A computational study of the effects of adherent leukocytes on secondary recruitment

Leukocyte rolling is known to be mediated by the selectin family of adhesion molecules and their corresponding ligands and is characterized by the formation and breakage of receptor-ligand bonds. Selectin mediated rolling is associated with the initial stages of the leukocyte adhesion cascade (LAC) in which the cell passes through several stages including chemo-attraction, rolling adhesion, tight adhesion and transmigration before moving out of the circulatory system towards the site of injury. This thesis studies the initial stages of the leukocyte adhesion cascade through a direct numerical simulation based on boundary element techniques. Besides, cell deformation during rolling is believed to further enhance rolling interactions. This feature is accounted for by implementing a constitutive model that qualitatively represents the morphology of white blood cells in the early stages of selectin mediated rolling. This research describes the contribution of contact mechanics towards modulation in the contact area for cell substrate interactions. The results predict that compliant cells could roll slower (~ 25%) as compared to their stiffer counterparts. The effect of variations in cell size and bond compression on the decrease in the translational velocity as well as the inherent noisiness in the translational velocity is investigated. The latter part of this thesis attempts to quantify hydrodynamic recruitment of leukocytes with a view to providing insights into cell trafficking in physiological phenomena such as the homing of stem cells towards bone marrow. In vivo experimental data of leukocyte accumulations during exposure to Zymosan-Activated Serum (ZAS) in rabbits and immune response in hamster cheek pouches appears to reinforce this hypothesis. The influence of deformation on the attachment of free-stream cells through collisions and near wall interactions with adherent cells is numerically investigated. The trajectories of free-stream cells colliding with deformed adherent cells are computed through a series of glancing collisions in order to study the influence of cell shape on secondary recruitment

Multi-scale interaction of flow and the artery wall

We discuss, from the perspective of basic science, the physical and biological processes which underlie atherosclerotic (plaque) initiation at the vascular endothelium, identifying their widely separated spatial and temporal scales which participate. We draw on current, related models of vessel wall evolution, paying particular attention to the role of flow, and proceed to propose, then validate (in practical, qualitative terms, at least) a multiply coupled, multi-scale modeling strategy, which, eventually, aims at a quantitative, patient-specific understanding of the coupling between the flow and the endothelial state

Computational Biorheology of Human Blood Flow in Health and Disease

Hematologic disorders arising from infectious diseases, hereditary factors and environmental influences can lead to, and can be influenced by, significant changes in the shape, mechanical and physical properties of red blood cells (RBCs), and the biorheology of blood flow. Hence, modeling of hematologic disorders should take into account the multiphase nature of blood flow, especially in arterioles and capillaries. We present here an overview of a general computational framework based on dissipative particle dynamics (DPD) which has broad applicability in cell biophysics with implications for diagnostics, therapeutics and drug efficacy assessments for a wide variety of human diseases. This computational approach, validated by independent experimental results, is capable of modeling the biorheology of whole blood and its individual components during blood flow so as to investigate cell mechanistic processes in health and disease. DPD is a Lagrangian method that can be derived from systematic coarse-graining of molecular dynamics but can scale efficiently up to arterioles and can also be used to model RBCs down to the spectrin level. We start from experimental measurements of a single RBC to extract the relevant biophysical parameters, using single-cell measurements involving such methods as optical tweezers, atomic force microscopy and micropipette aspiration, and cell-population experiments involving microfluidic devices. We then use these validated RBC models to predict the biorheological behavior of whole blood in healthy or pathological states, and compare the simulations with experimental results involving apparent viscosity and other relevant parameters. While the approach discussed here is sufficiently general to address a broad spectrum of hematologic disorders including certain types of cancer, this paper specifically deals with results obtained using this computational framework for blood flow in malaria and sickle cell anemia.National Institutes of Health (U.S.)Singapore-MIT Alliance for Research and Technology (SMART)United States. Dept. of Energy. Collaboratory on Mathematics for Mesoscopic Modeling of MaterialsUnited States. Dept. of Energy (INCITE Award

Nano-motion Dynamics are Determined by Surface-Tethered Selectin Mechanokinetics and Bond Formation

