Shear-induced mixing and demixing in aqueous methyl hydroxypropyl cellulose solutions

The influence of shear flow on the phase separation of aqueous methyl hydroxypropyl cellulose solutions was investigated by means of rheoturbidity and online rheo small angle light scattering (SALS) experiments. In semidilute solution shear-induced mixing was observed and the cloud curve was shifted to higher temperatures with increasing shear rate. With higher concentrated solutions, however, shear-induced demixing was found. The shear-induced mixing is interpreted as being a disruption of slightly entangled clusters under the influence of the shear energy. The shear demixing appears in line with the observation with other systems. A characteristic butterfly pattern was observed in rheo-SALS.     

The effects of elongational stress exposure on the activation and aggregation of blood platelets.

Hemodynamic shear is known to stimulate blood and endothelial cells and induce platelet activation. Many studies of shear-induced platelet stimulation have employed rotational viscometers in which secondary flow effects are assumed to be negligible. Shear induced platelet activation occurs at elevated shear rates where secondary flows may contribute a significant percentage of the total hydrodynamic force experienced by the sample. Elongational stress, one component of this secondary flow, has been shown to alter transmembrane ion flux in intact cell and the permeability of synthetic membrane preparations. Elongational flow also occurs in the vasculature at sites of elevated shear stress. Secondary flow components may contribute to platelet activation induced during shear stress application in rotational viscometry. A unique 'constrained convergence' elongational flow chamber was designed and fabricated to study platelet response to elongational stress exposure. The elongational flow chamber was capable of producing an elongation rate of 2.1 s-1 with a corresponding volume averaged shear rate of 58.33 s-1. Significant changes were observed in the total platelet volume distribution and measured response to added chemical antagonists after elongational stress exposure. The total platelet volume histogram shifted toward larger particle sizes, suggesting the formation of large aggregates as a result of elongational stress exposure. Platelets exposed to elongational stress demonstrated a dose dependent decrease in added ADP-induced aggregation rate and extent of aggregation.     

Visualizing protein motion in Couette flow by all-atom molecular dynamics

In living cells, biomacromolecules are exposed to a highly crowded environment. The cytoplasm, the nucleus, and other organelles are highly viscous fluids that differ from dilute in vitro conditions. Viscosity, a measure of fluid internal friction, directly affects the forces that act on immersed macromolecules. Although active motion of this viscous fluid – cytoplasmic streaming – occurs in many plant and animal cells, the effect of fluid motion (flow) on biomolecules is rarely discussed. Recently NMR experiments that apply a shearing flow in situ have been used for protein studies. While these NMR experiments have succeeded in spectroscopically tracking protein aggregation in real time, they do not provide a visual picture of protein motion under shear. To fill this gap, here we have used molecular dynamics simulations to study the motion of three proteins of different size and shape in a simple shearing flow. The proteins exhibit a superposition of random diffusion and shear-flow-induced rotational motion. Random rotational diffusion dominates at lower shear stresses, whereas an active “rolling motion” along the axis of the applied flow occurs at higher shear stress. Even larger shear stresses perturb protein secondary structure elements resulting in local and global unfolding. Apart from shear-induced unfolding, our results imply that, in an ideal Couette flow field biomolecules undergo correlated motion, which should enhance the probability of inter-molecular interaction and aggregation. Connecting biomolecular simulation with experiments applying shear flow in situ appears to be a promising strategy to study protein alignment, deformation, and dynamics under shear.     

Beta-lactoglobulin fibers under capillary flow

We describe the capillary flow behavior of gels of beta-lactoglobulin (beta-lg) containing droplets of fibrils and the shear flow alignment of beta-lg fibers in dilute aqueous solutions. Polarized optical microscopy and laser scanning confocal microscopy are used to show that capillary shear flow does not affect the fibril droplet sizes in the beta-lg gels, the system behaving in this respect as a solution of compact colloidal particles under shear flow. Small-angle X-ray scattering (SAXS) on dilute aqueous solutions indicates that the fibers can be initially aligned under capillary shear, but this alignment is lost after 18 min of shear. Transmission electron microscopy experiments on the samples studied by SAXS suggest that the loss of orientation is due to a shear-induced breakup of the swollen fibril network. Dynamic and static light scattering on dilute beta-lg fibril aqueous solutions are used to show that before shear beta-lg fibrils behave as strongly interacting semiflexible polymers, while they behave as weakly interacting rods after 18 min of capillary shear.     

