Regulation of Vascular Smooth Muscle Cell Stiffness and Adhesion by [Ca2+]i: An Atomic Force Microscopy-Based Study

Abstract

The intracellular concentration of calcium ion ([Ca2+]i) is a critical regulator of cell signaling and contractility of vascular smooth muscle cells (VSMCs). In this study, we employed an atomic force microscopy (AFM) nanoindentation-based approach to investigate the role of [Ca2+]i in regulating the cortical elasticity of rat cremaster VSMCs and the ability of rat VSMCs to adhere to fibronectin (Fn) matrix. Elevation of [Ca2+]i by ionomycin treatment increased rat VSMC stiffness and cell adhesion to Fn-biofunctionalized AFM probes, whereas attenuation of [Ca2+]i by 1,2-Bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) treatment decreased the mechanical and matrix adhesive properties of VSMCs. Furthermore, we found that ionomycin/BAPTA-AM treatments altered expression of α 5 integrin subunits and α smooth muscle actin in rat VSMCs. These data suggest that [Ca2+]i regulates VSMC elasticity and adhesion to the extracellular matrix by a potential mechanism involving changing dynamics of the integrin-actin cytoskeleton axis.

Keywords: adhesion; atomic force microscopy; calcium; elasticity; vascular smooth muscle cell.

Figure 1:. AFM nanoindentation analyses of topography, stiffness, and adhesion probability of rat VSMCs treated with or without Ionomycin/BAPTA-AM.

Rat cremaster VSMCs at passage 0 were cultured in the presence or absence of 0.2 μmol/L Ionomycin or BAPTA-AM. Representative AFM deflection images are shown for A, the control VSMCs, B, VSMCs treated by Ionomycin, and C, VSMCs treated by BAPTA-AM. Graphs show comparisons of D, stiffness and E, adhesion probability in the control, Ionomycin-treated, and BAPTA-AM-treated rat VSMCs. Results are expressed as mean ± SEM; n = 5 cells from 3 rats per group. ^**p<0.01 and *p<0.05 for comparisons between the control vs. drug-treated cells.

Figure 2:. Mathematical analysis of the elasticity waveforms in the control and Ionomycin/BAPTA-AM-treated rat VSMCs by three principle components of oscillation.

The oscillatory waveforms of elastic modulus in a comprehensive format (top panel) and three oscillatory components (lower three panels) are shown for A, the control VSMCs, n = 9 cells from 3 rats, B, Ionomycin-treated VSMCs, n = 5 cells from 3 rats, and C, BAPTA-AM-treated VSMCs, n = 11 cells from 3 rats. The apparent values of frequency, amplitude and phase are shown in Table 2.

Figure 3:. Effects of Ionomycin/BAPTA-AM treatments on expression of α₅ integrin subunits and αSMA in rat VSMCs.

Representative confocal immunofluorescent images show A, α₅ integrin expression and C, αSMA expression in the control, Ionomycin-treated, and BAPTA-AM-treated rat VSMCs. Graphs show comparisons of the intensities of B, α₅ integrin-positive signals and D, αSMA-positive signals among different cell groups. Results are expressed as mean ± SEM; n = 40 cells from 3 rats in the control group; n = 32 cells from 3 rats in Ionomycin-treated group; and n = 34 from 3 rats in BAPTA-AM-treated group. ^**p<0.01 and *p<0.05 for comparisons between the control vs. drug-treated cells.