2 on shear stress-induced EPC differentiation.

Medroxyprogesterone Acetate Enhances Monocyte-Endothelial Interaction Under Flow Conditions by Stimulating the Expression of Cell Adhesion Molecules

Monocyte adhesion to endothelial cells is an important initial event in atherosclerosis and is partially mediated by adhesion molecule expression on the cell surface. Although estrogens inhibit atherosclerosis development, effects of coadministered progestogen remain controversial. We examined the effects of progestogen on cytokine-stimulated human umbilical venous endothelial cell (HUVEC) expression of adhesion molecules. In HUVECs, adhesion molecule mRNA levels were measured by real-time PCR. Protein expression was quantified by immunocytochemistry and ELISAs. To mimic the monocyte adherence to endothelial cells, we used a flow chamber system to assess progestogen effects on U937 monocytoid cell adherence to HUVEC monolayers. We also examined the suppression effects of adhesion molecules with small interference RNAs. mRNA levels of adhesion molecules in HUVECs treated with medroxyprogesterone acetate (MPA) or 17β-estradiol + MPA were 1.7- to 2.5-fold higher than those in the control. MPA increased the protein expression of E-selectin, P-selectin, and intercellular adhesion molecule-1 compared with that for the control (83.0 ± 0.7, 34.8 ± 1.2, and 5.4 ± 0.0 ng/mL, respectively), whereas other progestogens or 17β-estradiol additive to progestogens did not significantly change expression. MPA significantly increased U937 monocytoid cell adherence compared with the control (56.0 ± 1.5 vs 46.5 ± 3.5 adherent cells per 10 fields) but did not increase adherence to HUVECs with knocked down intercellular adhesion molecule-1. MPA increases cell adhesion molecule expression on HUVECs, causing increased numbers of monocytoid cells to adhere to HUVECs. These MPA effects may be a risk factor for atherogenesis on endothelial cells in postmenopausal women receiving hormone replacement therapy.

Abstract W P114: Assessment of Platelet Thrombus Formation in Patients with Carotid Disease Administered Clopidogrel

Background: Many studies have demonstrated that high residual platelet function in patients administered clopidogrel is associated with cardiovascular events, although, the majority of those studies examined the patients with coronary artery disease. We studied platelet function in patients with ischemic stroke treated by clopidogrel, and compared residual platelet function according to the severity of carotid disease. Methods: We measured platelet aggregation induced by ADP, VerifyNow P2Y12 assay (PRU), phosphorylation of vasodilator-stimulated phosphoprotein (PRI), platelet-leukocyte complex (PLC), and platelet p-selectin expression (PS) in 30 patients with ischemic stroke administered clopidogrel (21 males and 9 females, mean age was 65 years). We also measured platelet thrombus formation under arterial flow conditions using newly developed microchip-based flow chamber system. In this system, platelet thrombus formation was analyzed by measuring flow pressure changes due to occlusion of microchip and was quantified by calculating area under the flow pressure curve (AUC). Each marker was compared between patients with severe carotid stenosis (15 cases) and those with mild to moderate carotid stenosis (15 cases). Results: Platelet function tests such as percentages of platelet aggregation induced by ADP, PRU, PRI, and PS was not significantly different between different severities of carotid stenosis. On the other hand, AUC values and PLC of patients with severe major carotid stenosis were significantly higher than those with mild to moderate stenosis. Conclusions: These results suggested that clopidogrel inhibits platelet aggregation irrespective of the severity of carotid stenosis. On the other hand, platelet thrombus formation under arterial flow conditions and platelet leukocyte interactions were higher in patients with severe carotid stenosis than those with mild to moderate stenosis.