Shear-Induced Unfolding of Lysozyme Monitored In Situ

Conformational changes due to externally applied physiochemical parameters, including pH, temperature, solvent composition, and mechanical forces, have been extensively reported for numerous proteins. However, investigations on the effect of fluid shear flow on protein conformation remain inconclusive despite its importance not only in the research of protein dynamics but also for biotechnology applications where processes such as pumping, filtration, and mixing may expose protein solutions to changes in protein structure. By combining particle image velocimetry and Raman spectroscopy, we have successfully monitored reversible, shear-induced structural changes of lysozyme in well-characterized flows. Shearing of lysozyme in water altered the protein's backbone structure, whereas similar shear rates in glycerol solution affected the solvent exposure of side-chain residues located toward the exterior of the lysozyme α-domain. The results demonstrate the importance of measuring conformational changes in situ and of quantifying fluid stresses by the three-dimensional shear tensor to establish reversible unfolding or misfolding transitions occurring due to flow exposure.     

Phase separation in a sheared gelatin/maltodextrin mixture studied by small-angle light scattering

The influence of shear on the structure of a gelatin/maltodextrin mixture was investigated using small-angle light scattering both during phase separation and after phase separation was allowed to occur quiescently. In all cases, phase separation occurred via spinodal decomposition to form a droplet morphology, and a characteristic length scale was formed in the structure that was prevalent during shear, as well as in quiescent conditions. Below the critical shear rate for droplet breakup, shear accelerated the coarsening rate of the droplets. A transient regime of rapid hydrodynamic coarsening was present when shear was initiated after phase separation and at late times in all cases once the droplets attained a certain size. At the critical shear rate for droplet breakup (1 s(-1)), the rapid repetition of breakup and coarsening was postulated to occur, which enabled a microstructure consisting of elongated droplets with a narrow size distribution to form. When the shear rate enabled droplets to extend to such an extent that a percolated structure could form (10 s(-1)), then the structure was relatively stable and changed very slowly over time. At very high shear rates (100 s(-1)), droplet breakup was suppressed and a highly fibrillar morphology formed that was stable only while the system was under shear. Cessation of shear at high rates led to fiber breakup and the formation of many small droplets. For a given shear rate, the final microstructure appeared to be independent of the time that shear was started when the structure consisted of discrete droplets or fibers. When a percolated structure could form, however, the shear history appeared to be important.     

Biorheological views of endothelial cell responses to mechanical stimuli

Vascular endothelial cells are located at the innermost layer of the blood vessel wall and are always exposed to three different mechanical forces: shear stress due to blood flow, hydrostatic pressure due to blood pressure and cyclic stretch due to vessel deformation. It is well known that endothelial cells respond to these mechanical forces and change their shapes, cytoskeletal structures and functions. In this review, we would like to mainly focus on the effects of shear stress and hydrostatic pressure on endothelial cell morphology. After applying fluid shear stress, cultured endothelial cells show marked elongation and orientation in the flow direction. In addition, thick stress fibers of actin filaments appear and align along the cell long axis. Thus, endothelial cell morphology is closely related to the cytoskeletal structure. Further, the dynamic course of the morphological changes is shown and the related events such as changes in mechanical stiffness and functions are also summarized. When endothelial cells were exposed to hydrostatic pressure, they exhibited a marked elongation and orientation in a random direction, together with development of centrally located, thick stress fibers. Pressured endothelial cells also exhibited a multilayered structure with less expression of VE-cadherin unlike under control conditions. Simultaneous loading of hydrostatic pressure and shear stress inhibited endothelial cell multilayering and induced elongation and orientation of endothelial cells with well-developed VE-cadherin in a monolayer, which suggests that for a better understanding of vascular endothelial cell responses one has to take into consideration the combination of the different mechanical forces such as exist under in vivo mechanical conditions.     