The interaction of proteins at cellular interfaces is critical for many biological processes, from intercellular signaling to cell adhesion. For example, the selectin family of adhesion receptors plays a critical role in trafficking during inflammation and immunosurveillance. Quantitative measurements of binding rates between surface-constrained proteins elicit insight into how molecular structural details and post-translational modifications contribute to function. However, nano-scale transport effects can obfuscate measurements in experimental assays. We constructed a biophysical simulation of the motion of a rigid microsphere coated with biomolecular adhesion receptors in shearing flow undergoing thermal motion. The simulation enabled in silico investigation of the effects of kinetic force dependence, molecular deformation, grouping adhesion receptors into clusters, surface-constrained bond formation, and nano-scale vertical transport on outputs that directly map to observable motions. Simulations recreated the jerky, discrete stop-and-go motions observed in P-selectin/PSGL-1 microbead assays with physiologic ligand densities. Motion statistics tied detailed simulated motion data to experimentally reported quantities. New deductions about biomolecular function for P-selectin/PSGL-1 interactions were made. Distributing adhesive forces among P-selectin/PSGL-1 molecules closely grouped in clusters was necessary to achieve bond lifetimes observed in microbead assays. Initial, capturing bond formation effectively occurred across the entire molecular contour length. However, subsequent rebinding events were enhanced by the reduced separation distance following the initial capture. The result demonstrates that vertical transport can contribute to an enhancement in the apparent bond formation rate. A detailed analysis of in silico motions prompted the proposition of wobble autocorrelation as an indicator of two-dimensional function. Insight into two-dimensional bond formation gained from flow cell assays might therefore be important to understand processes involving extended cellular interactions, such as immunological synapse formation. A biologically informative in silico system was created with minimal, high-confidence inputs. Incorporating random effects in surface separation through thermal motion enabled new deductions of the effects of surface-constrained biomolecular function. Important molecular information is embedded in the patterns and statistics of motion

Direct simulations of cells motions and deformations in flow

Direct numerical simulations (DNS) are used to study the motions and deformations of blood cells, especially leukocytes, in pressure driven flows in parallel plate channels with both smooth and uneven walls under adhesion force between the leukocytes and the channel wall. Leukocytes are represented by two composite fluid models. The first model is the compound-drop model in which the cytoplasm and the nucleus are modeled as fluids, and the second one is the drop-rigid-particle model in which the cytoplasm is modeled as a fluid and the nucleus as a rigid particle. The adhesion force is computed using two adhesion force models. In the first model, the adhesion force is given by a potential, and in the second model it is given by Dembo\u27s kinetic adhesion model. The numerical code is based on the finite element method and the level-set technique is used to track the cell membrane position. In the absence of the adhesion force, in a pressure driven flow the leukocyte moves away from the wall to an equilibrium location. In presence of the adhesion force, provided it is located within the range of the force, the leukocyte is attracted to the layer of endothelial cells and it flattens under the action of hydrodynamic forces. It is found that for the normal parameter values and flow rates the adhesive force given by the kinetic model is too small to capture the leukocyte. The time at which all bonds are broken and the leukocyte moves away from the wall increases when the capillary number is increased, and decreases with increasing Reynolds number. The former suggests that the adhesion tendency of a leukocyte increases as its cortical tension is reduced. The distance traveled by a leukocyte before all bonds are broken increases with the Reynolds and capillary numbers. The rolling velocity of the leukocyte near an uneven wall varies in the sense that it appears to slip when its lower surface is in the gap between the spheres and stick when it comes close to the spheres\u27 surfaces, which is in qualitative agreement with the experimental data

Interplay of Ethanol and Cholesterol in the Neutrophil Membrane: Effects on Membrane and Cell Mechanics and Neutrophil Adhesion.

