Abstract
ORAL PRESENTATION 1: FLOW VISUALIZATION AND MODELING
O1-1 Model studies on the role of vibration in the development of cerebral aneurysms
Dieter Walter Liepscha, Andrea Balassob and Sergej Frolovc
a Munich University of Applied Sciences, Germany
b Technical University of Munich, Germany
c Tambov University, Russian Federation
In investigations into human blood flow, especially pertaining to the development of aneurysms, the vibrational activity of the blood vessel wall has been neglected. When considering the development and rupture of an aneurysm, many complex factors have to be taken into account, including shear stresses due to flow acting on the vessel wall, wall elasticity, blood pressure, pulsatility, the non-Newtonian flow behavior of blood and vibrations. Vibrational activity arises under a variety of circumstances (e.g., in small aneurysms, vortices are created). These vortices cause velocity fluctuations which act on the aneurysm wall causing it to vibrate. Initially, the intensity of vibrations is smaller than the average pulse intensity. However, as the aneurysm increases in size, smaller, additional vortices arise. The energy exchange in these smaller areas increases, resulting in higher wall vibration. One to one, true-to-scale, physiologically accurate models were used for flow visualization techniques: dyes for steady flow and a birefringent solution with photo elasticity apparatus for unsteady flow. In addition, velocity measurements with a laser-Doppler-anemometer were carried out and mural vibrations were measured with a high-resolution laser vibrometer. After insertion of a stent, the vibrations were measured again. To confirm the results found in models, we also measured wall vibrations of 2,200 Hz in a rabbit AV fistula. The vibrations immediately formed an aneurysm; addition of stents to arterial models with aneurysms showed a decrease of vibration. Recorded vibration activity with frequencies up to and exceeding 550 Hz are important and must be investigated in more detail in the cerebral arterial system, especially in areas prone to aneurysm. Low shear inside aneurysms may provide the conditions for the formation of thrombogenic areas; it remains to be investigated how these areas are affected by stent placement.
O1-2 Ex vivo measurement of blood flows using X-ray PIV technique
Hanwook Park, Eunseop Yeom and Sang Joon Lee
Pohang University of Science and Technology (POSTECH), Republic of Korea
X-ray imaging techniques have been used to visualize various bio-fluid flow phenomena in a nondestructive manner. To obtain hemodynamic information related to circulatory vascular diseases, a time-resolved X-ray PIV (particle image velocimetry) technique with high temporal resolution was developed. In this study, to examine actual pulsatile blood flows in a circular conduit without changes in hemorheological properties, a bypass loop was established by connecting a micro-tube between the jugular vein and femoral artery of a rat. Biocompatible CO2 microbubbles were used as tracer particles. After mixing with whole blood, CO2 microbubbles were injected into the bypass loop. Particle images of the pulsatile blood flow in the bypass loop were consecutively captured by the time-resolved X-ray PIV system. The velocity field information was then obtained with varying flow rate and pulsatility. To determine the feasibility of the use of CO2 microbubbles under in vivo conditions, the effects of the surrounding-tissues were also investigated, because these effects contribute significantly to deterioration of the image contrast of the CO2 microbubbles. Thus, the velocity information of blood flow in the abdominal aorta was obtained to demonstrate the visibility and usefulness of CO2 microbubbles under ex vivo conditions.
O1-3 Lattice Boltzmann-immersed boundary approach for vesicle motion and trajectory in bifurcated vessel
Ji Young Moona,b, Young Woo Kima and Joon Sang Leea
a School of Mechanical Engineering, Yonsei University, Republic of Korea
b The University of Sydney, Sydney, Australia
Vesicle motion and trajectory in bifurcated micro vessels are simulated using a three-dimensional lattice Boltzmann immersed boundary method (LB-IBM). The lattice Boltzmann method is used to determine incompressible fluid flow with a regular Eulerian grid. The immersed boundary method is used to study a vesicle with a Lagrangian grid. We investigate the behaviour of single vesicle and multi vesicles in bifurcated micro vessels. For the single vesicle, the trajectories of vesicle according to the changing offset point are calculated in symmetric bifurcation model. For the multi vesicles, distribution of vesicles is simulated in bifurcation to calculate concentration. Effect of the flow rate ratio, vesicle deformability and critical offset point are considered in simulation. We analyze the change in hydrodynamic resistance caused by the movement of a vesicle. To quantify the hydrodynamic resistance due to a single moving vesicle, flow rates of each channel are measured. Hydrodynamic resistance in micro vessel systems are calculated using Ohm’s law as an electric circuit. The simulation results show that increased vesicle rigidity effects on vesicle trajectory and hydrodynamic resistance in a bifurcation. Simulation results are similar to those predicted by a previous published result of resultant velocity. This study has implications for relevant research in fundamental biology, biomedical and homology application.
O1-4 A comparative study of centrifugal blood pumps
Wonjung Kima , Sung-Gil Kima, Seokbin Honga, Taehong Kima, Sungmin Honga, Minwook Changa, Mohammad Moshfeghia, Seongwon Kanga, Shin-Hyoung Kangb and Nahmkeon Hura
a Department of Mechanical Engineering, Sogang University, Republic of Korea
b Department of Mechanical and Aerospace Engineering, Seoul National University, Republic of Korea
Centrifugal pumps are widely used in blood circulatory mechanical assist systems, such as cardiopulmonary bypass (CPB), ventricular assist devices (VAD), percutaneous cardiopulmonary support (PCPS) and extracorporeal membrane oxygenation (ECMO). Various forms of centrifugal pumps in terms of the design of the rotor and blade have been developed. Here we present a comparative study of a few centrifugal blood pumps available on the market. We experimentally measure the total pressure head and flow rate of the pumps, and assess the hemolysis of each pump using the computational fluid dynamic (CFD) techniques. The comparative study leads us to suggest a new design to minimize the hemolysis without the loss of pumping performance.
