A Mechanostimulation System for Revealing Intercellular Calcium Communication in HUVEC Networks

Abstract -This paper reports a mechanostimulation system for studying mechanically induced intercellular calcium signaling in networks of human umbilical vein endothelial cells (HUVECs). By incorporating a capacitive (comb drive) force probe and plasma lithography cell patterning, the roles of biophysical factors, including force, duration, and network architecture, in calcium intercellular communication can be investigated systematically. Particularly, we observed cancellation of calcium waves in linear networks and bi-directional splitting in cross junctions. The effects of key biophysical factors on intercellular calcium wave propagation were studied. These results demonstrate the applicability of the mechanostimulation system in studying intercellular calcium signaling and reveal the robustness of calcium signaling in HUVEC networks, which mimics the vasculature

Theoretical Investigation of Architecture-Dependent Calcium Signaling in Multicellular Network

Calcium signal can be found in many types of cell. It has been treated as a life and death signal in cell-level for triggering life at fertilization, controlling the development and differentiation of cells into specialized types, mediating the subsequent activity, and finally affecting the cell death. In tissues, intercellular calcium wave is thought to serve as a long-range signaling, affected by the cell architecture. The aim of this thesis is to provide insight into the intercellular calcium waves in multicellular complex structures subjected to mechano- or chemical-stimuli. In the mechano-stimulated study, we combine the development of theoretical and experimental study of the propagation of calcium signals in multicellular structures composed of human endothelial cells. This analysis provides evidence for an effect of architecture on the propagation of calcium signals and the effect of single and dual stimulation on the multicellular structures. A simple model was established based on the calcium release/intake reaction and diffusion through gap junction from stimulated cell to the downstream cells. The simulation result shows similar results as what is shown in experiments. In the chemical-stimulated model, we studied computationally the interdependence between intracellular calcium and inositol-1,4,5-trisphosphate (IP₃) pathway and cell-cell communication via gap junction. We investigate the influence of the microenvironment of cells on the frequency of intracellular calcium oscillation. The simulation result shows that the oscillation frequency of an isolated cell is lower than that of a cell embedded in a cell-chain. This phenomenon is attributed to retrograde diffusion of calcium and IP₃ originating from a widening range of cells in the chain undergoing oscillations. It further demonstrates the important influence of microenvironment on the bio-signaling propagation.Release 13-Aug-201

Calcium Wave Propagation in Networks of Endothelial Cells: Model-based Theoretical and Experimental Study

<div><p>In this paper, we present a combined theoretical and experimental study of the propagation of calcium signals in multicellular structures composed of human endothelial cells. We consider multicellular structures composed of a single chain of cells as well as a chain of cells with a side branch, namely a “T” structure. In the experiments, we investigate the result of applying mechano-stimulation to induce signaling in the form of calcium waves along the chain and the effect of single and dual stimulation of the multicellular structure. The experimental results provide evidence of an effect of architecture on the propagation of calcium waves. Simulations based on a model of calcium-induced calcium release and cell-to-cell diffusion through gap junctions shows that the propagation of calcium waves is dependent upon the competition between intracellular calcium regulation and architecture-dependent intercellular diffusion.</p> </div

Schematic representation of the non-linear intracellular calcium reaction dynamics as a loop in calcium concentration space.

<p>UC₁ and UC₂ are lower and upper thresholds of intracellular calcium concentration, respectively, which determine the value of calcium release/intake rate constant, <i>k</i>.</p

Experiment image and normalized intensity of cells in single fine line subjected to single stimulus.

<p>(A) Image of a finite single fine line of cells. Cells are labeled 1 through 9. The stimulating probe is clearly visible on the right of the stimulated cell (cell 4 labeled with a red circle). The time (in sec) at which the normalized fluorescence reaches its maximum positive rate of change is indicated for every cell. The uncertainty for each one of these times is 0.6 s. The origin of time is the time at which fluorescence in the stimulated cell attains its maximum rate of change. (B) and (C) show the normalized intensity of fluorescence of cells 4 through 9 and cells 3 through 1, respectively, as functions of time. The vertical axis is the dimensionless normalized intensity of fluorescence and the horizontal axis is the time in seconds in intervals of 1.2 s between recordings.</p

Experiment image 2 and normalized intensity of cells in “T” structure subjected to a single mechano-stimulation.

<p>(A) Image of a T structure of cells subjected to single mechano-stimulation. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002847#pcbi-1002847-g008" target="_blank">Figure 8</a> for detail. (B–D) shows the normalized intensity of fluorescence of representative cells as functions of time. The vertical axis is the dimensionless normalized intensity of fluorescence and the horizontal axis is the time in seconds in intervals of 1.2 s between recordings.</p

Experiment image and normalized intensity of cells in single fine line subjected to double stimulus.

<p>(A) Image of a finite single fine line of cells subjected to dual mechano-stimulation. The stimulating probes are visible at the top-left and bottom-right of the image. The response time of cells labeled in red was calculated from the maximum rate of change of the fluorescence intensity with uncertainties 0.6 s. The response time of cells labeled in green was calculated from a running average of the fluorescence intensity with uncertainties 1.2 s. The first response time for cell 4 (1), represents the time of the first sharp rise in fluorescence versus time. The second response time of cell 4 (2), indicates the time when the fluorescence intensity increases a second time. Cells 4 and 5 represent the region where the calcium pulses are anticipated to meet. (B) and (C) present the normalized intensity of fluorescence of individual cells as a function of time.</p

Plasma lithography for cell patterning.

<p>(A) Photolithography is used to form a template of the desired multicellular network. (B) PDMS is poured over the photoresist pattern to create an initial plasma shielding model. (C) PDMS mold is transformed onto a Petri Dish (polystyrene). (D) The plasma surface treatment is used to produce cell-sensitive chemical pattern on the area of PDMS mold. (E) Cell seeding. (F) Cell stimulation.</p

Value of dimensionless parameters used in the reaction-diffusion model of intercellular and intracellular reaction-diffusion dynamics.

<p>Value of dimensionless parameters used in the reaction-diffusion model of intercellular and intracellular reaction-diffusion dynamics.</p

Schematic illustration and simulation results of the model “T” structure subjected to single stimulus with .

<p>(A) Schematic illustration of the model “T” structure subjected to a single stimulus. Red cell is the stimulated cell. Orange arrows represent the edge-to-edge diffusion. Green arrows represent vertex-to-vertex diffusion. Purple cells highlight the junction cell cluster. (B) Calcium concentration of cells in backbone as a function of time. (C) Calcium concentration of cells 1', 2' and cells in the side branch.</p