Previous work from our lab has demonstrated that ethanol and cholesterol each have significant effects on neutrophil tethering, rolling, and adhesion. Since ethanol and cholesterol are often present within the body simultaneously, we endeavored to characterize their combined effects on neutrophil membrane mechanics and adhesion. The effect of ethanol on membrane cholesterol loading was ascertained by incubating neutrophils with 0.3% ethanol before or after cholesterol manipulation with methyl-beta-cyclodextrin (MβCD) or MβCD/cholesterol complexes. Microcapillary flow chamber assays at a wall shear rate of 100 s-1 were used to examine the effects of ethanol on cholesterol-loaded neutrophils with respect to: (1) collision efficiency and membrane tethering to P-selectin-coated microbeads, (2) rolling on P-selectin-coated surfaces, and (3) primary and secondary interactions with neutrophils preadhered to intercellular adhesion molecule-1 (ICAM-1). We performed flow cytometry experiments to assess the effects of ethanol and cholesterol together on adhesion molecule expression. Microcapillary flow chambers presenting chamber-adherent, non-adhesive and P-selectin-coated 10 μm beads were used to assess neutrophil whole-cell deformation changes with separate ethanol and cholesterol treatment, and varied shear rate. The individual effects of ethanol and cholesterol on membrane fluidity, measured by membrane lipid diffusivity, were determined using fluorescence recovery after photobleaching (FRAP) experiments to obtain lipid diffusion coefficients. We found that membrane cholesterol was increased over control in the absence of ethanol; ethanol pre- or post- loading reduced this increase. Cholesterol enrichment did not alter CD11b expression; however, PSGL-1 and L-selectin expression were lowered by cholesterol enrichment plus or minus ethanol. Ethanol appeared to abrogate the pro-adhesive effects of cholesterol, but it did not impact cholesterol\u27s ability to enhance tethering probability. Ethanol enhanced membrane fluidity of cholesterol-enriched neutrophils as indicated by tethering metrics. Ethanol and cholesterol enrichment both increased neutrophil deformation, while cholesterol depletion decreased neutrophil deformation. Neutrophil deformation increased in a dose-dependent manner with shear rate. Membrane lipid diffusivity was increased by ethanol administration and cholesterol enrichment, and decreased by cholesterol depletion. Our research shows that ethanol and cholesterol interact in a complex manner in the neutrophil membrane, sometimes in concert, and sometimes in conflict, to influence neutrophil adhesion via biomechanical and biochemical effects

Unraveling the vascular fate of deformable circulating tumor cells via a hierarchical computational model

Distant spreading of primary lesions is modulated by the vascular dynamics of circulating tumor cells (CTCs) and their ability to establish metastatic niches. While the mechanisms regulating CTC homing in specific tissues are yet to be elucidated, it is well documented that CTCs possess different size, biological properties and deformability. A computational model is presented to predict the vascular transport and adhesion of CTCs in whole blood. A Lattice-Boltzmann method, which is employed to solve the Navier-Stokes equation for the plasma flow, is coupled with an Immersed Boundary Method. The vascular dynamics of a CTC is assessed in large and small microcapillaries. The CTC shear modulus k ctc is varied returning CTCs that are stiffer, softer and equally deformable as compared to RBCs. In large microcapillaries, soft CTCs behave similarly to RBCs and move away from the vessel walls; whereas rigid CTCs are pushed laterally by the fast moving RBCs and interact with the vessel walls. Three adhesion behaviors are observed, firm adhesion, rolling and crawling over the vessel walls, depending on the CTC stiffness. On the contrary, in small microcapillaries, rigid CTCs are pushed downstream by a compact train of RBCs and cannot establish any firm interaction with the vessel walls; whereas soft CTCs are squeezed between the vessel wall and the RBC train and rapidly establish firm adhesion. These findings document the relevance of cell deformability in CTC vascular adhesion and provide insights on the mechanisms regulating metastasis formation in different vascular districts

