Mechano-sensing and cell migration: a 3D model approach.

Cell migration is essential for tissue development in different physiological and pathological conditions. It is a complex process orchestrated by chemistry, biological factors, microstructure and surrounding mechanical properties. Focusing on the mechanical interactions, cells do not only exert forces on the matrix that surrounds them, but they also sense and react to mechanical cues in a process called mechano-sensing. Here, we hypothesize the involvement of mechano-sensing in the regulation of directional cell migration through a three-dimensional (3D) matrix. For this purpose, we develop a 3D numerical model of individual cell migration, which incorporates the mechano-sensing process of the cell as the main mechanism regulating its movement. Consistent with this hypothesis, we found that factors, such as substrate stiffness, boundary conditions and external forces, regulate specific and distinct cell movements.

Effective pulmonary ventilation with small-volume oscillations at high frequency.

At high oscillation frequencies (4 to 30 hertz), effective alveolar ventilation can be achieved with tidal volumes much smaller than the anatomic dead space. An explanation of this phenomenon is given in terms of the combined effects of diffusion and convection and in terms of data consistent with the hypothesis. Theory and experimental results both show that the significant variable determining the effectiveness of gas exchange is the amplitude of the oscillatory flow rate independent of the individual values of frequency and stroke volume.

The effects of monocytes on tumor cell extravasation in a 3D vascularized microfluidic model.

Metastasis is the leading cause of cancer-related deaths. Recent developments in cancer immunotherapy have shown exciting therapeutic promise for metastatic patients. While most therapies target T cells, other immune cells, such as monocytes, hold great promise for therapeutic intervention. In our study, we provide primary evidence of direct engagement between human monocytes and tumor cells in a 3D vascularized microfluidic model. We first characterize the novel application of our model to investigate and visualize at high resolution the evolution of monocytes as they migrate from the intravascular to the extravascular micro-environment. We also demonstrate their differentiation into macrophages in our all-human model. Our model replicates physiological differences between different monocyte subsets. In particular, we report that inflammatory, but not patrolling, monocytes rely on actomyosin based motility. Finally, we exploit this platform to study the effect of monocytes, at different stages of their life cycle, on cancer cell extravasation. Our data demonstrates that monocytes can directly reduce cancer cell extravasation in a non-contact dependent manner. In contrast, we see little effect of monocytes on cancer cell extravasation once monocytes transmigrate through the vasculature and are macrophage-like. Taken together, our study brings novel insight into the role of monocytes in cancer cell extravasation, which is an important step in the metastatic cascade. These findings establish our microfluidic platform as a powerful tool to investigate the characteristics and function of monocytes and monocyte-derived macrophages in normal and diseased states. We propose that monocyte-cancer cell interactions could be targeted to potentiate the anti-metastatic effect we observe in vitro, possibly expanding the milieu of immunotherapies available to tame metastasis.

Molecular dynamics study of talin-vinculin binding.

Cells can sense mechanical force in regulating focal adhesion assembly. One vivid example is the force-induced recruitment of vinculin to reinforce initial contacts between a cell and the extracellular matrix. Crystal structures of the unbound proteins and bound complex between the vinculin head subdomain (Vh1) and the talin vinculin binding site 1 (VBS1) indicate that vinculin undergoes a conformational change upon binding to talin. However, the molecular basis for this event and the precise nature of the binding pathway remain elusive. In this article, molecular dynamics is used to investigate the binding mechanism of Vh1 and VBS1 under minimal constraints to facilitate binding. One simulation demonstrates binding of the two molecules in the complete absence of external force. VBS1 makes early hydrophobic contact with Vh1 by positioning the critical hydrophobic residues (L608, L615, and L622) in the groove formed by helices 1 and 2 of Vh1. The solvent-exposed hydrophobic residues (V619 and L623) then gradually penetrate the hydrophobic core of Vh1, thus further separating helix 1 from helix 2. These critical residues are highly conserved as large hydrophobic side groups in other vinculin binding sites; studies also have demonstrated that these residues are essential in Vh1-VBS1 binding. Similar binding mechanisms are also demonstrated in separate molecular dynamics simulations of Vh1 binding to other vinculin binding sites both in talin and alpha-actinin.

Effects of frequency, tidal volume, and lung volume on CO2 elimination in dogs by high frequency (2-30 Hz), low tidal volume ventilation.

