Measurements of endothelial cell-to-cell and cell-to-substrate gaps and micromechanical properties of endothelial cells during monocyte adhesion

Abstract

The interaction between monocytes and endothelial cells is considered to play a major role in the early stage of atherosclerosis, and the involved endothelial cell micromechanics may provide us with important aspects of atherogenesis. In the present study, we evaluated (i) the endothelial cell-to-cell and cell-to-substrate gaps with the electric cell-substrate impedance sensing system, which can detect the nanometer order changes of cell-to-cell and cell-to-substrate distances separately, and (ii) the endothelial cell micromechanical properties with an atomic force microscope after application of monocytes to endothelial cells. Application of monocytic THP-1 cells to IL-1beta-stimulated human umbilical vein endothelial cells immediately decreased the electrical resistance of the endothelial cell-to-substrate (increase of the cell-to-substrate gap), whereas the endothelial cell-to-cell resistance (cell-to-cell gap) did not change. The elastic modulus of the endothelial cells decreased after 2-h monocyte application, indicating an increase of endothelial cell deformability. In conclusion, the interaction of the monocytes to the endothelial cells reduced the adhesiveness to the substrate and increased the deformability of endothelial cells. These changes in the adhesiveness and the deformability may facilitate migration of monocytes, a key process of atherogenesis in the later stage.

Fig 1.

Photomicrographs of monocytes remaining on the surface of IL-1β-unstimulated (Left) and stimulated (Right) HUVEC after washing off the nonadherent monocytes.

Fig 2.

Typical examples and summary of the total electrical impedance changes of the IL-1β-stimulated and unstimulated HUVEC after application of monocytes. (Upper) Black line, stimulated HUVEC; gray line, unstimulated HUVEC. Arrow indicates the application of monocytes. (Lower) ○, Unstimulated HUVEC (n = 17); •, stimulated HUVEC, (n = 15). *, P < 0.05 stimulated HUVEC vs. unstimulated HUVEC.

Fig 3.

Typical real-time traces of the total (double line), cell-to-substrate (gray line), and cell-to-cell (black line) resistance in the IL-1β-stimulated HUVEC.

Fig 4.

Summary of the normalized values of α (cell-to-substrate) and Rb (cell-to-cell) after 1 h of monocyte application. *, P < 0.05; N.S., not significant.

Fig 5.

Computer-reconstructed 3D images of GFP-expressed monocytes (green) and endothelial cells (red) after 2 h of application.

Fig 6.

Typical force-curves for the peripheral region of control (Left) and monocyte-adhered (Right) HUVEC measured with AFM. Black lines, measured force-curve; gray lines, fitted parabolic curve.

Fig 7.

Elastic modulus of HUVEC measured in the separated dishes (Left; nonapplication and application of monocytes). Elastic modulus of HUVEC with and without monocytes adhesion in the same dish (Right). Open bars, without monocyte adhesion; filled bars, with monocyte adhesion.

Fig 8.

Photomicrographs of F-actin filaments and p125^FAK in HUVEC before and after application of monocytes.