Microcirculation and inflammation in a numerical simulation approach

Inflammation is the response of the organism to eradicate the agent of lesion or infection in order to achieve hemostasis. This response requires the migration of specific leukocyte populations from the blood circulation towards the inflamed area. Leukocyte recruitment constitutes a complex cellular process by which leukocytes are first recruited to the endothelial vascular wall of post-capillary venules across which they further extravasate into the interstitial tissue. Recruitment is mediated via cell-cell interactions between the leukocyte and the endothelium and occurs through a multi-step cascade: tethering, rolling, slow rolling, arrest, crawling, adhesion and transmigration. However, whether or not the leukocytes adhere to the endothelium depends not only on the chemical forces generated by adhesion molecules on leukocytes and endothelial cells, but also on the physical forces that act on those cells. It has been suggested that fluid shear stress resulting from blood flow also regulates leukocyte activity which makes the fluid dynamic environment of the circulation to be considered an important aspect for leukocyte recruitment and migration during the inflammatory response. Most of the studies on the inflammatory response and in particular on leukocyte recruitment are based on animal models and involve, among others, the quantification of inflammatory mediators and cellular players, and/or the analysis of the leukocyte-endothelial cell interactions by intravital microscopy. However, the contribution of hemodynamics for leukocyte recruitment has been seldom addressed in those studies. This is mostly due to the fact that the study of hemodynamics in in vivo animal models is not straightforward and moreover, that several hemodynamic parameters cannot be experimentally determined due to technical constraints. In this work, we reasoned that these limitations could be circumvented by the development and use of numerical simulations to describe leukocyte recruitment. Many of the processes, which take place in living organisms, can be expressed as mathematical equations. This applies to leukocyte recruitment, for which scarce numerical models existed before the beginning of this work. Importantly, these mathematical simulations were performed without considering simultaneously all the players in the process, namely the vessel, the blood flow and the leukocytes. Moreover, most of these studies were two dimensional, assumed blood as a Newtonian fluid with constant viscosity and did not take into account in vivo experimental data. Taken this, our major goal with this work was to understand the contribution of hemodynamics to leukocyte recruitment in inflammation. For such purpose, we aimed here at developing numerical simulations that more adequately reproduced this process. For such, we set up animal models of inflammation to obtain the experimental data required for the development of those numerical simulations. Finally, we used these models to investigate the role of hemodynamics in leukocyte recruitment in inflammation. First, we considered the simpler case of a numerical simulation that assumed leukocytes to be rigid spheres and blood, a non-Newtonian fluid. For such, we initially developed an animal model of inflammation in Wistar rats using a lipopolysaccharide (LPS) as an inflammatory agent. Blood samples were collected for determination of TNF-α levels to ensure the triggering of the inflammatory process. Importantly, the number of rolling and adherent leukocytes in post-capillary venules was monitored using an intravital microscopy approach. As expected, our results showed that there is an increase in TNF-α concentrations after 15 minutes of LPS administration and a significant increase in the number of rolling and adherent leukocytes. The recorded intravital microcopy images, along with other recorded parameters, were then used, in collaboration with a group of mathematicians, to develop a numerical model capable of describing leukocyte recruitment in the microcirculation. To evaluate the contribution of hemodynamics, the localized velocity fields and shear stresses on the surface of leukocytes and near the vessel wall contact points have been computed in two discrete situations, namely as a single leukocyte or when a cluster of them are recruited towards the vessel wall. In the first situation, our numerical results showed the presence of one region of maximum shear stress on the surface of the leuko- cyte close to the endothelial wall and of two regions of minimum shear stress on the op- posite side of the cell. The different areas of shear stress observed in the surface of the leukocyte may be important in directing it towards the endothelial wall during an inflammatory response. The identification of a region of maximum shear stress is consistent with the molecular mechanisms that govern leukocyte rolling because it may actually cor- respond to the area that supports the interaction with the endothelium. On the other hand, the relatively lower shear stress regions may correlate with leukocyte surface areas where binding to the endothelium is not occurring at the moment, thus enabling the roll- ing of the cell along the endothelium. It was also observed that the shear stress at the endothelium gets higher as a cluster of leukocytes moves in the main stream. This sug- gests that the presence of a cluster of leukocytes may potentiate leukocyte rolling, as the increase in the shear stress promoted by the recruited leukocytes may support the migra- tion and recruitment of additional cells. Despite closely simulating leukocyte recruitment, our initial numerical simulation