Recent studies have shown that effective pulmonary ventilation is possible with tidal volumes (VT) less than the anatomic dead-space if the oscillatory frequency (f) is sufficiently large. We systematically studied the effect on pulmonary CO2 elimination (VCO2) of varying f (2-30 Hz) and VT (1-7 ml/kg) as well as lung volume (VL) in 13 anesthetized, paralyzed dogs in order to examine the contribution of those variables that are thought to be important in determining gas exchange by high frequency ventilation. All experiments were performed when the alveolar PCO2 was 40 +/- 1.5 mm Hg. In all studies, VCO2 increased monotonically with f at constant VT. We quantitated the effects of f and VT on VCO2 by using the dimensionless equation VCO2/VOSC = a(VT/VTo)b(f/fo)c where: VOSC = f X VT, VTo = mean VT, fo = mean f and a, b, c, are constants obtained by multiple regression. The mean values of a, b, and c for all dogs were 2.12 X 10(-3), 0.49, and 0.08, respectively. The most important variable in determining VCO2 was VOSC; however, there was considerable variability among dogs in the independent effect of VT and f on VCO2, with a doubling of VT at a constant VOSC causing changes in VCO2 ranging from -13 to +110% (mean = +35%). Increasing VL from functional residual capacity (FRC) to the lung volume at an airway opening minus body surface pressure of 25 cm H2O had no significant effect on VCO2.

The stiffness of three-dimensional ionic self-assembling peptide gels affects the extent of capillary-like network formation.

Improving our ability to control capillary morphogenesis has implications for not only better understanding of basic biology, but also for applications in tissue engineering and in vitro testing. Numerous biomaterials have been investigated as cellular supports for these applications and the biophysical environment biomaterials provide to cells has been increasingly recognized as an important factor in directing cell function. Here, the ability of ionic self-assembling peptide gels to support capillary morphogenesis and the effect of their mechanical properties is investigated. When placed in a physiological salt solution, these oligopeptides spontaneously self-assemble into gels with an extracellular matrix (ECM)-like microarchitecture. To evaluate the ability of three-dimensional (3D) self-assembled peptide gels to support capillary-like network formation, human umbilical vein endothelial cells (HUVECs) were embedded within RAD16-I ((RADA)4) or RAD16-II ((RARADADA)2) peptide gels with various stiffness values. As peptide stiffness is decreased cells show increased elongation and are increasingly able to contract gels. The observation that capillary morphogenesis is favored in more malleable substrates is consistent with previous reports using natural biomaterials. The structural properties of peptide gels and their ability to support capillary morphogenesis in vitro make them promising biomaterials to investigate for numerous biomedical applications.

PO-12 - The key role of talin-1 in cancer cell extravasation dissected through human vascularized 3D microfluidic model.

INTRODUCTION: Metastases are responsible for more than 90% of cancer related mortality. The hematogenous metastatic invasion is a complex process in which the endothelium plays a key role. Extravasation is a dynamic process involving remodeling and change in cell shape and in cytoskeleton whereby a series of strongly dependent interactions between CTCs and endothelium occurs [1]. Talins are proteins regulating focal adhesions and cytoskeleton remodeling. Talin-1 seems to be involved in the aggressiveness, motility, survival and invadopodia formation of cancer cells throughout the entire metastatic cascade [2], being up-regulated in breast cancer cells and mutated in sarcomas. Understand the implication of talin-1 in extravasation could facilitate the design of new therapies and finally fight cancer. AIM: We hypothesized that Talin-1 could be specifically involved in extravasation driving each of its steps. MATERIALS AND METHODS: We developed a human 3D microfluidic model that enables the study of human cancer cell extravasation within a perfusable human microvascularized organ specific environment[3]. For the study of extravasation we applied microfluidic approach through the development of a microfluidic device in which endothelial cells and fibroblasts generated a 3D human functional vascular networks. Microvessel characterization was performed with immunofluorescence and permeability assays. We knocked-down talin-1 in triple negative breast cancer cell line MDA-MB231 and metastatic fibro-sarcoma cell line HT1080 with SiRNA and verified by Western-blot. Cancer cells were then perfused in the vessels and extravasation monitored through confocal imaging. RESULTS: We developed a human vascularized 3D microfluidic device with human perfusable capillary-like structures embedded in fibrin matrix, characterized by mature endothelium markers and physiological permeability (1.5±0.76)×10(-6) cm/s. We focused on the role of Talin-1 in adhesion to endothelium, trans-endothelial migration (TEM) and early invasion. Adhesion to the endothelium, TEM and migration within the ECM were monitored through confocal analyses. We demonstrated that Talin-1 KD significantly reduced the adhesion efficiency and TEM in both cell lines. Early invasion was also strongly and statistically reduced by the SiRNA treatment in both cell lines. CONCLUSIONS: We proved Talin-1 function in driving the extravasation mechanism in a human 3D vascularized environment. We demonstrated that Talin-1 is involved in each part of extravasation significantly affecting adhesion, TEM and the invasion stages. Targeting this protein could thus be an effective strategy to block metastasis.

