Biomedical subjects

Elliot L Elson

Publications and source records attributed to Elliot L Elson.

At least 19 recordsLinked to original sources

PubMed2006

Cellular and matrix mechanics of bioartificial tissues during continuous cyclic stretch.

Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 microM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001-0.25 Hz. Cell stiffness decreased with stretch amplitude (5-25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch.

Animals↗

PubMed2006

Cellular and matrix contributions to tissue construct stiffness increase with cellular concentration.

The mechanics of bio-artificial tissue constructs result from active and passive contributions of cells and extracellular matrix (ECM). We delineated these for a fibroblast-populated matrix (FPM) consisting of chick embryo fibroblast cells in a type I collagen ECM through mechanical testing, mechanical modeling, and selective biochemical elimination of tissue components. From a series of relaxation tests, we found that contributions to overall tissue mechanics from both cells and ECM increase exponentially with the cell concentration. The force responses in these relaxation tests exhibited a logarithmic decay over the 3600 second test duration. The amplitudes of these responses were nearly linear with the amplitude of the applied stretch. The active component of cellular forces rose dramatically for FPMs containing higher cell concentrations.

Animals↗

PubMed2006

Reconstitution of the Frank-Starling mechanism in engineered heart tissues.

According to the Frank-Starling mechanism, as the heart is stretched, it increases its contraction force. Reconstitution of the Frank-Starling mechanism is an important milestone for producing functional heart tissue constructs. Spontaneously contracting engineered heart tissues (EHTs) were reconstituted by growing dissociated chicken embryo cardiomyocytes in collagen matrices. Twitch and baseline tensions were recorded at precisely controlled levels of tissue strain. The EHTs showed a steep increase in twitch tension from 0.47 +/- 0.02 to 0.91 +/- 0.02 mN/mm2 as they were stretched at a constant rate (2.67% per min) from 86% to 100% of the length at which maximum twitch force was exerted. In response to a sudden stretch (3.3%), the twitch tension increased gradually (approximately 60 s) in a Gd3+-sensitive manner, suggesting the presence of stretch-activated Ca2+ channels. A large difference in baseline tension between lengthening (loading) and shortening (unloading) was also recorded. Disruption of nonsarcomeric actin filaments by cytochalasin D and latrunculin B decreased this difference. A simple mechanical model interprets these results in terms of mechanical connections between myocytes and nonmuscle cells. The experimental results strongly suggest that regulation of twitch tension in EHTs is similar to that of natural myocardium.

Animals↗

PubMed2006

Myosin-based contraction is not necessary for cardiac c-looping in the chick embryo.

During the initial phase of cardiac looping, known as c-looping, the heart bends and twists into a c-shaped tube with the convex outer curvature normally directed toward the right side of the embryo. Despite intensive study for more than 80 years, the biophysical mechanisms that drive and regulate looping remain poorly understood, although some investigators have speculated that differential cytoskeletal contraction supplies the driving force for c-looping. The purpose of this investigation was to test this hypothesis. To inhibit contraction, embryonic chick hearts at stages 10-12 (10-16 somites, 33-48 h) were exposed to the myosin inhibitors 2,3-butanedione monoxime (BDM), ML-7, Y-27632, and blebbistatin. Experiments were conducted in both whole embryo culture and, to focus on bending alone, isolated heart culture. Measurements of heart stiffness and phosphorylation of the myosin regulatory light chains showed that BDM, Y-27632, and blebbistatin significantly reduced myocardial contractility, while ML-7 had a lesser effect. None of these drugs significantly affected looping during the studied stages. These results suggest that active contraction is not required for normal c-looping of the embryonic chick heart between stages 10 and 12.

Amides↗

PubMed2005

On the application of strain factors for approximation of the contribution of anisotropic cells to the mechanics of a tissue construct.