consid- ered the simple case of leukocytes as rigid spheres. However, while circulating leukocytes maintain an approximately spherical shape, rolling leukocytes are known to deform. In order to account for the leukocyte deformability changes that occur during its recruit- ment in inflammation, we needed to assess the deformability profile of the leukocytes under flow and therefore, to “directly” observe them regardless of the other blood cells. For such, intravital microscopy was performed in the mouse cremaster of a transgenic mice strain (Lys-EGFP-ki) in which fluorescent neutrophils can be individually tracked. By using PAF as an inflammatory agent, the analysis of the leukocyte-endothelial cell interac- tions showed a continuous increase in the number of rolling and adherent neutrophils up to 4 hours after the introduction of the inflammatory stimuli, thus confirming the devel- opment of an inflammatory response. As the properties of the red blood cells modulate blood flow properties, erythrocyte deformability was also addressed in this model. A con- tinuous decrease of this parameter was observed throughout time. The decrease in the erythrocyte deformability will most probably lead to an increase in the blood viscosity and to the decrease of the blood flow velocity. These conditions should facilitate the mi- gration of leukocytes from the mainstream to the endothelial wall and promote leukocyte slow rolling and adhesion during the inflammatory response. Importantly, in the intravital microcopy images obtained with this latter model, we clearly observed the deformation of neutrophils along the endothelial wall during rolling, as well as the formation of tethers. As such, in these images, leukocyte trajectories were tracked and their velocities and diameters were measured and further applied to the numerical simulations. Using a recent validated mathematical model describing the coupled defor- mation-flow of an individual leukocyte and the respective experimental results, numerical simulations of the recruitment of an individual leukocyte and of two leukocytes under different velocities were performed, considering a constant blood viscosity. The mathe- matical models obtained showed that under conditions of increased velocity the cell movement is accelerated along the endothelial layer, favouring the dissociation of leuko- cyte-endothelium interactions at designated attraction points. These observations lead us to propose that, in order to attain an efficient inflammatory response, the blood flow ve- locity needs so as to decrease to facilitate slow rolling and subsequent adhesion. These results are corroborated by the decrease in the erythrocyte deformability observed in our animal model, which will ultimately have an impact on the blood flow velocity. Our results further showed that in the vicinity of an adherent leukocyte there is an early slight decel- eration of the rolling leukocyte when compared with the case of an individual leukocyte. As such, these observations strongly suggest that the presence of an adherent cell in the vicinity should decrease the velocity of another leukocyte that is being recruited, thus promoting its slow rolling, and contributing to its capture by the endothelial cells. Altogether, our experimental data and numerical simulations support our working hy- pothesis that the hemodynamic properties of the flow and of the cells in circulation should play an essential role in the margination and rolling of the leukocytes to the endo- thelial wall, which in turn will impact the success of the inflammatory response. In partic- ular, our results strongly suggest that changes in hemodynamic conditions, such as de- creased flow velocities and the increase of the shear stress, will contribute to target leu- kocytes to the endothelial wall. Given our results, we propose that any change in the he- modynamic properties will certainly influence the outcome of the inflammatory response. As such, the adherence of the leukocytes to the endothelium should depend not only on the relative magnitude of the chemical forces generated by the interaction of adhesion molecules between leukocytes and endothelial cells, but also on the physical forces that act on the leukocytes. In this respect, our results suggest that alterations in the blood flow, for example in the flow velocity, will occur during an inflammatory process, thus potentiating the recruitment of more leukocytes towards the inflamed area and contrib- uting to a successful inflammatory response. Overall, the numerical simulations allowed us to better understand the contribution of the hemodynamic properties of the flow to the progression of an inflammatory response and to deepen our knowledge on leukocyte recruitment in inflammation. Importantly, our work provided numerical tools that can be used for the subsequent study and modulation of the hemodynamic parameters involved in an inflammatory response. In particular, these numerical simulations will surely enable us, in the near future, to determine or es- timate a large set of parameters which are unlikely to be recoverable by in vivo experi- ments. Moreover, our methods will allow us to analyze how the parameters evolve over time. Altogether our results further reinforce the notion that the improvement and de- velopment of animal models and numerical tools will certainly provide the medical and biological community with useful tools to study leukocyte recruitment in inflammation. By closely reproducing the microcirculation and the inflammatory process, these tools will be critical for a better comprehension of the inflammatory process and of the mecha- nisms underlying a multitude of inflammatory pathological conditions