Enhanced concanavalin a agglutination of trypsinised erythrocytes is due to a specific class of aggregation

Using the output of a rotational viscometer as a continuous index of aggregation, we have shown previously that the concanavalin A agglutination of native human erythrocytes can be resolved into three distinct classes of aggregation, static, type I and type II. Static aggregation occurs in the absence of shear forces while both type I and II aggregations are shear-induced. We now report that the increased concanavalin A agglutination of trypsinised erythrocytes is attributable to a specific enhancement in the development of type II aggregation. While type II formation in native cell suspensions requires high concanavalin A concentrations and continual shearing, an indistinguishable type of aggregation develops in suspensions of trypsinised red cells at considerably lower lectin concentrations and in the absence of applied shear forces.     

Von Willlebrand Adhesion to Surfaces at High Shear Rates Is Controlled by Long-Lived Bonds

Von Willebrand factor (vWF) adsorbs and immobilizes platelets at sites of injury under high-shear-rate conditions. It has been recently demonstrated that single vWF molecules only adsorb significantly to collagen above a threshold shear, and here we explain such counterintuitive behavior using a coarse-grained simulation and a phenomenological theory. We find that shear-induced adsorption only occurs if the vWF-surface bonds are slip-resistant such that force-induced unbinding is suppressed, which occurs in many biological bonds (i.e., catch bonds). Our results quantitatively match experimental observations and may be important to understand the activation and mechanical regulation of vWF activity during blood clotting.     

Von Willlebrand Adhesion to Surfaces at High Shear Rates Is Controlled by?Long-Lived Bonds

Von Willebrand factor (vWF) adsorbs and immobilizes platelets at sites of injury under high-shear-rate conditions. It has been recently demonstrated that single vWF molecules only adsorb significantly to collagen above a threshold shear, and here we explain such counterintuitive behavior using a coarse-grained simulation and a phenomenological theory. We find that shear-induced adsorption only occurs if the vWF-surface bonds are slip-resistant such that force-induced unbinding is suppressed, which occurs in many biological bonds (i.e., catch bonds). Our results quantitatively match experimental observations and may be important to understand the activation and mechanical regulation of vWF activity during blood clotting.     

Mechanical degradation of polyacrylamide solutions as a model for flow induced blood damage in artificial organs

Human or animal blood is normally used as a test fluid for the in vitro evaluation of hemolysis by artificial organs. However, blood has some disadvantages (large biological variability and problems with cleaning the devices). For that reason, we searched for a reproducible technical fluid with blood-like flow characteristics that exhibits similar shear depending destruction. In this study, a direct comparison between erythrocyte damage of bovine blood and shear-induced degradation of polyacrylamide solution is given. A uniform shear field was applied to the fluids using a shear device with a plate-plate geometry. It was shown that similarities exist between erythrocytes disaggregation and breakdown of super molecular structures in polymer solutions, caused by mechanical stress. In both cases steady low shear viscositity was diminished and the elastic component of complex viscosity of blood and polymer solutions has been reduced. There is a correlation between shear-induced hemolysis of bovine blood and mechanical polymer-degradation, which depends on the applied shear stresses.     

The unfolding of the P pili quaternary structure by stretching is reversible, not plastic

P pili are protein filaments expressed by uropathogenic Escherichia coli that mediate binding to glycolipids on epithelial cell surfaces, which is a prerequisite for bacterial infection. When a bacterium, attached to a cell surface, is exposed to external forces, the pili, which are composed of approximately 10(3) PapA protein subunits arranged in a helical conformation, can elongate by unfolding to a linear conformation. This property is considered important for the ability of a bacterium to withstand shear forces caused by urine flow. It has hitherto been assumed that this elongation is plastic, thus constituting a permanent conformational deformation. We demonstrate, using optical tweezers, that this is not the case; the unfolding of the helical structure to a linear conformation is fully reversible. It is surmised that this reversibility helps the bacteria regain close contact to the host cells after exposure to significant shear forces, which is believed to facilitate their colonization.     