The impact of calcification on the biomechanical stability of atherosclerotic plaques.

BACKGROUND: Increased biomechanical stresses in the fibrous cap of atherosclerotic plaques contribute to plaque rupture and, consequently, to thrombosis and myocardial infarction. Thin fibrous caps and large lipid pools are important determinants of increased plaque stresses. Although coronary calcification is associated with worse cardiovascular prognosis, the relationship between atheroma calcification and stresses is incompletely described. METHODS AND RESULTS: To test the hypothesis that calcification impacts biomechanical stresses in human atherosclerotic lesions, we studied 20 human coronary lesions with techniques that have previously been shown to predict plaque rupture locations accurately. Ten ruptured and 10 stable lesions derived from post mortem coronary arteries were studied using large-strain finite element analysis. Maximum stress was not correlated with percentage of calcification, but it was positively correlated with the percentage of lipid (P:=0.024). When calcification was eliminated and replaced with fibrous plaque, stress changed insignificantly; the median increase in stress for all specimens was 0.1% (range, 0% to 8%; P:=0.85). In contrast, stress decreased by a median of 26% (range, 1% to 78%; P:=0.02) when lipid was replaced with fibrous plaque. CONCLUSIONS: Calcification does not increase fibrous cap stress in typical ruptured or stable human coronary atherosclerotic lesions. In contrast to lipid pools, which dramatically increase stresses, calcification does not seem to decrease the mechanical stability of the coronary atheroma.

Vascular mechanics for the cardiologist.

Many common problems in clinical cardiology are due to disturbances in vascular mechanics. The terminology and basic principles of vascular mechanics, including fundamentals of the relation of stress and strain, are described in this review. Approaches to measuring vessel wall stiffness and the mechanical basis for vascular catastrophes are introduced.

Exploring the molecular basis for mechanosensation, signal transduction, and cytoskeletal remodeling.

Living cells respond to mechanical stimulation in a variety of ways that affect nearly every aspect of their function. Such responses can range from changes in cell morphology to activation of signaling cascades and changes in cell phenotype. Although the biochemical signaling pathways activated by mechanical stimulus have been extensively studied, little is known of the basic mechanisms by which mechanical force is transduced into a biochemical signal, or how the cell changes its behavior or properties in response to external or internal stresses. One hypothesis is that forces transmitted via individual proteins either at the site of cell adhesion to its surroundings or within the stress-bearing members of the cytoskeleton cause conformational changes that alter their binding affinity to other intracellular molecules. This altered equilibrium state can subsequently either initiate a biochemical signaling cascade or produce more immediate and local structural changes. To understand the phenomena related to mechanotransduction, the mechanics and chemistry of single molecules that form the signal transduction pathways must be examined. This paper presents a range of case studies that seek to explore the molecular basis of mechanical signal sensation and transduction, with particular attention to their macroscopic manifestation in the cell properties, e.g. in focal adhesion remodeling due to local application of force or changes in cytoskeletal rheology and remodeling due to cellular deformation.

Using microfluidics to investigate tumor cell extravasation and T-cell immunotherapies.

Understanding the mechanism of tumor cell extravasation, cell migration and the role of the immunosystem is crucial in creating targeted and patient-specific cancer therapies. We created an in-vitro microfluidic cell extravasation assay, incorporating a microvascular network and demonstrated its use to study cancer cells extravasation. Separately, we developed an assay for screening T-cell migration and cytotoxicity as a means to evaluate the efficiency of adoptive immunotherapies against cancer. Similar devices using a similar platform can be used to recreate a tumor liver microenvironment, taking in consideration the hypoxic and inflammatory conditions in the liver. These platforms show considerable potential as efficient pre-clinical models for testing the efficiency of cancer drugs and engineered T-cell functionality for personalized medicine.