ORAL PRESENTATION 2: RED BLOOD CELL MECHANICS
O2-1 Study on RBC (red blood cell) deformability under various shear rate condition using two plane parallel gold electrodes
Ji-chul Hyun, Taekeon Jung, Hanbyul Kim and Sung Yang
Gwangju Institute of Science and Technology (GIST), Republic of Korea
Deformability of RBCs is an important parameter for diagnosing the initial state of diseases during clinical investigation. In this paper, a printed circuit board (PCB) which has two parallel gold electrodes patterned on opposite sides of its engraved channel has been used to measure whole blood impedance (frequency range 10 kHz–10 MHz) for examining RBC deformability under different shear rate conditions. Whole blood can be modeled as an equivalent circuit since plasma and RBC cytoplasm have different electrical properties, and these two fluids are separated by an RBC membrane whose conductivity changes according to the frequency. The electrical properties of blood can be modeled by three different parameters; plasma resistance (Rp), RBC resistance (Ri) and RBC membrane capacitance (Cm). With the measured impedance data (Nyquist plot), curve fitting was done to extract the above three parameters. When the RBCs are subjected to fluidic shear rate in the PCB channel, RBCs start to deform resulting in a decrease in Ri. The shear rate conditions of 1 s−1–100 s−1 have been applied to whole blood to measure the change of Ri. RBC deformation has been studied for two different samples: Rigid RBCs (0.8% (vol/vol) glutaraldehyde solution for 60 min) and normal RBCs. The hematocrit of the blood was 45% for both samples. To compare difference between the two samples, the initial Ri value (shear rate condition: 1 s−1) was subtracted from the Ri value at different shear rate conditions. When shear rate condition was 100 s−1, the difference of Ri was about 17 Ω and 7.6 Ω for normal and rigid RBCs, respectively. From these results, we conclude that the rigid RBCs deform less than normal RBCs. The proposed method for measuring the RBC deformation has a potential to be used for diagnosing the blood pathological state.
O2-2 Evaluation of sub-hemolytic red blood cell damage based on changes of cell deformability
M. Turkaya , B. Eglenena, G. Yavasa, M.J. Simmondsb, H.J. Meiselmanc and O. Yalcina
a Koc University School of Medicine, Istanbul, Turkey
b Heart Foundation Research Centre, Griffith Health Institute, Griffith University, Queensland, Australia
c Department of Physiology and Biophysics, Keck School of Medicine, Los Angeles, CA, USA
Erythrocyte exposure to high shear stress (⩾100 Pa) induces mechanical damage to the red blood cell and may cause hemolysis. This study was conducted to analyze erythrocyte deformability following exposure to constant shear stress and to assess whether a “sub-hemolytic threshold” could be detected. Three damage models were employed to determine whether the sub-hemolytic threshold was sensitive to stressors that are known to impair cellular deformability: (1) heat treatment (48°C – 30 minutes); (2) oxidative damage by 1 mM hydrogen peroxide; (3) metabolic depletion. Subsequently, various levels of shear stress (0–100 Pa) were applied to erythrocytes in a Couette-type shearing system for 300 seconds. Erythrocyte deformability was measured immediately following shear stress exposure via ektacytometry. Impaired deformability was observed in the oxidative damage model, while a slight improvement was observed for heat treatment and metabolic depletion. Hemolysis was observed at each level of shear stress in the samples with oxidative damage, whereas metabolic depletion and heat treatment caused mechanical hemolysis at high shear stress conditions. The effects of shear stress on erythrocyte mechanical properties were observed to be biphasic in both the heat treatment and oxidative damage groups. This biphasic response disappeared after 48 hours of incubation at room temperature. In summary, the sub-hemolytic threshold for erythrocytes was found to be sensitive to the damage models employed in the present study.
O2-3 Shear stress induces F-actin remodeling in erythroid cells by regulating E-Tmod isoforms
Weiyun Mua , Lanping Amy Sungb and Weijuan Yaoa
a Hemorheology Center, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
b Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA
Shear stress can promote the differentiation of erythroid cells and this process is accompanied by the cytoskeletal remodeling. How shear stress regulates the cytoskeletal remodeling remains elusive. Erythrocyte tropomodulin (E-Tmod) is a protein of 41 kDa (E-Tmod41) that caps the pointed ends of actin filaments (F-actin) on the erythrocyte membrane, and its short isoform of 29 kDa (E-Tmod29) binds to tropomyosin (TM) or G-actin in the cytosol. Here we study the roles of E-Tmod isoforms in shear stress induced F-actin remodeling in erythroid cells. Murine erythroleukemia (MEL) cells or primary erythroid cells isolated from the yolk sacs of E9.5-10.5 mouse embryos were subjected to shear stress (5 dyn/cm2 for 3–24 hours). Flow cytometry and confocal microscopy showed that 24 hours of shearing induced ∼1.4 fold increase in F-actin content in MEL cells. Similar result was seen in DMSO-treated MEL cells. Quantitative PCR (qPCR) showed that in MEL cells the transcript for E-Tmod41 was upregulated by 3, 6, 12 and 24 hours of shearing, while that of E-Tmod29 was downregulated. Western Blot showed that the protein level of E-Tmod41 indeed was augmented by 24 hours of shearing in both MEL and primary erythroid cells. Adenovirus-mediated overexpression of E-Tmod41 resulted in the significant increase of F-actin content in MEL cells. To explain how shear stress regulates E-Tmod isoforms, luciferase vectors driven by E-Tmod41-promoter PE0 and E-Tmod29-promoter PE1, respectively, were transfected to cells and the cells were subjected to shear stress for 12 hours. Data showed that the activity of PE0 was elevated but that of PE1 was reduced. Furthermore, our miRNA array data showed that E-Tmod 3′-UTR targeting miRNA, miR-23b-3p, was downregulated by shear stress, which may explain the upregulation of E-Tmod41. Our data indicates that shear stress causes F-actin remodeling in erythroid cells by differentially regulating the E-Tmod isoforms. (This work is supported by National Natural Science Foundation of China Grant No. 31170886.)