TNF α‐induced leukocyte–endothelial cell interactions show marked interindividual differences independent of the clinical response to adalimumab

The responses of patients with psoriasis to TNFα-antagonists show interindividual differences. Here, TNFα-incubation induced endothelial adhesion molecules, a prerequisite for leucocyte recruitment to inflamed tissues. Using a standardized flow chamber system equipped with near-confluent endothelial cells and a mobile phase containing human leucocytes, proportions of leucocytes interacting with endothelial cells were remarkably different between individual donors (up to 3.5-fold), regardless of whether the leucocytes originated from patients with psoriasis (n=10) or healthy donors (n=10). Adalimumab abrogated adhesion molecule induction and interactions with leucocytes when present prior to or simultaneously with, but not after exposure of the cultures to TNFα. This pattern was seen similarly with leucocytes from healthy donors (n=4), and patients whose psoriasis responded well to adalimumab (n=5) and non-responders (n=5). Thus, although considerable interindividual differences of leucocyte-endothelial cell interactions were demonstrated ex vivo in a TNFα-governed microenvironment, such differences are not associated with individual responses to treatment with adalimumab in vivo.

PECAM-1 phosphorylation and tissue factor expression in HUVECs exposed to uniform and disturbed pulsatile flow and chemical stimuli

We analyzed the relationship between platelet endothelial cell adhesion molecule-1 (PECAM-1) activation and tissue factor (TF) RNA expression in human umbilical vein endothelial cells (HUVECs) exposed to mechanical and chemical stimulation. Fifty percent confluent and 100% confluent HUVEC cultures were exposed to pulsatile forward flow, as a model for uniform flow, or pulsatile to-and-fro flow, as a model for disturbed flow, using a parallel-plate flow chamber system for up to 4 hours in the presence or absence of 4 U/mL thrombin. Protein lysates were immunoprecipitated for PECAM-1 and then immunoblotted with anti-phospho-tyrosine antibody. TF RNA expression was determined using quantitative reverse transcription polymerase chain reaction. HUVECs exposed to disturbed flow induced higher TF expression at 4 hours than HUVECs exposed to uniform flow in sparse cultures (16.8 ± 5.8 vs 5.1 ± 1.2; P < .05). HUVECs exposed to disturbed flow and thrombin induced higher TF RNA expression at 4 hours than cultures exposed to uniform flow and thrombin in both confluent (47.0 ± 6.0 vs 30.2 ± 4.9; P < .05) and sparse (72.3 ± 10.7 vs 49.8 ± 4.7; P < .05) cultures. In confluent HUVEC cultures, PECAM-1 is minimally phosphorylated by disturbed and uniform flow, while in sparse HUVEC cultures, PECAM-1 phosphorylation at 15 minutes is greater in both disturbed and uniform flow (2.0 ± 0.2 and 2.1 ± 0.4 respectively; P < .05). Thrombin treatment of static HUVECs exhibited greater PECAM-1 phosphorylation at 15 minutes in confluent compared with sparse cultures (3.0 ± 0.5 vs 2.3 ± 0.1; P < .05). PECAM-1 phosphorylation of HUVECs exposed to both flow and thrombin is significantly higher in sparse cultures compared with either flow or thrombin stimulation alone but was suppressed in confluent cultures. The significantly higher TF RNA expression induced by disturbed flow and cell confluence indicates that suppression of PECAM-1 phosphorylation may be an important contributory mechanical signal pathway that promotes TF expression when HUVECs are exposed to disturbed flow.

Haemodilution-induced changes in coagulation and effects of haemostatic components under flow conditions

Blood flow patterns are important modifiers of platelet interactions with plasma coagulation factors. However, it is not feasible to evaluate rheological effects of haemodilution on coagulation using conventional coagulation testing. We evaluated thrombus formation with a microchip-based flow-chamber system using whole blood from 12 healthy volunteers (with/without 40% dilution with saline), and 15 cardiac patients [before/after cardiopulmonary bypass (CPB)] in parallel with thromboelastometry. The in vitro additions of von Willebrand factor (vWF, 1.5 U ml(-1)), prothrombin complex concentrate (PCC, 0.3 U ml(-1)), fibrinogen (2 g litre(-1)), or combined PCC (0.3 U ml(-1)) and fibrinogen (1 g litre(-1)) were examined. Recalcified whole-blood samples were perfused over the microchip coated with collagen and tissue thromboplastin at flow rates of 10 and 3 µl min(-1). Dilution of whole blood led to delayed onset of thrombus formation (Ton), and thrombus growth (T80). Changes relative to baseline values were more extensive at 10 µl min(-1) (≥85% prolongation for Ton and T80) than at 3 µl min(-1) (≥40% prolongation for Ton and T80). Adding vWF accelerated thrombus formation only at 10 µl min(-1), while PCC increased thrombin generation in the thrombus at both flow rates. Fibrinogen increased mural thrombus formation at 3 µl min(-1). Decreased clot strength after dilution was restored by fibrinogen, but not by vWF or PCC on thromboelastometry. Additive effects of fibrinogen and PCC in post-CPB blood were demonstrated by both flow chamber and thromboelastometry. Blood flow affects thrombus formation after haemodilution and subsequent haemostatic component interventions, with differential effects at low and high flow.