Bio-artificial tissue constructs consisting of fibroblast cells embedded in a collagenous matrix are valuable in vitro systems in which to study cellular mechanics. Deriving cellular mechanics from the results of experimentation on tissue constructs requires a mathematical relationship that delineates amongst the contributions of the constituents of a tissue construct. A scaling between the average strain in a uniformly stretched tissue and the axial strain in isotropic cells was used in earlier work to study relations between cell mechanics and the overall mechanics of a tissue construct. That work showed that a scaling factor called a "strain factor" provided an accurate representation of the average axial strain in isotropic cells. The present study analyzes such relationships for anisotropic cells. We incorporate Eshelby's (1957; Proceedings of the Royal Society of London A 241, 376; 1959; Proceedings of the Royal Society of London A 252, 561) exact solution for the strain field in isolated ellipsoidal inclusions into the Zahalak (Biophysical journal 79, 2369) constitutive model for tissue constructs. Results showed that, for the case of prolate cells, the strain along the major cell axis is mostly influenced by the remote strain projected along that axis; off-axis cell mechanics plays only a small role in most tissues. The strain factor approximation is shown to be accurate for anisotropic cells to within a few percent for the vast majority of tissues. The results presented in this paper provide an explicit measure of the effects of cellular anisotropy, and a mechanism for calculating the contributions of these effects to overall tissue mechanics when these effects are important.

Anisotropy↗

PubMed2005

The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods.

The simplest dynamic model for an unfolded protein is a statistical coil that continually undergoes substantial conformational fluctuations. A growing number of studies indicate that the unfolded protein is not a simple random coil but rather forms transient structures. We have directly measured the rate of conformational fluctuations of unfolded intestinal fatty acid binding protein (131 aa, 15 kDa) by using fluorescence self-quenching in combination with fluorescence correlation spectroscopy. The conformational fluctuations in this state have an apparent relaxation time, tauR, of 1.6 microsec in 3 M guanidine-HCl at pH 7 and 20 degrees C. The value of tauR increases with increasing solution viscosity, suggesting a diffusive process. In the molten globule state at pH 2, tauR is 2.5 microsec, increasing further with the formation of salt-induced secondary structure. These measurements, which should be widely applicable to other systems, can provide important information about the still incompletely understood conformational properties of unfolded proteins and the mechanism of protein folding.

Biophysical Phenomena↗

PubMed2005

Role of actin polymerization in bending of the early heart tube.

During cardiac c-looping, the heart transforms from a straight tube into a c-shaped tube, presenting the first evidence of left-right asymmetry in the embryo. C-looping consists of two primary deformation components: ventral bending and dextral rotation. This study examines the role of actin polymerization in bending of the heart tube. Exposure of stage 9-11 chick embryos to low concentrations of the actin polymerization inhibitors cytochalasin D (5 nM-2.0 microM) and latrunculin A (LA; 25 nM-2.0 microM) suppressed looping in a stage- and concentration-dependent manner in both whole embryos and isolated hearts. Local exposure of either the dorsal or ventral sides of isolated hearts to LA also inhibited looping, but less than global exposure, indicating that both sides contribute to the bending mechanism. Taken together, these data suggest that ongoing actin polymerization is required for the bending component of cardiac c-looping, and we speculate that polymerization-driven myocardial cell shape changes cause this deformation.

Abnormalities, Drug-Induced↗

PubMed2004

Thin bio-artificial tissues in plane stress: the relationship between cell and tissue strain, and an improved constitutive model.

Constitutive models are needed to relate the active and passive mechanical properties of cells to the overall mechanical response of bio-artificial tissues. The Zahalak model attempts to explicitly describe this link for a class of bio-artificial tissues. A fundamental assumption made by Zahalak is that cells stretch in perfect registry with a tissue. We show this assumption to be valid only for special cases, and we correct the Zahalak model accordingly. We focus on short-term and very long-term behavior, and therefore consider tissue constituents that are linear in their loading response (although not necessarily linear in unloading). In such cases, the average strain in a cell is related to the macroscopic tissue strain by a scalar we call the "strain factor". We incorporate a model predicting the strain factor into the Zahalak model, and then reinterpret experiments reported by Zahalak and co-workers to determine the in situ stiffness of cells in a tissue construct. We find that, without the modification in this article, the Zahalak model can underpredict cell stiffness by an order of magnitude.

Animals↗

PubMed2004

The relationship between cell and tissue strain in three-dimensional bio-artificial tissues.

Continuum constitutive laws are needed to ensure that bio-artificial tissue constructs replicate the mechanical response of the tissues they replace, and to understand how the constituents of these constructs contribute to their overall mechanical response. One model designed to achieve both of these aims is the Zahalak model, which was modified by Marquez and co-workers to incorporate inhomogeneous strain fields within very thin tissues. When applied to reinterpret previous measurements, the modified Zahalak model predicted higher values of the continuum stiffness of fibroblasts than earlier estimates. In this work, we further modify the Zahalak model to account for inhomogeneous strain fields in constructs whose cell orientations have a significant out-of-plane component. When applied to reinterpret results from the literature, the new model shows that estimates of continuum cell stiffness might need to be revised upward. As in this article's companion, we updated the average cell strain by defining a correction factor ("strain factor"), based upon the elastic response. Three different cell orientation distributions were studied. We derived an approximate scaling model for the strain factor, and validated it against exact and self-consistent (mean-field) solutions from the literature for dilute cell concentrations, and Monte Carlo simulations involving three-dimensional finite element analyses for high cell concentrations.