O2-4 Variation in red blood cell deformability within whole blood using a sinusoidally changing shear flow
Nobuo Watanabe, Tatsuya Tsuzuki and Yusuke Suzuki
Department of Bio-science and Engineering, College of Systems Engineering and Science, Shibaura Institute of Technology, Japan
We have developed an experimental system that enabled us to observe the time series deformation behavior of a single red blood cell (RBC) under sinusoidal shear flow. The experimental system incorporated a sinusoidal counter-oscillating mechanism of two parallel glass plates with the flow field in between. Using this system, this study aimed to quantify the variation degree of RBC deformability within whole blood. Ten levels of density-classified RBCs were prepared by centrifugation of slaughterhouse acquired porcine whole blood with sodium citrate as an anticoagulant. The density classified RBCs were inserted in a 31 wt% Dextran Phosphate Buffered saline solution with a mixture ratio of 1–200, following which the RBC suspension fluid was exposed to a 2 Hz sinusoidal shear fluctuation using the developed experimental system. The time series of RBC deformation behavior was recorded in a 464 × 1008 pixel image using a 40× objective lens and a high speed video camera with a frame rate of 500 fps with an effective field size of 139 × 302 square micrometers. The image analysis was then performed to acquire the response in major and minor axis and major-to-minor axis ratio of ellipsoidally deformed red blood cells. In total, one hundred cycles were examined. Both the maximal and minimal deformation level decreased almost monotonically with RBC density level. Variation of the maximum deformation level showed an almost normal distribution with several well defined deformation levels. Additionally, no significant difference was found between middle and top density RBCs, whereas great significance between the top three layers and the other layers were found. These data represent a standard normal healthy diagnostic condition. As the negative control study, RBCs hardened by treatment with glutaradehyde always tumbled.
ORAL PRESENTATION 3: FLOW AND RED BLOOD CELL INTERACTION IN MICROCHANNELS AND THE MICROCIRCULATION
O3-1 Investigation of the viscoelastic property of flowing erythrocyte suspensions with oscillatory flow rate using a wall-patterned electrode configuration
Byung Jun Kima, Sulaiman Khanb and Sung Yanga,b
a Department of Medical System Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea
b School of Mechatronics, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea
We have evaluated the viscoelastic property of RBCs in a suspension flowing through a channel equipped with wall-patterned electrodes. A fluidic channel (1.2 × 2.0 mm2 in cross-sectional area) made of glass and PCB board was used. The two types of samples, normal and hardened RBCs (by adding 0.8% glutaraldehyde) in PBS were used with a hematocrit of 45%. The samples were injected into the channel with the oscillatory flow profile (±3 ml/min for deformation and ±0.1 ml/min for relaxation periods) using a syringe pump. The viscoelastic behavior of RBCs was observed for a deformation period of 1 second and for relaxation periods of 10 and 20 seconds. The electrodes measured the electrical impedance at 200 kHz frequency using an impedance analyzer under different flow conditions. The resultant impedance shows the periodic behavior for both normal and hardened RBC suspensions. For normal RBCs, the range for impedance change was 512–515 Ω and 504–513 Ω for a relaxation period of 10 seconds and 20 seconds, respectively. For hardened RBCs, it was 455–459 Ω and 454–459 Ω for 10 seconds and 20 seconds. The magnitude of impedance changes for a relaxation period of 10 seconds were 3 and 4.3 Ω for normal and hardened RBC, respectively. For a 20 seconds relaxation period, a drastic increase in impedance (6.3 Ω) for normal RBCs took place; however the hardened RBCs did not change greatly (0.7 Ω). Since normal RBCs can be deformed and recover well, there was a huge amount of the increment in impedance. In hardened RBCs, however, a lesser change in impedance occurred because they could not be deformed nor recover. In conclusion, this method appears capable of distinguishing between normal and hardened RBCs, and is potentially applicable for investigating rheological characteristics of blood flow in conjunction with other blood physical properties.