Efavirenz induces interactions between leucocytes and endothelium through the activation of Mac-1 and gp150,95

The potential cardiovascular (CV) toxicity associated with combined antiretroviral therapy (cART) has been attributed mainly to the nucleoside reverse transcriptase inhibitors abacavir and didanosine. However, the other two components of cART--non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs)--may also be implicated, either directly or by influencing the action of the other drugs. This study evaluates the acute direct effects of the NNRTIs efavirenz and nevirapine and one of the most widely employed PIs, lopinavir, on leucocyte-endothelium interactions, a hallmark of CV disease. Drugs were analysed in vitro in human cells (interactions of peripheral blood polymorphonuclear or mononuclear cells with human umbilical vein endothelial cells) using a flow chamber system, and in vivo in rat mesenteric vessels by means of intravital microscopy. The expression of adhesion molecules in leucocytes and endothelial cells was studied by flow cytometry, and the role of these molecules in white cell recruitment was evaluated by pre-treating human cells or rats with blocking antibodies. Efavirenz and nevirapine, but not lopinavir, increased the rolling flux and adhesion of leucocytes in vitro and in vivo while inducing emigration in rat venules. Efavirenz, but not nevirapine, augmented the levels of CD11b, CD11c and CD18 in neutrophils and monocytes. The actions of efavirenz, but not of nevirapine, were reversed by antibodies against Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18) or ICAM-1 (CD54). NNRTIs, but not PIs, interfere with leucocyte-endothelial interactions. However, differences between efavirenz and nevirapine suggest a specific CV profile for each compound.

Free Thiol Groups In Von Willebrand Factor (VWF) Are Required For Its Proper Function Under Physiological Flow Conditions

BackgroundThe multimeric plasma glycoprotein von Willebrand factor (VWF) is exceptionally rich in cysteine, and its structure is largely determined by inter- and intramolecular disulfide bonding. Additionally, VWF was shown to contain unpaired cysteine residues potentially affecting protein function. The significance of free thiols on the surface of plasmatic VWF has been confirmed previously with respect to platelet binding under pathologically high shear stress. Furthermore their potential involvement in functional VWF self-association occurring at elevated shear stress has been suggested. AimsObjective of the present study was to investigate whether free thiol groups of plasma VWF contribute to the physiological VWF function under high physiological arterial shear stress conditions. Furthermore, we aimed to elucidate possible underlying mechanisms involved in this regulation. MethodsFree and accessible thiol groups of plasma-derived VWF were blocked with N-ethylmaleimide (NEM). Derivatization was followed by detailed structural and functional examination including multimer analysis (MMA) and Fourier transform infrared spectroscopy (FTIR). Functional differences between the NEM-derivatized sample and the control sample were detected using an in vitro flow chamber system with respect to VWF-mediated platelet adhesion to collagen. Interactions with collagen type III and platelet glycoprotein (GP)Ib receptor were investigated using surface plasmon resonance (SPR). Identification of accessible cysteine residues was accomplished using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation as well as mass spectrometry. ResultsBlocking free thiol groups provoked substantial loss of VWF activity with respect to platelet recruitment to collagen type III under flow. The lowered platelet adhesion to collagen type III was shown to be a combined effect of inhibition of (i) the initial VWF binding to collagen type III as well as (ii) VWF-platelet GPIb interaction. Free thiol groups were accessible for derivatization solely on the surface of coiled multimers. Domain incorporation studies revealed a high level of derivatization in VWF N- and C-terminus. This was in accordance with the mass spectrometric analysis, where 19 MPB-derivatized peptides, predominantly located at the N- and C-terminus, could be identified. ConclusionBlocking free thiol groups in VWF significantly impaired mediation of platelet adhesion under physiological shear stress conditions. This result suggests an essential functional role of free thiol groups in VWF regarding binding to subendothelial matrix as well as platelet recruitment. Disclosures:Solecka:Octapharma AG: Employment. Fuchs:Octapharma AG: Employment. Kannicht:Octapharma AG: Employment.