Modeling the Blood-Brain Barrier in a 3D triple co-culture microfluidic system.

The need for a blood-brain barrier (BBB) model that accurately mimics the physiological characteristics of the in-vivo situation is well-recognized by researchers in academia and industry. However, there is currently no in-vitro model allowing studies of neuronal growth and/or function influenced by factors from the blood that cross through the BBB. Therefore, we established a 3D triple co-culture microfluidic system using human umbilical vein endothelial cells (HUVEC) together with primary rat astrocytes and neurons. Immunostaining confirmed the successful triple co-culture system consisting of an intact BBB with tight intercellular junctions in the endothelial monolayer. The BBB selective permeability was determined by a fluorescent-based assay using dextrans of different molecular weights. Finally, neuron functionality was demonstrated by calcium imaging.

The role of Schlemm's canal in aqueous outflow from the human eye.

A mathematical model of Schlemm's canal is developed to simulate collapse of the canal and its effect on resistance to flow through the aqueous outflow network. Schlemm's canal is modeled as a porous, compliant channel that is held open by the trabecular meshwork. The trabecular meshwork is modeled as a series of linear springs that allow the inner wall of Schlemm's canal to deform in proportion to the local pressure drop across it. Based on comparisons between the model and results in the literature, the following tentative conclusions are reached: (1) Most of the resistance in the aqueous outflow network occurs in the inner wall of Schlemm's canal. (2) Glaucoma is not caused by a weakening of the trabecular meshwork and a resultant collapse of Schlemm's canal alone. Instead, glaucoma probably results from an increased flow resistance of the inner wall of the canal.

Measurements of the compressive properties of scleral tissue.

Tests were conducted on small samples of sclera taken from cattle eyes and human eyes to determine the mechanical properties of the tissue in compression. It was found that the elastic modulus for radial compressive stress of human sclera was more than a factor of 100 less than the modulus for circumferential tensile stress. The stress-strain relationship was found to be mildly nonlinear and exhibited hysteresis when cycled through a pattern of increasing-then-decreasing stress. Given sufficient time, however, the sample returned to its original state and, therefore, exhibited no permanent deformation. Measurements of Poisson's ratio yielded values of 0.46 to 0.50, indicating that these samples were essentially incompressible.

Modulation of outflow resistance by the pores of the inner wall endothelium.

The juxtacanalicular connective tissue (JCT) is widely believed to generate the bulk of aqueous humor outflow resistance, while the pores of the inner wall endothelium are thought to generate at most 10% of this resistance in humans. However, the hydrodynamic interaction of these two components of the aqueous outflow system, which arises because of their spatial proximity, has only recently been considered. Modelling the JCT as a homogeneously distributed porous material upstream of a low porosity filter (the inner wall endothelium), the pores of the inner wall are found to cause a "funneling effect," in which the aqueous humor flows preferentially through those regions of the JCT nearest the inner wall pores. The bulk of the pressure drop occurs in the immediate proximity of the pores (within three pore radii). This greatly increases the apparent flow resistance of the JCT. For a set of parameters characterizing the normal eye, this enhancement is approximately 30-fold. The conclusion of this study is that changes in inner wall porosity may greatly affect aqueous outflow resistance, despite the low flow resistance of the inner wall pores themselves.

Calculations of flow resistance in the juxtacanalicular meshwork.

The structure of the juxtacanalicular meshwork (JCM) was analyzed morphometrically, and the resulting data were used to calculate the resistance to flow through this tissue. Two models of the JCM were presented and compared. In the first (Model A), aqueous humor was assumed to flow via open channels within a solid framework, while, in the second (Model B), these open spaces were assumed to be filled with extracellular matrix gel. An expression giving the resistance of such a gel as a function of gel concentration was presented and tested on corneal and scleral stroma. Morphometry of normal and glaucomatous human eyes showed that Model A underpredicted the resistance of the JCM by factors of 10-100, suggesting that a GAG or proteoglycan gel may control the flow resistance of this tissue. This was supported by Model B, which showed that measured bulk concentrations of GAGs were consistent with gel concentrations needed to account for the estimated resistance of the JCM in vivo. Some limitations and implications of Model B were discussed.