Importance of intrinsic properties of dense caseinate dispersions for structure formation

Rheological measurements of dense calcium caseinate and sodium caseinate dispersions (> or =15%) provided insight into the factors determining shear-induced structure formation in caseinates. Calcium caseinate at a sufficiently high concentration (30%) was shown to form highly anisotropic structures during shearing and concurrent enzymatic cross-linking. In contrast, sodium caseinate formed isotropic structures using similar processing conditions. The main difference between the two types of caseinates is the counterion present, and as a consequence, the size of structural elements and their interactions. The rheological behavior of calcium caseinate and sodium caseinate reflected these differences, yielding non-monotonic and shear thinning flow behavior for calcium caseinate whereas sodium caseinate behaved only slightly shear thinning. It appears that the intrinsic properties of the dense caseinate dispersions, which are reflected in their rheological behavior, affect the structure formation that was found after applying shear. Therefore, rheological measurements are useful to obtain an indication of the structure formation potential of caseinate dispersions.     

The shear stress of it all: the cell membrane and mechanochemical transduction

As the inner lining of the vessel wall, vascular endothelial cells are poised to act as a signal transduction interface between haemodynamic forces and the underlying vascular smooth-muscle cells. Detailed analyses of fluid mechanics in atherosclerosis-susceptible regions of the vasculature reveal a strong correlation between endothelial cell dysfunction and areas of low mean shear stress and oscillatory flow with flow recirculation. Conversely, steady shear stress stimulates cellular responses that are essential for endothelial cell function and are atheroprotective. The molecular basis of shear-induced mechanochemical signal transduction and the endothelium's ability to discriminate between flow profiles remains largely unclear. Given that fluid shear stress does not involve a traditional receptor/ligand interaction, identification of the molecule(s) responsible for sensing fluid flow and mechanical force discrimination has been difficult. This review will provide an overview of the haemodynamic forces experienced by the vascular endothelium and its role in localizing atherosclerotic lesions within specific regions of the vasculature. Also reviewed are several recent lines of evidence suggesting that both changes in membrane microviscosity linked to heterotrimeric G proteins, and the transmission of tension across the cell membrane to the cell-cell junction where known shear-sensitive proteins are localized, may serve as the primary force-sensing elements of the cell.     

Integrin mechanotransduction stimulates caveolin-1 phosphorylation and recruitment of Csk to mediate actin reorganization

To identify the role of caveolin-1 in integrin mechanotransduction, we exposed bovine aortic endothelial cells to 10 dyn/cm2 of laminar shear stress. Caveolin-1 was acutely and transiently phosphorylated with shear, occurring downstream of beta1-integrin activation as the beta1-integrin blocking antibody JB1A was inhibitory. In manipulating Src family kinase (SFK) activity with knockdown of Csk or type 1 protein phosphatase (PP1) treatment, we observed coordinate increase and decrease in shear-induced caveolin-1 phosphorylation, respectively. Hence, shear-stimulated caveolin-1 phosphorylation is regulated by SFKs. Shear-induced recruitment and phosphorylation of caveolin-1 occurred at beta1-integrin sites in a beta1-integrin- and SFK-dependent manner. Csk, described to interact with pY14-caveolin-1 and integrins, bound to an increased pool of phosphorylated caveolin-1 after shear corresponding with elevated Csk at beta1-integrin sites. Like caveolin-1, treatment with JB1A and PP1 attenuated shear-induced Csk association with beta1-integrins. Csk function was assayed with transfection of a caveolin-1 phosphorylation domain peptide. The peptide attenuated shear-induced association of Csk at beta1-integrin sites, as well as colocalization of Csk with paxillin and phosphorylated caveolin-1. Because integrin and Csk activity regulate cytoskeletal reorganization, we evaluated the role of this mechanism in shear-induced myosin light chain (MLC) phosphorylation. Knockdown of Csk expression was sufficient to reduce MLC diphosphorylation due to shear. Disruption of Csk-integrin association by peptide treatment was also inhibitory of the MLC diphosphorylation response. Together these data indicate that integrin activation with shear stress results in SFK-regulated caveolin-1 phosphorylation that, in turn, mediates Csk association at integrin sites, where it plays a role in downstream, shear-stimulated MLC diphosphorylation.     