The N-terminal Flanking Region of the A1 Domain Regulates the Force-dependent Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

Binding of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF) initiates platelet adhesion to disrupted vascular surface under arterial blood flow. Flow exerts forces on the platelet that are transmitted to VWF-GPIbα bonds, which regulate their dissociation. Mutations in VWF and/or GPIbα may alter the mechanical regulation of platelet adhesion to cause hemostatic defects as found in patients with von Willebrand disease (VWD). Using a biomembrane force probe, we observed biphasic force-decelerated (catch) and force-accelerated (slip) dissociation of GPIbα from VWF. The VWF A1 domain that contains the N-terminal flanking sequence Gln(1238)-Glu(1260) (1238-A1) formed triphasic slip-catch-slip bonds with GPIbα. By comparison, using a short form of A1 that deletes this sequence (1261-A1) abolished the catch bond, destabilizing its binding to GPIbα at high forces. Importantly, shear-dependent platelet rolling velocities on these VWF ligands in a flow chamber system mirrored the force-dependent single-bond lifetimes. Adding the Gln(1238)-Glu(1260) peptide, which interacted with GPIbα and 1261-A1 but not 1238-A1, to whole blood decreased platelet attachment under shear stress. Soluble Gln(1238)-Glu(1260) reduced the lifetimes of GPIbα bonds with VWF and 1238-A1 but rescued the catch bond of GPIbα with 1261-A1. A type 2B VWD 1238-A1 mutation eliminated the catch bond by prolonging lifetimes at low forces, a type 2M VWD 1238-A1 mutation shifted the respective slip-catch and catch-slip transition points to higher forces, whereas a platelet type VWD GPIbα mutation enhanced the bond lifetime in the entire force regime. These data reveal the structural determinants of VWF activation by hemodynamic force of the circulation.

Antithrombotic effects of PAR1 and PAR4 antagonists evaluated under flow and static conditions

Thrombin-mediated activation of human platelets involves the G-protein-coupled protease-activated receptors PAR1 and PAR4. Inhibition of PAR1 and/or PAR4 is thought to modulate platelet activation and subsequent procoagulant reactions. However, the antithrombotic effects of PAR1 and PAR4 antagonism have not been fully elucidated, particularly under flow conditions. A microchip-based flow chamber system was used to evaluate the influence of SCH79797 (PAR1 antagonist) and YD-3 (PAR4 antagonist) on thrombus formation mediated by collagen and tissue thromboplastin at shear rates simulating those experienced in small- to medium-sized arteries (600s(-1)) and large arteries and small veins (240s(-1)). At a shear rate of 600s(-1), SCH79797 (10μM) efficiently reduced fibrin-rich platelet thrombi and significantly delayed occlusion of the flow chamber capillary (1.44 fold of control; P<0.001). The inhibitory activity of SCH79797 was diminished at 240s(-1). YD-3 (20μM) had no significant effect at either shear rate. The antithrombotic effects of SCH79797 were significantly augmented when combined with aspirin and AR-C66096 (P2Y12 antagonist), but not with YD-3. In contrast, no significant inhibition of tissue factor-induced clot formation under static conditions was observed in blood treated with SCH79797 and YD-3, although thrombin generation in platelet-rich plasma was weakly delayed by these antagonists. Our results suggest that the antithrombotic activities of PAR1 and/or PAR4 antagonism is influenced by shear conditions as well as by combined platelet inhibition with aspirin and a P2Y12-antagonist.