O3-2 S22 is required during the regulation of blood flow for the vascular development in zebrafish
Xiang Xie, Ting Sun, Daoxi Lei, Yongfei Liu, Lu Huang, Tian Zhou, Yi Wang and Guixue Wang
College of Bioengineering, Chongqing University, Chongqing, China
Embryonic vascular system development is directed or modulated by genetic and epigenetic factors. Much has been learned about the genetic mechanisms, however, few details are known about the combinatorial roles of genetic and hemodynamic forces underlying the development of vascular system. In this study, we used the Flk1:GFP × Gata1:DsRed transgenic zebrafish (blood vessel being marked with GFP and blood cells with Dsred) to investigate the involvement of blood flow in vascular development and study the molecular mechanism of S22 involved in the process of vascular development. Separately, zebrafish embryos were exposed to Tricaine (0.09 mg/ml) and Nifedipine (0.006 mg/ml) to reduce blood flow. The results showed that chemical blood flow modulators regulate vascular development. The tnnt2a morphant embryos, lacking blood circulation, exhibited severely reduced angiogenesis. Morpholino knockdown of S22 blocked vascular development, and overexpression of S22 in tnnt2a morphant embryos can rescue vascular defects, suggesting that S22 is the downstream mediator of blood flow. Taken together, we have demonstrated that blood flow is essential for in vivo angiogenesis and S22 plays a critical role in zebrafish vascular development.
O3-3 Hybrid capillary-inserted microchannel for sheathless viscoelastic particle focusing and separation
Jeonghun Nam, Justin Kok Soon Tan and Sangho Kim
Department of Biomedical Engineering, National University of Singapore, Singapore
Particle manipulation in a viscoelastic non-Newtonian fluid has been increasingly studied due to its potential range of uses. In viscoelastic flows, elastic forces are induced due to non-uniform distribution of the first normal stress difference. Suspended particles tend to migrate laterally toward the equilibrium positions, for example, 4 corners and the center of a square channel. However, in a circular channel, 3-dimensional particle focusing along the centerline can be achieved. In this study, we propose a novel hybrid device containing a circular capillary and polydimethylsiloxane (PDMS) channel for sheathless particle focusing and separation using a non-Newtonian fluid. The PDMS channel has one inlet, bifurcation and expansion region connected to two outlets. To fabricate the capillary-inserted device, the capillary (O.D. 360 µm, I.D. 50 µm) was inserted in the inlet and fixed by liquid PDMS applied at the inlet. A tygon tube connected the capillary to a syringe. To avoid the abrupt expansion at the interconnection between the capillary tube and the PDMS channel, UV-curable material was filled in the capillary to the connection part and exposed to UV light to be cured. To confirm the particle focusing at the end of the capillary, distribution of fluorescent polystyrene particles (5 and 10 µm) suspended in 5% polyvinylpyrrolidone (PVP) solution was monitored. After flowing through the capillary, randomly distributed particles became focused due to the elasticity-driven migration. At the expansion, 5 and 10 µm particles showed different degrees of lateral migration, so that particles could be separated into two different outlets. The separation efficiency of each outlet was evaluated as ∼99% using the automated cell counter. A major advantage of this technique is the tunability of separable particle size. By using commercial capillaries with different sizes, the proposed technique can be developed as a versatile particle separation tool for various biological samples with different size ranges.
O3-4 Yield stress to assess the risk of microcirculatory impairment in acute coronary syndromes
Euiho Leea , Uiyun Leea,b and Jinmu Jungc,d
a Department of Bionanosystem Engineering, Chonbuk National University, Jeonju, Chonbuk, Republic of Korea
b BK 21 Plus Program, Chonbuk National University, Jeonju, Chonbuk, Republic of Korea
c Hemorheology Research Institute, Chonbuk National University, Jeonju, Chonbuk, Republic of Korea
d Division of Mechanical Design Engineering, Chonbuk National University, Jeonju, Chonbuk, Republic of Korea
In the microvasculature with highly complex networks of micro-sized capillary vessels, the most important challenge is to maintain sufficient perfusion to provide oxygen to tissues. In acute events, increase in WBV has been reported to increase flow resistance, resulting in the elevated risk of flow stagnation in the capillary vessels. Yield stress related to blood viscosity is a rheological parameter in evaluating the risk of circulatory impairment in the microvasculature, which can be defined as a limit of shear stress at a shear rate of zero. The present study determined yield stress using a U-shaped scanning capillary tube viscometer and compared the results between acute coronary syndrome (ACS) patients (
ORAL PRESENTATION 4: CELLULAR AND MOLECULAR BIOMECHANICS
O4-1 Altered membrane skeleton protein binding affinity by protein 4.1 phosphorylation plays a potential role in the deformability of vertebrate erythrocytes
Fuzhou Tang, Yang Ren, Ruofeng Wang, Xueru Deng and Xiang Wang
Department of Biomedical Engineering, Chongqing University, Chongqing, China
In vitro studies have shown that phosphorylation of protein 4.1 (4.1R) alters the affinity of 4.1R for spectrin–actin binding and that this modulates the deformability of human erythrocytes. The main purpose of the present study was to investigate in vertebrate erythrocytes whether alteration affinity by 4.1R phosphorylation is related to erythrocyte deformability. Phorbol 12-myristate-13-acetate (PMA)-induced phosphorylation of 4.1R was tested, erythrocyte deformability was determined and the erythrocyte elastic modulus was evaluated for human, chick, frog and fish. In addition, amino acid sequences of the functionally important domains of 4.1R were analyzed. PMA-induced phosphorylation of 4.1R decreased erythrocyte deformability. The Young’s modulus alterations gradually decreased from human to fish (0.388 ± 0.035, 0.219 ± 0.022, 0.191 ± 0.036 and 0.141 ± 0.007 kPa). Ser-312 and Ser-331 are located within the consensus sequence recognized by protein kinase C (PKC); however, Ser-331 in zebrafish was replaced by Ala-331. The sequence of the eight aa motif from vertebrate 4.1R showed only one amino acid mutation in frog and numerous substitutions in fish. Analysis of Young’s modulus suggested that the interaction between 4.1R and the spectrin–actin binding domain may have a special relationship with the development of erythrocyte deformability. In addition, amino acid mutations in 4.1R further support this relationship. Thus, we hypothesize that alteration of membrane skeleton protein binding affinity may play a potential role in the development of erythrocyte deformability, and alteration of Young’s modulus values may provide a method for determining the deformability development of vertebrate erythrocytes. Acknowledgments: Dr. Xiang Wang was supported by grants from the National Natural Science Foundation of China (NSFC 11072275 and 31271229) and the National High Technology Research and Development Program of China (863 Program No. 2011AA02A103).