Animals↗

PubMed2004

Measuring unfolding of proteins in the presence of denaturant using fluorescence correlation spectroscopy.

IFABP is a small (15 kDa) protein consisting mostly of antiparallel beta-strands that surround a large cavity into which ligands bind. We have previously used FCS to show that the native protein, labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time of 35 micros. Here we report the use of FCS to study the unfolding of the protein induced by guanidine hydrochloride. Although the application of this technique to measure diffusion coefficients and molecular dynamics is straightforward, the FCS results need to be corrected for both viscosity and refractive index changes as the guanidine hydrochloride concentration increases. We present here a detailed study of the effects of viscosity and refractive index of guanidine hydrochloride solutions to calibrate FCS data. After correction, the increase in the diffusion time of IFABP corresponds well with the unfolding transition monitored by far ultraviolet circular dichroism. We also show that the magnitude of the 35 micros phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. Although FCS experiments indicate that the unfolded state at pH 2 is rather compact and native-like, the radius in the presence of guanidine hydrochloride falls well within the range expected for a random coil.

Algorithms↗

PubMed2004

Interstitial collagenase is a Brownian ratchet driven by proteolysis of collagen.

We show that activated collagenase (MMP-1) moves processively on the collagen fibril. The mechanism of movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. Inactivation of the enzyme by a single amino acid residue substitution in the active center eliminates the bias without noticeable effect on rate of diffusion. Monte Carlo simulations using a model similar to a "burnt bridge" Brownian ratchet accurately describe our experimental results and previous observations on kinetics of collagen digestion. The biological implications of MMP-1 acting as a molecular ratchet tethered to the cell surface suggest new mechanisms for its role in tissue remodeling and cell-matrix interaction.

Adenosine Triphosphate↗

PubMed2004

Fluorescence correlation spectroscopy with high-order and dual-color correlation to probe nonequilibrium steady states.

In living cells, biochemical reaction networks often function in nonequilibrium steady states. Under these conditions, the networks necessarily have cyclic reaction kinetics that are maintained by sustained constant input and output, i.e., pumping. To differentiate this state from an equilibrium state without flux, we propose a microscopic method based on concentration fluctuation measurements, via fluorescence correlation spectroscopy, and statistical analyses of high-order correlations and cross correlations beyond the standard fluorescence correlation spectroscopy autocorrelation. We show that, for equilibrium systems with time reversibility, the correlation functions possess certain symmetries, the violation of which is a measure of steady-state fluxes in reaction cycles. This result demonstrates the theoretical basis for experimentally measuring reaction fluxes in a biochemical network in situ and the importance of single-molecule measurements in providing fundamental information on nonequilibrium steady-states in biochemistry.

Enzymes↗

PubMed2004

The biochemical response of the heart to hypertension and exercise.

Mechanical stress on the heart can lead to crucially different outcomes. Exercise is beneficial because it causes heart muscle cells to enlarge (hypertrophy). Chronic hypertension also causes hypertrophy, but in addition it causes an excessive increase in fibroblasts and extracellular matrix (fibrosis), death of cardiomyocytes and ultimately heart failure. Recent research shows that stimulation of physiological (beneficial) hypertrophy involves several signaling pathways, including those mediated by protein kinase B (also known as Akt) and the extracellular-signal-regulated kinases 1 and 2 (ERK1/2). Hypertension, beta-adrenergic stimulation and agonists such as angiotensin II (Ang II) activate not only ERK1/2 but also p38 and the Jun N-terminal kinase (JNK), leading to pathological heart remodeling. Despite this progress, the mechanisms that activate fibroblasts to cause fibrosis and those that differentiate between exercise and hypertension to produce physiological and pathological responses, respectively, remain to be established.

Animals↗

PubMed2004

Cell orientation influences the biaxial mechanical properties of fibroblast populated collagen vessels.