The flow of aqueous humor through micro-porous filters.

Flow resistance was measured as bovine and primate aqueous humor was passed through Nuclepore polycarbonate filters having flow dimensions similar to those found within the juxtacanalicular meshwork of the aqueous outflow network. The results indicate that aqueous humor has a greater flow resistance than isotonic saline; this greater resistance is attributable to proteins or glycoproteins in aqueous humor that obstruct the filters. If the same phenomenon is operative in the aqueous outflow network, it would help to explain discrepancies between calculated and measured aqueous outflow resistance.

The source of protein in the aqueous humor of the normal monkey eye.

In vivo aqueous fluorophotometry, morphology, and computational modeling were combined to examine the source of protein and the pathway by which protein enters the aqueous humor of monkeys. A computational model was developed to determine the likelihood of a diffusional route for delivering plasma proteins from ciliary body capillaries via the iris to anterior chamber aqueous humor, bypassing the posterior chamber. Model predictions were compared to aqueous fluorophotometric data obtained from monkeys following a single intravenous injection of fluoresceinated horseradish peroxidase (F-HRP, 250 mg/kg body mass). Model predictions of the magnitude and time course of anterior chamber F-HRP concentration agree with the fluorophotometric measurements. For example, of anterior chamber F-HRP concentration as a percentage of initial plasma F-HRP concentration at 90 min and 180 min post-injection was predicted to be 0.02% and 0.05%, respectively, and was measured to be 0.01-0.03% and 0.03-0.06%, respectively. In addition, model predictions in the case of a constant plasma protein level also are consistent with experimental data. The steady-state anterior chamber total protein concentration as a percentage of plasma protein concentration was predicted to be 0.2% and was assayed to be 0.05-0.2%. As in our previous study of the normal rabbit eye, morphologic and tracer localization evidence combined with the good agreement between model predictions and experimental data lead to the conclusion that a significant amount of the plasma protein normally present in monkey aqueous humor originates in ciliary body capillaries and diffuses anteriorly through the iris and into the anterior chamber.

Morphologic correlations with fluorophotometric data from monkey eyes with anterior uveitis.

Acute anterior uveitis was induced in monkeys by unilateral intravitreal injection of 1.0 ng of Escherichia coli endotoxin. Twenty-four hours later, each animal received an intravenous injection of 250 mg/kg body weight of fluoresceinated horseradish peroxidase (F-HRP), and fluorophotometric measurements were taken for 90 min. The animals were killed, and both eyes were processed for HRP demonstration. In the anterior chamber aqueous humor of normal control eyes, F-HRP concentrations were less than 0.002 mg/ml at 90 min. The F-HRP concentration was elevated consistently in the endotoxin-injected eyes; however, the magnitude of the effect varied. By fluorophotometry, inflamed eyes fell into two distinct groups. At 90 min, most had an anterior chamber F-HRP concentration of 0.014-0.06 mg/ml, although others had 0.39 mg/ml. In the latter group, an appreciably shorter latency was observed between the time of tracer injection and its detection in the anterior chamber. Aqueous humor protein concentrations, although highest in the most F-HRP-permeable eyes, followed more of a continuum in their distribution and identified less clearly the subpopulations seen by fluorophotometry. Normal eyes had no tracer leakage across either the ciliary epithelial or iris vascular endothelial barriers. All inflamed eyes had HRP leakage across the ciliary epithelium, but the subpopulation of eyes with shorter latencies and higher F-HRP concentrations by fluorophotometry also had iris vascular leakage.

Further studies on the flow of aqueous humor through microporous filters.

It has recently been shown that aqueous humor is able to obstruct flow through Nuclepore polycarbonate filters having flow dimensions similar to those found in the juxtacanalicular tissue (JCT). We undertook studies designed to identify the component(s) of aqueous humor responsible for this obstruction and to determine the mechanism of blockage. We conclude that aqueous humor contains two components (one of which is specific to aqueous) which, when simultaneously present, hydrophobically bind to microporous filters and lead to filter blockage. Some implications for aqueous humor flow through the JCT are briefly discussed.