O4-2 Investigating the molecular mechanisms of membrane vesiculation and cell deformability after microvesiculation
Duangdao Palasuwan and Attakorn Palasuwan
Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand
The importance of microparticles (MPs) has been explored in the cerebral malaria (CM), and showed that this increase was restricted to impairment of blood rheology. A murine model demonstrated that a down-production of MPs by the deletion of the ATP Binding Cassette Transporter A1 (ABCA1) gene completely protects the CM. Therefore, to interfere with the production of MPs represents a new approach to improve the design of new therapeutic tools for blocking the pathogenic MPs. First, we have investigated the molecular variation of ABAC1 gene in malaria infected patient with known concentration of MPs and studied the effect of citicoline, a molecule that mimics the action of ABCA1 transporter, on reducing the production of MPs. It was found that the ABCA1-102G/G (rs2246298) located in the putative promoter region was significantly associated with susceptibility to severe malaria and directly correlated with MPs concentration. In order to study the modulating the process of vesiculation by blocking the action of ABCA1 transporter, membrane vesiculation was induced in RBCs by incubating with calcium ionophore (0–10 µM) in concomitantly presence or absence of 10 µM of citicoline. Calcium ionophore (>8 µM) induced RMPs formation. These effects were inhibited by a molecule that mimics the action of ABCA1 transporter, citicoline (10 µM). In order to investigate the deformability, the intact RBCs were spun down and filtered through 0.2 µm milli-pore membrane filter and their deformability was examined by Atomic Force Microscope (AFM) (XE-70; Park system). Deformation of RBC was observed using the force curves, 100 RBCs were selected at random and the stiffness of each one was probed in 30 arbitrarily chosen points. Using AFM, it was revealed that the average stiffness and the deformation index of MPs and their vesiculating RBCs were significantly higher than those of non-vasiculating RBCs (
O4-3 Mechanical regulation of kinetics and structural bases of beta2 integrin–ICAM-1 interaction
Shouqin Lü, Debin Mao, Ning Li, Xiao Zhang and Mian Long
Center of Biomechanics and Bioengineering and Key Laboratory of Microgravity (National Microgravity Laboratory), Institute of Mechanics, Chinese Academy of Sciences, Beijing, China
Beta2 integrins are heterodimeric transmembrane proteins that mediate cellular adhesion (via binding to ICAM-1 ligand) to mediate leukocyte–endothelial cell adhesion in inflammation. Beta2 integrin–ICAM-1 interactions exhibit two-dimensional (2D) binding in that they are anchored onto two opposing surfaces of cells and their interaction kinetics are regulated by the external forces of blood flow. The structural characteristics of outside-in activation upon ligand binding, or external forces, determine the binding kinetics of beta2 integrin and further cellular adhesion dynamics between leukocytes and endothelium. Therefore, the mechanical regulation of binding kinetics and corresponding micro-structural mechanisms are crucial for understanding their structure–function relationship. In this study, both biomechanical measurements and molecular dynamics simulations were employed to quantify the force-regulated binding kinetics and to investigate the conformational allosteric dynamics, respectively. Our data indicate that two major beta2 integrin members, LFA-1 and Mac-1, regulate distinctly their binding to ICAM-1, since the on-rate, off-rate and binding affinity of the interacting pair yield different values under various biochemical stimuli for multiple cell types. It was also found that internal ligand binding is critical for allosteric pathways from I domain to hybrid domain, and that the external ligand binding greatly facilitates the internal ligand binding and vice versa. These results further an understanding of outside-in activation of beta2 integrins.
O4-4 Force generation and morphology of actomyosin machinery
T. Kim
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA
Actomyosin machinery generates mechanical forces required for various biological processes of cells such as migration, cytokinesis and morphogenesis. Although microscopic properties of key constituents of the actomyosin machinery have been well characterized, it still remains elusive how force generation and morphology of the machinery are governed by the microscopic properties of individual components and their local interactions. To bridge such a gap between macroscopic and microscopic scales, we developed a three-dimensional agent-based computational model of actomyosin bundles and networks with minimal components: actin filaments, passive cross-linkers and active motors. The model considers key features neglected by other studies despite their potential significance, such as the mechanics, kinetics and geometry of the components. We systematically studied how interplay between actin filaments, motors and cross-linkers leads to force generation and morphology of actomyosin bundles and networks. We showed the force buildup is governed largely by the density and stall force of motors as well as how stably motors walk on actin filaments. It was also found that passive cross-linkers can help force buildup by increasing stability and connectivity but can also act as dampers due to transient binding to actin filaments, limiting increase of generated forces. Therefore, the ratio of motor density to cross-linker density is found to be the most significant factor governing force generation and morphology; with numerous motors, unbinding of the cross-linkers from actin filaments is accelerated due to high load concentrated on the cross-linkers, resulting in fast relaxation of the generated force and significant increase in network mesh size over time. By contrast, with a relatively small amount of motors, force is sustained after generation with relatively constant mesh size because the cross-linkers do not undergo the accelerated unbinding. We also demonstrated how rigidity and dynamics of actin filaments regulate the force generation and morphology.