A novel flow chamber for biodegradable alloy assessment in physiologically realistic environments

In order to better understand the in vivo corrosion of biodegradable alloys, it is necessary to replicate the physiological environment as closely as possible. In this study, a novel flow chamber system is developed that allows the investigation of biodegradable alloy corrosion in a simulated physiological environment. The system is designed to reproduce flow conditions encountered in coronary arteries using a parallel plate setup and to allow the culturing of cells. Computational fluid dynamics and analytical methods are used as part of the design process to ensure that suitable flow conditions are maintained in the test region. The system is used to investigate the corrosion behavior of AZ31 alloy foils of different thickness, in test media with and without proteins and in static and dynamic solutions. It is observed that pulsatile flows, similar to those in the coronary arteries, significantly increase corrosion rates and lead to a different corrosion surface morphologies relative to static immersion tests.

Studies of a microchip flow-chamber system to characterize whole blood thrombogenicity in healthy individuals

A whole blood flow-chamber system, the Total Thrombus-formation Analysis System (T-TAS), was developed for quantitative analysis of platelet thrombus formation (PTF) using microchips with thrombogenic surfaces (collagen, PL chip; collagen plus tissue thromboplastin, AR chip) under shear stress conditions. We evaluated the usefulness of the T-TAS for assessing individual thrombogenicity compared with other platelet function tests. Blood samples from 31 healthy volunteers were applied to the T-TAS to measure PTF starting time (T10: time to reach 10 kPa), occlusion time (T60 for PL chip; T80 for AR chip), and area under the curve (AUC10, area under curve until 10 min for PL chip; AUC30, 30 min for AR chip) under various shear rates (1000, 1500, 2000s(-1) for PL chip; 300 s(-1) for AR chip). Platelet functions were also tested using platelet aggregometry, the PFA-100 (collagen and epinephrine [C/EPI], collagen and adenosine diphosphate [C/ADP]), and the VerifyNow P2Y12 assay. Individual pressure waveforms, including PTF starting and ending points, varied among healthy subjects. In the PL chip, T10 and AUC10 showed a shear-dependent correlation with C/EPI or C/ADP. VerifyNow P2Y12 values were not significantly associated with the parameters of the T-TAS. Platelet counts were correlated with all AR measurements, and mostly with PL measurements. The results of the T-TAS were associated with those of the PFA-100 in many respects, indicating that its characteristics are related to shear-induced PTF. The T-TAS showed few correlations with platelet aggregometry and the VerifyNow P2Y12 assay. The T-TAS may allow for the measurement of comprehensive parameters of individual thrombogenicity under whole blood flow conditions.

Phenotypic and functional traits of peripheral blood mononuclear cells retained by controlled cryopreservation: implications for reliable sequential studies of dynamic interactions with endothelial cells

Many immunological experiments would be greatly facilitated if peripheral blood mononuclear cells (PBMC) preserved important functional properties over longer periods of time. Regarding adhesive interactions with endothelial cells, a crucial step of inflammatory processes, this had not been investigated yet. We demonstrate that PBMC subsets subjected to controlled cryopreservation retain their phenotypic traits inasmuch as the proportion of viable T cells, monocytes/macrophages and B cells was comparable with their freshly isolated counterparts. More importantly, we demonstrate for the first time that the procedure does not impede crucial adhesion-mediated dynamic interactions with endothelial cells. Using a flow chamber system, freshly isolated and cryopreserved PBMC showed similar rolling and firm adhesion on TNF-α-activated endothelial cells under shear flow as compared to freshly isolated PBMC from the same donors. Thus, our observation is an important prerequisite for functional studies of leucocyte recruitment when sequential investigations with material from the same patients are required.