Correspondence of low mean shear and high harmonic content in the porcine iliac arteries

BACKGROUND: Temporal variations in shear stress have been suggested to affect endothelial cell biology. To better quantify the range of dynamic shear forces that occur in vivo, the frequency content of shear variations that occur naturally over a cardiac cycle in the iliac arteries was determined. METHOD OF APPROACH: Computational fluid dynamic calculations were performed in six iliac arteries from three juvenile swine. Fourier analysis of the time-varying shear stress computed at the arterial wall was performed to determine the prevalence of shear forces occurring at higher frequencies in these arteries. RESULTS: While most of each artery experienced shear forces predominantly at the frequency of the heart rate, the frequency spectra at certain regions were dominated by shear forces at higher frequencies. Regions whose frequency spectra were dominated by higher harmonics generally experienced lower mean shear stress. The negative correlation between shear and dominant harmonic was significant (p=0.002). CONCLUSIONS: Since lesion development typically occurs in regions experiencing low time-average shear stress, this result suggests that the frequency content of the shear exposure may also be a contributing factor in lesion development. A better understanding of the vascular response to shear components of different frequencies might help rationalize the notion of "disturbed flow" as a hemodynamic entity.     

Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression

Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm² in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (^·OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H₂O₂), ^·OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H₂O₂ was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow. J. Cell. Physiol. 175:156–162, 1998. © 1998 Wiley-Liss, Inc.     

Experimental measurement of dynamic fluid shear stress on the aortic surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. Although exact causes and mechanisms of AV calcification are unclear, previous studies suggest that mechanical forces play a role. Since calcium deposits occur almost exclusively on the aortic surfaces of AV leaflets, it has been hypothesized that adverse patterns of fluid shear stress on the aortic surface of AV leaflets promote calcification. The current study characterizes AV leaflet aortic surface fluid shear stresses using Laser Doppler velocimetry and an in vitro pulsatile flow loop. The valve model used was a native porcine valve mounted on a suturing ring and preserved using 0.15% glutaraldehyde solution. This valve model was inserted in a mounting chamber with sinus geometries, which is made of clear acrylic to provide optical access for measurements. To understand the effects of hemodynamics on fluid shear stress, shear stress was measured across a range of conditions: varying stroke volumes at the same heart rate and varying heart rates at the same stroke volume. Systolic shear stress magnitude was found to be much higher than diastolic shear stress magnitude due to the stronger flow in the sinuses during systole, reaching up to 20 dyn/cm² at mid-systole. Upon increasing stroke volume, fluid shear stresses increased due to stronger sinus fluid motion. Upon increasing heart rate, fluid shear stresses decreased due to reduced systolic duration that restricted the formation of strong sinus flow. Significant changes in the shear stress waveform were observed at 90 beats/min, most likely due to altered leaflet dynamics at this higher heart rate. Overall, this study represents the most well-resolved shear stress measurements to date across a range of conditions on the aortic side of the AV. The data presented can be used for further investigation to understand AV biological response to shear stresses.     

Experimental measurement of dynamic fluid shear stress on the ventricular surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. The exact causes and mechanisms of AV calcification are unclear, although previous studies suggest that mechanical forces play a role. It has been clinically demonstrated that calcification preferentially occurs on the aortic surface of the AV. This is hypothesized to be due to differences in the mechanical environments on the two sides of the valve. It is thus necessary to characterize fluid shear forces acting on both sides of the leaflet to test this hypothesis. The current study is one of two studies characterizing dynamic shear stress on both sides of the AV leaflets. In the current study, shear stresses on the ventricular surface of the AV leaflets were measured experimentally on two prosthetic AV models with transparent leaflets in an in vitro pulsatile flow loop using two-component Laser Doppler Velocimetry (LDV). Experimental measurements were utilized to validate a theoretical model of AV ventricular surface shear stress based on the Womersley profile in a straight tube, with corrections for the opening angle of the valve leaflets. This theoretical model was applied to in vivo data based on MRI-derived volumetric flow rates and valve dimension obtained from the literature. Experimental results showed that ventricular surface shear stress was dominated by the streamwise component. The systolic shear stress waveform resembled a half-sinusoid during systole and peaks at 64–71 dyn/cm², and reversed in direction at the end of systole for 15–25?ms, and reached a significant negative magnitude of 40–51 dyn/cm². Shear stresses from the theoretical model applied to in vivo data showed that shear stresses peaked at 77–92 dyn/cm² and reversed in direction for substantial period of time (108–110?ms) during late systole with peak negative shear stress of 35–38 dyn/cm².