ORAL PRESENTATION 5: MECHANICS OF BLOOD COMPONENTS
O5-1 Effects of secoisolariciresinol on blood viscosity, aggregation and deformability of RBCs in ovariectomised rats
T.M. Plotnikovaa, A.M. Anishchenkob, O.I. Alievb, N.E. Nifantievc and M.B. Plotnikovb
a Siberian State Medical University, Tomsk, Russia
b E.D. Goldberg Institute of Pharmacology and Regenerative Medicine, Tomsk, Russia
c N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
Natural and postoperative estrogen insufficiency occur under expressed oxidative stress and disturbance of RBC properties (deformability and aggregation). Secoisolariciresinol (SECO) is a natural antioxidant, belonging to phytoestrogens – selective estrogen receptor modulators. This study aimed to investigate the effect of SECO on the processes of lipid peroxidation in erythrocyte membranes, deformability, aggregation of RBCs and blood viscosity in ovariectomized rats. The study was performed on 38 female Wistar rats, divided into 3 groups: sham operated rats (Sham), ovariectomized rats (OVX), OVX-treated with SECO (obtained from larch wood, purity 99.7%) in dose of 20 mg/kg/day during 2 weeks (OVXSECO). Whole blood viscosity was measured using rotational viscometer LVDV-II+P (USA). RBC aggregation (T1/2) and deformability (elongation index, EI) were measured using RheoScan AnD-300 (Korea). In erythrocyte membranes we evaluated the contents of dienic conjugates (DC) and TBA-reactive substances (TBARS). The content of DC and TBARS in erythrocyte membranes was higher by 1.5 and 2 times, respectively, in OVX than in Sham. In OVX the EI was lower by 16–29%, T1/2 was lower by 65% and whole blood viscosity was higher by 12–30% than in Sham. In OVXSECO the level of DC and TBARS in erythrocyte membranes was lower by 47% and 40%, respectively, T1/2 was more by 32%, EI was higher by 4–17%, blood viscosity was lower by 9–18% compared with OVX. It can be concluded that SECO in OVX rats reduces the intensity of lipid peroxidation and improves the microrheological parameters, provided an additional mechanism to decrease cardiovascular risk in menopausal women.
O5-2 Diversity of biomechanical and nanostructural changes to human and cynomolgus monkey red cells infected with malaria parasites
Rou Zhanga , Eric Lombardinib, Amirah Amirc, Georges Snounoud, Laurent Reniae, Brian M. Cookef, Mun Yik Fongc , Yee Ling Lauc, Francois Nosteng and Bruce Russella
a National University of Singapore, Singapore
b Armed Forces Research Institute of the Medical Sciences (USAMC-AFRIMS), Thailand
c University of Malaya, Malaysia
d Sorbonne Universités, UPMC Université Paris 06, UPMC UMRS CR7, F-75005, France
e A*STAR, Singapore
f Monash University, Australia
g Shoklo Malaria Research Unit, Mae Sod, Thailand
Malaria remains the most important mosquito-borne infectious disease, threatening the well-being of 3.2 billion people. Historically, four important human malaria species have been identified, Plasmodium falciparum, P. vivax, P. ovale and P. malariae. However, it has now been recognized that human populations in Asia are also at risk of zoonotic malaria (natural host Macaca fascicularis) caused by P. knowlesi and P. cynomolgi. The malaria parasites can drastically changes the host red cells’ nanostructures and biomechanical properties after infection. Most studies have focused on only P. falciparum where increased membrane rigidity and cytoadhesion (modulated by knob like excrescences on the membrane), cause P. falciparum infected red cells to hinder normal blood flow, often leading to severe malaria symptoms. In our study, we compare and contrast the biomechanical properties and nanostructural changes among Plasmodium spp. that cause malaria in humans (with the exception of P. ovale). Plasmodium falciparum and P. malariae rapidly increase the membrane rigidity of their host cells and also cause excrescences formation of the surface of the RBC. Whereas, P. vivax, P. knowlesi and P. cynomolgi parasites either decrease or maintain the shear modulus of the infected red cell relative to the uninfected comparator. Interestingly we observed rosetting in all species except for P. cynomolgi and P. knowlesi. In those species that rosette, the cell–cell adhesion forces of rosettes in different species were not significantly different. In all cases rosette formation increased membrane rigidity, and infected red cells travelled much slower than normal cells in microfluidic devices.
