Francisco J. Tovar-Lopez

A shear gradient–dependent platelet aggregation mechanism drives thrombus formation

Platelet aggregation at sites of vascular injury is essential for hemostasis and arterial thrombosis. It has long been assumed that platelet aggregation and thrombus growth are initiated by soluble agonists generated at sites of vascular injury. By using high-resolution intravital imaging techniques and hydrodynamic analyses, we show that platelet aggregation is primarily driven by changes in blood flow parameters (rheology), with soluble agonists having a secondary role, stabilizing formed aggregates. We find that in response to vascular injury, thrombi initially develop through the progressive stabilization of discoid platelet aggregates. Analysis of blood flow dynamics revealed that discoid platelets preferentially adhere in low-shear zones at the downstream face of forming thrombi, with stabilization of aggregates dependent on the dynamic restructuring of membrane tethers. These findings provide insight into the prothrombotic effects of disturbed blood flow parameters and suggest a fundamental reinterpretation of the mechanisms driving platelet aggregation and thrombus growth.

Dielectrophoretic manipulation and separation of microparticles using curved microelectrodes

This paper presents the development and experimental analysis of a dielectrophoresis (DEP) system, which is used for the manipulation and separation of microparticles in liquid flow. The system is composed of arrays of microelectrodes integrated to a microchannel. Novel curved microelectrodes are symmetrically placed with respect to the centre of the microchannel with a minimum gap of 40 μm. Computational fluid dynamics method is utilised to characterise the DEP field and predict the dynamics of particles. The performance of the system is assessed with microspheres of 1, 5 and 12 μm diameters. When a high‐frequency potential is applied to microelectrodes a spatially varying electric field is induced in the microchannel, which creates the DEP force. Negative‐DEP behaviour is observed with particles being repelled from the microelectrodes. The particles of different dimensions experience different DEP forces and thus settle to separate equilibrium zones across the microchannel. Experiments demonstrate the capability of the system as a field flow fraction tool for sorting microparticles according to their dimensions and dielectric properties.

An Investigation on Platelet Transport during Thrombus Formation at Micro-Scale Stenosis

This paper reports on an investigation of mass transport of blood cells at micro-scale stenosis where local strain-rate micro-gradients trigger platelet aggregation. Using a microfluidic flow focusing platform we investigate the blood flow streams that principally contribute to platelet aggregation under shear micro-gradient conditions. We demonstrate that relatively thin surface streams located at the channel wall are the primary contributor of platelets to the developing aggregate under shear gradient conditions. Furthermore we delineate a role for red blood cell hydrodynamic lift forces in driving enhanced advection of platelets to the stenosis wall and surface of developing aggregates. We show that this novel microfluidic platform can be effectively used to study the role of mass transport phenomena driving platelet recruitment and aggregate formation and believe that this approach will lead to a greater understanding of the mechanisms underlying shear-gradient dependent discoid platelet aggregation in the context of cardiovascular diseases such as acute coronary syndromes and ischemic stroke.

Shear stress mediates exocytosis of functional TRPV4 channels in endothelial cells.

Mechanosensitive ion channels are implicated in the biology of touch, pain, hearing and vascular reactivity; however, the identity of these ion channels and the molecular basis of their activation is poorly understood. We previously found that transient receptor potential vanilloid 4 (TRPV4) is a receptor operated ion channel that is sensitised and activated by mechanical stress. Here, we investigated the effects of mechanical stimulation on TRPV4 localisation and activation in native and recombinant TRPV4-expressing cells. We used a combination of total internal reflection fluorescence microscopy, cell surface biotinylation assay and Ca2+ imaging with laser scanning confocal microscope to show that TRPV4 is expressed in primary vascular endothelial cells and that shear stress sensitises the response of TRPV4 to its agonist, GSK1016790A. The sensitisation was attributed to the recruitment of intracellular pools of TRPV4 to the plasma membrane, through the clathrin and dynamin-mediated exocytosis. The translocation was dependent on ILK/Akt signalling pathway, release of Ca2+ from intracellular stores and we demonstrated that shear stress stimulated phosphorylation of TRPV4 at tyrosine Y110. Our findings implicate calcium-sensitive TRPV4 translocation in the regulation of endothelial responses to mechanical stimulation.

Particle trapping using dielectrophoretically patterned carbon nanotubes.

This study presents the dielectrophoretic (DEP) assembly of multi‐walled carbon nanotubes (MWCNTs) between curved microelectrodes for the purpose of trapping polystyrene microparticles within a microfluidic system. Under normal conditions, polystyrene particles exhibit negative DEP behaviour and are repelled from microelectrodes. Interestingly, the addition of MWCNTs to the system alters this situation in two ways: first, they coat the surface of particles and change their dielectric properties to exhibit positive DEP behaviour; second, the assembled MWCNTs are highly conductive and after the deposition serve as extensions to the microelectrodes. They establish an array of nanoelectrodes that initiates from the edge of microelectrodes and grow along the electric field lines. These nanoelectrodes can effectively trap the MWCNT‐coated particles, since they cover a large portion of the microchannel bottom surface and also create a much stronger electric field than the primary microelectrodes as confirmed by our numerical simulations. We will show that the presence of MWCNT significantly changes performance of the system, which is investigated by trapping sample polystyrene particles with plain, COOH and goat anti‐mouse IgG surfaces.

