|
Angiogenesis, the transient formation of new blood vessels, is mainly observed under certain physiological conditions in the adult. Although recent in vitro models have been crucial in determining the effects of growth factors and inhibitors on cell migration and sprouting, the fundamental processes of cell adhesion kinetics and the interaction between endothelial cells (ECs) and extracellular matrix (ECM) during tumor induced angiogenesis have not been quantified. During early stages of angiogenesis, ECs migrate from pre-existing vessels through the ECM, adhere to each other, and form a specific pattern. Following this initial event, many ECs form new, small finger-like capillaries. The sprouts grow in length with the migration and further recruitment of endothelial cells, and move toward the tumor.
To study the network formation of endothelial cells (ECs) in an extracellular matrix (ECM) environment, we have devised an EC aggregation-type model based on a diffusion-limited-cluster-aggregation model (DLCA), where clusters of particles diffuse and stick together upon contact. We use this model to quantify EC differentiation into cord-like-structures by comparing experimental and simulation data. Approximations made with the DLCA model, when combined with experimental kinetics and cell concentration results, not only allow us to quantify cell differentiation by a pseudo diffusion coefficient, but also measure the effects of tumor angiogenic factors (TAF) on the formation of cord-like structures by ECs.
We have tested our model by using an in vitro assay, where we record EC aggregation by analyzing time-lapse images that provide us with the evolution of the fractal dimension measure through time. We performed these experiments for various cell concentrations and TAF (e.g. EVG, FGF-b, and VEGF). During the first six hours of an experiment, ECs aggregate quickly. The value of the measured fractal dimension decreases with time until reaching an asymptotic value that depends solely on the EC concentration. In contrast, the kinetics depend on the nature of TAF. The experimental and simulation results correlate with each other in regards to the fractal dimension and kinetics, allowing us to quantify the influence of each TAF by a pseudo diffusion coefficient.
We have shown that the shape, kinetic aggregation, and fractal dimension of the EC aggregates fit into an in vitro model capable of reproducing the first stage of angiogenesis. We conclude that the DLCA model, combined with experimental results, is a highly effective assay for the quantification of the kinetics and network characteristics of ECs embedded in ECM proteins. Finally, we present a new method that can be used for studying the effect of angiogenic drugs in in vitro assays.
Another project is the use of stochastic modeling to analyze the influence of the ECM's heterogeneity on tumor induced angiogenesis. Cell migration during angiogenesis guides the formation of new blood vessels. This migration is mainly directed by a chemotactic flux via a gradient of growth factors between the tumor and the cell. New studies have shown that the ECM substrate can influence cell migration and apoptosis. We have performed experiments showing the correlation between endothelial cell migration and the fiber distribution in the ECM. The endothelial cells follow the pathways created by the fibers in the ECM. We have seen through stochastic modeling that the heterogeneity of the fibers in the ECM create obstacles for the blood vessels to perfuse the tumor.
We are currently using fluorescence correlation spectroscopy (FCS) to measure locally the degradation of the ECM due to the ECs. During angiogenesis, when ECs migrate through the tumor to form new blood vessels, the ECs degrade the ECM by cell locomotion and the release of matrix metalloproteinase (MMP). FCS measures tiny variations in gel structures by measuring the diffusion coefficient. Some preliminary experiments have shown promising results. We are going to do a cordlike structure assay to measure how the cell migration and aggregation change locally the structure of the gel. Preliminary results show a variation in the gel structure during the cell aggregation. In the future we intend to study how MMP and anti-MMP could affect the degradation of the gel and quantify the cause of this degradation (cell locomotion and MMP).
Amyot, F., K. Camphausen, A. Siavosh, D. Sackett, and A. Gandjbakhche. Quantitative method to study the network formation of endothelial cells in response to tumor angiogenic factors. IEE Proc. - Systems Biology 152(2), 61-66 (June 2005).
|