Bioartificial tissues, composed of cells in a collagen matrix, can be fabricated with preferred cell orientations to mimic the histologic arrangement of biologic tissues. The influence of preferred cell orientations on the biaxial mechanical behavior of bioartificial tissues is unknown. Characterizing the biaxial mechanical behavior is necessary for better predicting the in vivo behavior of bioartificial tissues. Fibroblast populated collagen vessels (FPCVs) were fabricated with two different cell orientations by controlling the mechanical constraints during incubation. The cell orientation was verified by confocal microscopy and the collagen fiber organization was examined by confocal reflection and scanning electron microscopy (SEM). Pressure-diameter, force-length tests were performed to determine the influence of cell orientation on the biaxial mechanical behavior. FPCVs were more extensible in the direction perpendicular to the preferred cell orientation, than in the direction parallel to the cell orientation. Biaxial tests were also performed in the presence of Cytochalasin D (Cyto D) to minimize the mechanical contribution of the cells. After Cyto D treatment, the FPCVs remained more extensible in the direction perpendicular to the cell orientation, even though a preferred collagen fiber orientation was not observed in the microscopy images.

Animals↗

PubMed2003

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts.

Thus far, determining the relative contribution of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and Ca2+-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca2+, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 +/- 0.02 mol PO4/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 +/- 0.05 and 0.82 +/- 0.05 mol PO4/mol RLC, respectively, within 2.5 min. Ca2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.

Amides↗

PubMed2003

Mechanics of cell spreading: role of myosin II.

As it migrates over a substratum, a cell must exert different kinds of forces that act at various cellular locations and at specific times. These forces must therefore be coordinately regulated. The Rho-family GTPases Rac1 and Cdc42 promote actin polymerization that drives extension of the leading cell edge. Subsequently, RhoA regulates myosin-dependent contractile force, which is required for formation of adhesive contacts and stress fibers. During cell spreading, however, the activity of RhoA is reduced by a mechanism involving the tyrosine kinases c-Src and focal adhesion kinase (FAK), and the p190RhoGAP. It has been proposed that this reduction of RhoA activity facilitates edge extension by reducing myosin-dependent contractile forces that could resist this process. We have directly tested this hypothesis by correlating myosin activity with the rate of cell spreading on a substratum. The rate of spreading is inversely related to the myosin activity. Furthermore, spreading is inhibited by low concentrations of cytochalasin D, as expected for a process that depends on the growth of uncapped actin filaments. Cell indentation measurements show that a myosin-dependent viscoelastic force resists cell deformation.

Animals↗

PubMed2003

Statistical analysis of fluorescence correlation spectroscopy: the standard deviation and bias.

We present a detailed statistical analysis of fluorescence correlation spectroscopy for a wide range of timescales. The derivation is completely analytical and can provide an excellent tool for planning and analysis of FCS experiments. The dependence of the signal-to-noise ratio on different measurement conditions is extensively studied. We find that in addition to the shot noise and the noise associated with correlated molecular dynamics there is another source of noise that appears at very large lag times. We call this the "particle noise," as its behavior is governed by the number of particles that have entered and left the laser beam sample volume during large dwell times. The standard deviations of all the points on the correlation function are calculated analytically and shown to be in good agreement with experiments. We have also investigated the bias associated with experimental correlation function measurements. A "phase diagram" for FCS experiments is constructed that demonstrates the significance of the bias for any given experiment. We demonstrate that the value of the bias can be calculated and added back as a first-order correction to the experimental correlation function.

Artifacts↗

PubMed2003

Reciprocal interactions between cells and extracellular matrix during remodeling of tissue constructs.

Cells remodel extracellular matrix during tissue development and wound healing. Similar processes occur when cells compress and stiffen collagen gels. An important task for cell biologists, biophysicists, and tissue engineers is to guide these remodeling processes to produce tissue constructs that mimic the structure and mechanical properties of natural tissues. This requires an understanding of the mechanisms by which this remodeling occurs. Quantitative measurements of the contractile force developed by cells and the extent of compression and stiffening of the matrix describe the results of the remodeling processes. Not only do forces exerted by cells influence the structure of the matrix but also external forces exerted on the matrix can modulate the structure and orientation of the cells. The mechanisms of these processes remain largely unknown, but recent studies of the regulation of myosin-dependent contractile force and of cell protrusion driven by actin polymerization provide clues about the regulation of cellular functions during remodeling.

Animals↗