In Silico Vascular Modeling for Personalized Nanoparticle Delivery

To predict the deposition of nanoparticles in a patient-specific arterial tree as a function of the vascular architecture, flow conditions, receptor surface density and nanoparticle properties. The patient-specific vascular geometry is reconstructed from computed tomography angiography images. The isogeometric analysis framework integrated with a special boundary condition for the firm wall adhesion of nanoparticles is implemented. A parallel plate flow chamber system is used to validate the computational model in vitro. Particle adhesion is dramatically affected by changes in patient-specific attributes, such as branching angle and receptor density. The adhesion pattern correlates well with the spatial and temporal distribution of the wall shear rates. For the case considered, the larger (2.0 µm) particles adhere two-times more in the lower branches of the arterial tree, whereas the smaller (0.5 µm) particles deposit more in the upper branches. Our computational framework in conjunction with patient-specific attributes can be used to rationally select nanoparticle properties to personalize, and thus optimize, therapeutic interventions.

SIRT1 and Connexin40 Mediate the Normal Shear Stress-Induced Inhibition of the Proliferation of Endothelial Cells Co-Cultured with Vascular Smooth Muscle Cells

Background: Shear stress imposed by blood flow directly impacts endothelial cells (ECs), which are simultaneously influenced by neighboring vascular smooth muscle cells (VSMCs). However, the mechanisms by which shear stress and VSMCs modulate EC proliferation remain to be elucidated. Methods: ECs, cultured alone or co-cultured with VSMCs, were subjected to a normal level of laminar shear stress (NSS) of 15 dyne/cm² or kept under static conditions by using a parallel-plate flow chamber system, respectively. Results: BrdU incorporation assay and flow cytometry revealed that NSS inhibited EC proliferation with or without VSMCs. Western blot analysis demonstrated that NSS down-regulated the expression of Connexin40 (Cx40) in both ECs cultured alone and ECs co-cultured with VSMCs, accompanied by the increased expression of SIRT1. Moreover, salermide, an inhibitor of SIRT1, as well as SIRT1-specifc siRNA transfection inhibited the effect of NSS on EC proliferation and Cx40 expression. In contrast, resveratrol, a SIRT1 activator, induced an alteration in ECs similar to the application of NSS. Conclusion: NSS inhibits the proliferation of ECs via SIRT1 and Cx40 in the presence or absence of VSMCs. The data suggest that NSS plays a protective role in vascular homeostasis by maintaining EC proliferation at a normal level.

Analysing responses to aspirin and clopidogrel by measuring platelet thrombus formation under arterial flow conditions

High residual platelet aggregability and circulating platelet-monocyte aggregates in patients administered aspirin and clopidogrel are associated with ischaemic vascular events. To determine the relevance of these factors with residual thrombogenicity, we measured platelet thrombus formation using a microchip-based flow-chamber system in cardiac patients receiving aspirin and/or clopidogrel, and evaluated its correlation with agonist-inducible platelet aggregation and platelet-monocyte aggregates. Platelet thrombus formation was analysed by measuring flow pressure changes due to the occlusion of micro-capillaries and was quantified by calculating AUC 10 (area under the flow pressure curve. The growth and stability of platelet thrombi that formed inside microchips at shear rates of 1000, 1500, and 2000 s⁻¹ were markedly reduced in patients receiving aspirin and/or thienopyridine compared to healthy controls (n=33). AUC 10 values of aspirin therapy patients (n=20) were significantly lower and higher than those of healthy controls and dual antiplatelet therapy patients (n=19), respectively, and showed relatively good correlations with collagen-induced platelet aggregation and platelet-monocyte aggregates at 1000 and 1500 s⁻¹ (rs >0.59, p<0.01). In contrast, AUC 10 in dual antiplatelet therapy patients was significantly correlated with ADP-induced platelet aggregation at all examined shear rates (rs >0.59, p<0.01), but did not correlate with collagen-induced aggregation. Aspirin monotherapy patients with high residual platelet thrombogenicity also exhibited significant elevations in both collagen-induced platelet aggregation and platelet-monocyte aggregates. Our results, although preliminary, suggest that residual platelet thrombogenicity in aspirin-treated patients is associated with either collagen-induced platelet aggregation or circulating platelet-monocyte aggregates, but it is predominantly dependent on ADP-induced platelet aggregation in patients receiving dual antiplatelet therapy.

