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Microrheology and ROCK Signaling of Human Endothelial Cells Embedded in a 3D Matrix

Porntula Panorchan ^*, Jerry S. H. Lee ^*, Thomas P. Kole ^*, Yiider Tseng  and Denis Wirtz ^* 

^* Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218; Department of Chemical Engineering, University of Florida, Gainesville, Florida 32611; and Department of Material Science and Engineering, Howard Hughes Medical Institute Graduate Training Program, and Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland 21218

Correspondence: Address reprint requests to Denis Wirtz, Dept. of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles St., Baltimore, Maryland 21218. Tel.: 410-516-7006; Fax: 410-516-5510; E-mail: wirtz{at}jhu.edu.

Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

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