O5-3 Observation of morphological changes on platelets exposed to shear stress
Hoyoon Leea , Jeongho Kima, Jung Hun Kimb, Hye-Sun Parkb, Chae-Seung Limc and Sehyun Shina,c
a School of Mechanical Engineering, Korea University, Seoul, Republic of Korea
b Rheomeditech Inc., Seoul, Republic of Korea
c Department of Laboratory Medicine, Korea University Guro Hospital, Seoul, Republic of Korea
Activation of platelets, which is highly dependent on shear rate, plays a vital role in hemostasis. With shear induced platelet activation (SIPA) activated platelets start to aggregate and adhere at injured sites to stop bleeding. In our previous study, we suggested a promising method to quantify SIPA measuring with a migration distance (MD). Though the MD of blood was entirely proportional to shear rate, MD slightly decreased over 2800 rpm and the same tendency was verified through the flow cytometric analysis. The major objective of this research is to validate this tendency of SIPA through observation of morphological changes on platelets. Blood sample was collected from median cubital vein into EDTA. For a shear effect on platelet only, platelet rich plasma (PRP) was extracted from whole blood. To assay SIPA in a whole blood condition DIOC6 stained platelets were mixed with other blood cells. Platelets in the two samples were stimulated at various rotating speeds from zero to 2800 rpm by a rotating stirrer. Smeared samples on glass slides were observed with bright field and fluorescence modes of an inverted microscope. The morphological changes at different rotating speeds for platelets in PRP were shown in bright field images. In normal PRP there were small grain-like platelets. As shear rate increased, the number of platelets was diminished and their cluster size was increased. The morphological alteration of platelets became maximized at 2800 rpm with randomly aggregated and largely adhered shapes, which tended to diminish above this rate. The activation of stained platelets in whole blood observed in fluorescence images showed a similar trend despite the existence of erythrocytes. These results imply that SIPA tendency reaches a maximum at a rotating speed of 2800 rpm and tends to decrease above it, changes which can be verified by observation of morphological alterations on platelets.
O5-4 The influence of fluid shear stress on the Von Willebrand factor protein in an optical trap
Xavier J. Candelaa, Monica Corsettia, Peter J. Butlera and Keefe B. Manninga,b
a Department of Biomedical Engineering, The Pennsylvania State University, University Park, PA, USA
b Department of Surgery, Penn State Hershey Medical Center, Hershey, PA, USA
Many patients worldwide suffer from acquired von Willebrand Syndrome (AWS), a condition that arises when von Willebrand Factor (vWF) fails to function properly and spontaneous bleeding events occur. There has been an increase in patients suffering from AWS after left ventricular assist device (LVAD) implantation, and it is believed that the elevated shear stresses play a significant role. Determining the shear stress threshold for a conformational change of vWF would facilitate the development of LVADs to avoid creating certain flow regimes that are likely to induce vWF proteolysis. An experimental setup, which can test and quantify these thresholds for shear stress would thus be an invaluable resource to future medical device research and development. VWF is extracted from human plasma, purified through column chromatography, and attached to polystyrene beads. An optical trap, of known stiffness, is used to isolate beads, and a piezoelectric stage is moved to apply a shear stress to the bead. The phase shift between fluid velocity and bead motion and the produced amplitude are measured for the following bead groups: non-vWF coated beads, vWF coated beads with no shear, vWF coated beads with 3 cycles at a shear rate of 157 s−1, and vWF coated beads with 5 cycles at a shear rate of 157 s−1. Preliminary results show no difference in phase shift between the four groups, but a difference in amplitude is observed. This is presumably caused by an increase in the functional radius of the bead due to the characteristic unraveling of the vWF protein under significant amounts of shear stress. It is hoped that this research will ultimately aid in the establishment of threshold parameters of shear stresses and flow patterns that are dangerous to a patient, and thus, can be avoided in future designs of LVADs.
ORAL PRESENTATION 6: DISEASE AND HEMORHEOLOGY
O6-1 Comparative efficiency and hemorheological consequences of radiotherapy and chemotherapy in patients with solid nonmyeloid malignancies
I.A. Tikhomirovaa, A.V. Muravyova, S.V. Cheporovb, N.V. Kislovb and E.P. Petrochenkoa
a State Pedagogical University, Yaroslavl, Russia
b Regional Cancer Hospital, Yaroslavl, Russia
The aim of our study was to compare hemorheological consequences of chemotherapy and radiotherapy in cancer patients. Forty eight patients with solid nonmyeloid malignancies were enrolled in this prospective, open-label study. Prior to and following treatment (cisplatin for 4 weeks and radiotherapy the course of 44 Gy) hemorheological measurements were made: blood, plasma and red blood cell (RBC) suspension viscosity, Hct, Hb, RBC aggregation (RBCA), deformability (RBCD). It was found that the patients had reduced Hb, up to 85 g/l, and Hct 35% after cisplatin treatment. Under these conditions the whole blood viscosity was decreased by 13–25% (
O6-2 Autodigestion and proteolytic receptor cleavage in rheological and cardiovascular dysfunction
Geert W. Schmid-Schönbein, Frank. A. DeLano, Marco H. Santamaria, Angela Y. Chen, Edward E. Tran and Stephen F. Rodrigues
Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
Our evidence indicates that pancreatic digestive enzymes may leak out of the lumen of the small intestine in acute forms of experimental shock. This leads to the presence of unchecked digestive enzymes in the peritoneal cavity and in the systemic circulation, which in turn causes acute forms of cell dysfunction, multiple organ dysfunctions and possible death. We hypothesize that in chronic conditions of cardiovascular and rheological dysfunctions unchecked proteases may be present in the systemic circulation and involved in cell dysfunctions. We investigated the spontaneously hypertensive rat (SHR), a model of metabolic syndrome which beside elevated arterial blood pressure also exhibits multiple co-morbidities. Investigation of the selected tissue, cells and plasma shows an unchecked extracellular proteolytic activity (e.g. due matrix metalloproteinase and serine proteases) that causes extracellular domain cleavage and associated cell dysfunctions. For example, mechanotransduction regulation of pseudopod formation in circulating leukocytes in response to fluid shear stress is attenuated in the SHR and associated with proteolytic ectodomain cleavage of the FML receptor (FPR), the mechanosensor for pseudopod formation. The unchecked protease activity also causes cleavage of other membrane receptors, e.g. the insulin receptor, which leads lack of insulin signaling and thus to insulin resistance, the beta-2 adrenergic receptor in arterioles, which leads to their contraction and elevation of central blood pressure, or cleavage to of the VEGFR-2, which leads to endothelial apoptosis and at the level of the capillaries to rarefaction. There are other forms of cell dysfunctions caused by unchecked extracellular proteases. Pharmacological MMP inhibition in the SHR leads to attenuation of receptor cleavage and uniform restoration of cell dysfunctions. These experiments suggest that a pathophysiological mechanism responsible for cell dysfunctions in the SHR is due to an unchecked extracellular protease activity with increased cleavage of the ectodomain of multiple receptors, thus leading to its characteristic co-morbidities.