Size based separation of microparticles using a dielectrophoretic activated system

This work describes the separation of polystyrene microparticles suspended in deionized (DI) water according to their dimensions using a dielectrophoretic (DEP) system. The DEP system utilizes curved microelectrodes integrated into a microfluidic system. Microparticles of 1, 6, and 15 μm are applied to the system and their response to the DEP field is studied at different frequencies of 100, 200, and 20 MHz. The microelectrodes act as a DEP barrier for 15 μm particles and retain them at all frequencies whereas the response of 1 and 6 μm particles depend strongly on the applied frequency. At 100 kHz, both particles are trapped by the microelectrodes. However, at 200 kHz, the 1 μm particles are trapped by the microelectrodes while the 6 μm particles are pushed toward the sidewalls. Finally, at 20 MHz, both particles are pushed toward the sidewalls. The experiments show the tunable performance of the system to sort the microparticles of various dimensions in microfluidic systems.

Examination of the role of transient receptor potential vanilloid type 4 in endothelial responses to shear forces.

Shear stress is the major mechanical force applied on vascular endothelial cells by blood flow, and is a crucial factor in normal vascular physiology and in the development of some vascular pathologies. The exact mechanisms of cellular mechano-transduction in mammalian cells and tissues have not yet been elucidated, but it is known that mechanically sensitive receptors and ion channels play a crucial role. This paper describes the use of a novel and efficient microfluidic device to study mechanically-sensitive receptors and ion channels in vitro, which has three independent channels from which recordings can be made and has a small surface area such that fewer cells are required than for conventional flow chambers. The contoured channels of the device enabled examination of a range of shear stresses in one field of view, which is not possible with parallel plate flow chambers and other previously used devices, where one level of flow-induced shear stress is produced per fixed flow-rate. We exposed bovine aortic endothelial cells to different levels of shear stress, and measured the resulting change in intracellular calcium levels ([Ca(2+)]i) using the fluorescent calcium sensitive dye Fluo-4AM. Shear stress caused an elevation of [Ca(2+)]i that was proportional to the level of shear experienced. The response was temperature dependant such that at lower temperatures more shear stress was required to elicit a given level of calcium signal and the magnitude of influx was reduced. We demonstrated that shear stress-induced elevations in [Ca(2+)]i are largely due to calcium influx through the transient receptor potential vanilloid type 4 ion channel.

Structural and hydrodynamic simulation of an acute stenosis-dependent thrombosis model in mice

Platelet activation under blood flow is thought to be critically dependent on the autologous secretion of soluble platelet agonists (chemical activators) such as ADP and thromboxane. However, recent evidence challenging this model suggests that platelet activation can occur independent of soluble agonist signalling, in response to the mechanical effects of micro-scale shear gradients. A key experimental tool utilized to define the effect of shear gradients on platelet aggregation is the murine intravital microscopy model of platelet thrombosis under conditions of acute controlled arteriolar stenosis. This paper presents a computational structural and hydrodynamic simulation of acute stenotic blood flow in the small bowel mesenteric vessels of mice. Using a homogeneous fluid at low Reynolds number (0.45) we investigated the relationship between the local hydrodynamic strain-rates and the severity of arteriolar stensosis. We conclude that the critical rates of blood flow acceleration and deceleration at sites of artificially induced stenosis (vessel side-wall compression or ligation) are a function of tissue elasticity. By implementing a structural simulation of arteriolar side wall compression, we present a mechanistic model that provides accurate simulations of stenosis in vivo and allows for predictions of the effects on local haemodynamics in the murine small bowel mesenteric thrombosis model.

Dielectrophoresis with 3D microelectrodes fabricated by surface tension assisted lithography

This paper demonstrates the utilization of 3D semispherical shaped microelectrodes for dielectrophoretic manipulation of yeast cells. The semispherical microelectrodes are capable of producing strong electric field gradients, and in turn dielectrophoretic forces across a large area of channel cross‐section. The semispherical shape of microelectrodes avoids the formation of undesired sharp electric fields along the structure and also minimizes the disturbance of the streamlines of nearby passing fluid. The advantage of semispherical microelectrodes over the planar microelectrodes is demonstrated in a series of numerical simulations and proof‐of‐concept experiments aimed toward immobilization of viable yeast cells.

Hydrodynamic directional control of liquid metal droplets within a microfluidic flow focusing system

Here, we investigate the directional control of Galinstan liquid metal droplets when transferring from the high-viscosity glycerol core into the parallel low-viscosity NaOH sheath streams within a flow focusing microfluidic system. In the presence of sufficient flow mismatch between the sheath streams, the droplets are driven toward the higher velocity interface and cross the interface under the influence of surface tension gradient. A minimum flow mismatch of 125 μl/min is required to enable the continuous transfer of droplets toward the desired sheath stream. The response time of droplets, the time required to change the direction of droplet transfer, is governed by the response time of the syringe pump driven microfluidic system and is found to be 3.3 and 8.8 s when increasing and decreasing the flow rate of sheath stream, respectively.