Combined Dabigatran and Antiplatelet Agents Assessed by the Novel Microchip-Based Flow Chamber System

AbstractAbstract 1167Evaluation of the overall antithrombotic activity of dabigatran in combination with antiplatelet agents is difficult because plasma-based clotting for dabigatran, and platelet aggregometry in anticoagulated blood are two separate tests which do not reflect physiological interactions between soluble factors and platelets. The use of a flow chamber could be more suitable in evaluating a flow-dependent platelet activation and coagulation responses.The aim of the current study was to comparatively evaluate antithrombotic effects of dabigatran in combination with dual antiplatelet therapy (aspirin plus P2Y12 blockade) using the microchip-based flow chamber (T-TAS, Fujimori Kogyo, Japan)(1), and thrombin generation (TG) assay (Thrombinoscope, Maastricht, the Netherlands)(2). After the local ethics committee approval, blood samples were obtained from consented 5 healthy volunteers in the tubes containing 3.2% sodium citrate. Whole blood samples were mixed with dabigatran (250, 500, 1000 nM), aspirin (100 nM) plus ARC-66096 (P2Y12 inhibitor, 1000 nM) at 25¡C for 10 min. Corn trypsin inhibitor (50 μg/ml) was used to prevent contact activation. The whole blood sample was perfused in the capillary pre-coated with collagen and thromboplastin at the shear rate of 240 or 600 s−1. The process of thrombus formation was monitored by flow pressure increases inside the capillary; (i) lag time before it reaches 10 kPa (T10), (ii) occlusion time (OT) is the lag time before it reaches 80 kPa as thrombus completely occludes the capillary, and (iii) AUC30 is an area under the flow pressure curve (under 80 kPa) after 30 min of perfusion. For TG assay, platelet-rich plasma (platelet count 150 × 103/μl) was prepared from citrated whole blood. TG was triggered by adding 20 μl of CaCl2-fluorogenic substrate buffer to 80 μl of the sample mixed with tissue factor (1 pM) in each well. The lag time (min), and peak thrombin concentration (nM) were evaluated.In the flow chamber, dabigatran inhibited white thrombus formation in a concentration dependent manner at shear rates of 240 and 600 s−1(Fig. 1). At 500 nM of dabigatran, OT was prolonged by ∼2-fold from the (non-treated) control at both shear rates. The combination of aspirin and AR-C66096 only weakly suppressed thrombus formation, but it enhanced the antithrombotic efficacy of dabigatran at both shear rates (Fig. 1). In TG measurements using platelet-rich plasma, dabigatran at 500 nM prolonged the by 3.17-fold, and reduced the peak by 57.6% compared to the untreated control (Table 1). Aspirin and AR-C66096 weakly prolonged the lag time without affecting the peak height. There were relatively small changes in these parameters when antiplatelet agents were combined with dabigatran (Table 1).Our results suggest that combined antithrombotic effects of dabigatran, aspirin, and P2Y12inhibition can be demonstrated in the whole blood using the flow chamber system compared without additional plasma preparation required for TG assay. [Display omitted] The re-calcified whole blood was perfused at the shear rate of 240 s−1 or 600 s−1. Asp/AR-C=aspirin and AR-C66096Table 1Lag time (min)Peak (nM)NativeAsp/AR-CNativeAsp/AR-CControl6.8 ± 0.89.4 ± 3.292.1 ± 23.791.2 ± 29.5Dabi 250 nM18.6 ± 5.421.1 ± 4.569.3 ± 20.652.2 ± 13.6Dabi 500 nM21.6 ± 5.326.2 ± 10.253.0 ± 5.847.8 ± 9.1Dabi 1000 nM30.2 ± 5.635.1 ± 6.323.0 ± 6.922.0 ± 8.4Dabi=dabigatran, Native=no antiplatelet agents, Asp/AR-C=aspirin and AR-C66096 Disclosures:Hosokawa:Fujimori Kogyo: Employment. Ohnishi:Fujimori Kogyo: Employment. Sameshima:Fujimori Kogyo: Employment.