O6-3 Adhesion of mesenchymal stem cells from flowing blood: Effects of their tissue origin and of interactions with platelets
Asma Alanazia,b, Hafsa Munira, Helen M. McGettricka, N. Thin Luua, Steve P. Watsona and Gerard B. Nasha
a College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
b King Saud bin Abdulaziz University for Health Sciences, Riyadh, KSA
Mesenchymal stem cells (MSC) may have therapeutic uses in several diseases, some involving systemic delivery via injection into the blood. Adhesion from blood flow is then a critical step for their recruitment to injured tissue. However, the recruitment or ‘homing’ of circulating MSC is poorly understood, and may or may not be targeted to affected tissues. To assess the potential for recruitment of MSC from the circulation, MSC suspended in culture medium or added to whole blood were perfused at a range of wall shear rates through glass capillaries coated with different substrates (P-selectin, E-selectin, collagen, fibronectin or albumin). Cells were observed microscopically during perfusion or after washout and stably adherent cells were counted. MSC isolated from different sources (Wharton’s jelly, trabecular bone or bone marrow) were compared. We did not detect stable or intermittent adhesion of any type of isolated MSC to selectins or albumin at wall shear rates down to about 20 s−1. In contrast, all types of MSC adhered to collagen or fibronectin, with number decreasing in the range of shear rates 20–140 s−1. However, when MSC were perfused in whole blood they failed to bind to fibronectin, with the surface becoming covered in a single layer of spread platelets. When perfused over collagen, only MSC from Wharton’s jelly were found to attach, forming aggregates with platelets on the collagen surface. In separate experiments, MSC from both sources adhered efficiently to platelets alone. We hypothesize that MSC show origin-dependent interaction with platelets that may assist or inhibit adhesion to damaged vessels, depending on the matrix exposed and degree of activation of the platelet adhered to the MSC. It is not likely that capture from flow will occur for intact endothelium even if inflamed. Rather, these studies suggest that MSC will bind to frankly damaged endothelium, challenging current paradigms of MSC ‘homing’.
O6-4 Endothelial microparticles released in response to TNF-α vary in miRNA content and physical characteristics: Implications for their role as intercellular communicators
Tamas Alexy, Warren D. Gray, Kimberly Rooney, Martina Weber and Charles D. Searles
Emory University School of Medicine, Atlanta, GA and Atlanta VA Medical Center, Decatur, GA, USA
Endothelial microparticles (EMPs) are known to disrupt vascular homeostasis and contribute to the development and progression of atherosclerosis. New evidence suggests that they may also stimulate endothelial cell (EC) regeneration following injury and exert potent anti-inflammatory effects. The present study was designed to further investigate EMP physiology and explore the pathophysiological mechanisms responsible for the prior conflicting reports. Cultured ECs were treated with TNF-α and with or without inhibitors of distinct MP production pathways for 24 hours. The number, size and surface phosphatidylserine [PS] expression of the MPs was determined by flow cytometry. In addition, scanning electron microscopy was used to confirm their size, Zetasizer to determine surface charge and qRT-PCR to characterize their miRNA content. MPs were stained with Annexin V and calcein-AM and analyzed by flow cytometry. HAECs that were not treated produced MPs that had low Annexin V binding and low calcein-AM conversion, whereas MPs from TNF-α treated HAECs were positive for both calcein conversion and Annexin-V binding. Addition of caspase inhibitor changed the calcein–Annexin profile to resemble MPs from healthy HAECs. MPs were also incubated with otherwise untreated ECs and we assessed apoptosis (Caspase-3 ELISA) as well as the activity of various MP release pathways in these recipient cells. TNF-α induced the release of distinct MP populations through different signaling pathways. MPs produced through the ROCK pathway had significantly higher miRNA content, were smaller, had more positive surface charge and less PS on their outer membrane compared to MPs generated via the caspase pathway. In addition, ROCK-dependent MPs delivered an anti-apoptotic message to recipient cells while miRNA-poor MPs were pro-apoptotic. These findings emphasize the importance of MPs as intercellular communicators and shows that miRNAs can be efficiently transferred by these particles. Our data provide insight into the previously reported conflicting atheroprotective and pro-atherogenic roles of MPs.