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Research Papers

# Multi-Axial Mechanical Stimulation of HUVECs Demonstrates That Combined Loading is not Equivalent to the Superposition of Individual Wall Shear Stress and Tensile Hoop Stress Components

[+] Author and Article Information
Liam T. Breen

Department of Mechanical and Biomedical Engineering and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, University Road, Galway, Irelandliam.breen@nuigalway.ie

Peter E. McHugh

Department of Mechanical and Biomedical Engineering and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, University Road, Galway, Irelandpeter.mchugh@nuigalway.ie

Bruce P. Murphy1

Department of Mechanical and Biomedical Engineering, National University of Ireland, Galway, University Road, Galway, Irelandbruce.murphy@nuigalway.ie

1

Corresponding author.

J Biomech Eng 131(8), 081001 (Jun 17, 2009) (10 pages) doi:10.1115/1.3127248 History: Received June 11, 2007; Revised January 06, 2009; Published June 17, 2009

## Abstract

Over the past 25 years, many laboratory based bioreactors have been used to study the cellular response to hemodynamic forces. The vast majority of these studies have focused on the effect of a single isolated hemodynamic force, generally consisting of a wall shear stress (WSS) or a tensile hoop strain (THS). However, investigating the cellular response to a single isolated force does not accurately represent the true in vivo situation, where a number of forces are acting simultaneously. This study used a novel bioreactor to investigate the cellular response of human umbilical vein endothelial cells (HUVECs) exposed to a combination of steady WSS and a range of cyclic THS. HUVECs exposed to a range of cyclic THS (0–12%), over a 12 h testing period, expressed an upregulation of both ICAM-1 and VCAM-1. HUVECs exposed to a steady WSS ($0 dynes/cm2$ and $25 dynes/cm2$), over a 12 h testing period, also exhibited an ICAM-1 upregulation but a VCAM-1 downregulation, where the greatest level of WSS stimulus resulted in the largest upregulation and downregulation of ICAM-1 and VCAM-1, respectively. A number of HUVEC samples were exposed to a high steady WSS $(25 dynes/cm2)$ combined with a range of cyclic THS (0–4%, 0–8%, and 0–12%) for a 12 h testing period. The initial ICAM-1 upregulation, due to the WSS alone, was downregulated with the addition of a cyclic THS. It was observed that the largest THS (0–12%) had the greatest reducing effect on the ICAM-1 upregulation. Similarly, the initial VCAM-1 downregulation, due to the high steady WSS alone, was further downregulated with the addition of a cyclic THS. A similar outcome was observed when HUVEC samples were exposed to a low steady WSS combined with a range of cyclic THS. However, the addition of a THS to the low WSS did not result in an expected ICAM-1 downregulation. In fact, it resulted in a trend of unexpected ICAM-1 upregulation. The unexpected cellular response to the combination of a steady WSS and a cyclic THS demonstrates that such a response could not be determined by simply superimposing the cellular responses exhibited by ECs exposed to a steady WSS and a cyclic THS that were applied in isolation.

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## Figures

Figure 1

The bioreactor’s rheometer plate (shown as transparent for demonstrational purposes) with eight integrated flexible silicone substrates. The white arrows indicate the direction of applied WSS and the black arrows indicate the direction of applied THS. Two substrates were not cyclically stretched.

Figure 2

(a) The two levels of steady WSS applied to the cellular test substrates. (b) The range of cyclic saw tooth THS waveforms applied to the cellular test substrates.

Figure 3

An image of immunostained ECs, which were exposed to a cyclic saw tooth strain (0–6.5%) at a frequency of 1 Hz for 24 h. The adhesion molecules can be seen clearly in the high magnification view of one to the ECs.

Figure 4

The resulting normalized ICAM-1 expression, after 24 h of cyclic saw tooth stretching at a frequency of 1 Hz. A significant increase in ICAM-1 expression was observed with an increase in cyclic stretch magnitude. Four cellular samples were tested at each level of cyclic strain. The data are presented as mean±SDp∗<0.05 and p∗∗∗<0.001.

Figure 5

The flow cytometry results are shown as histograms of the ICAM-1(a) and VCAM-1 (b) expressions. Both adhesion molecules were upregulated after 24 h of VEGF stimulus. (1) Represents the negative control (nonspecific binding), (2) represents the basal level of expression (no VEGF), and (3) represents the positive control (VEGF stimulated). (c) The bar chart shows the quantified upregulation of the ICAM-1 and VCAM-1 MFIs after 24 h of VEGF stimulation under static conditions. The adhesion molecule expression was normalized against the basal level of expression. Data are represented as the mean of two substrates in one experiment.

Figure 6

ICAM-1 expression of HUVECs exposed to a combination of steady WSS and cyclic saw tooth THS mechanical stimuli for 12 h. The MFI was normalized against the static control samples using flow cytometry. Data are represented as mean±SD. Six cellular samples were tested for each combination of mechanical stimulus. p∗<0.05 versus static control.

Figure 7

VCAM-1 expression of HUVECs exposed to a combination of WSS and THS mechanical stimulus for 12 h. The MFI was normalized against the static control samples using flow cytometry. Data are represented as mean±SD. Six cellular samples were tested for each combination of mechanical stimulus. p∗<0.05, p∗∗<0.01 and p∗∗∗<0.001 versus static control.

Figure 8

Flow cytometry data for ICAM-1 and VCAM-1 upregulation in response to 12 h of cyclic saw tooth strain. Data are represented as mean±SD. Six cellular samples were tested at each level of cyclic strain. The test results are compared with ICAM-1 expression recorded in a previous study by Cheng (52). p∗<0.05 and p∗∗<0.01 versus static control.

Figure 9

Flow cytometry data for ICAM-1 and VCAM-1 expression of HUVECs exposed to a range of steady laminar WSS for 12 h. The results are compared against previous studies by Nagel (47) (ICAM-1) and Ohtsuka (58) (VCAM-1). The MFI was normalized against the static control samples in all studies. The bioreactor test results are represented as mean±SD. Six cellular samples were tested at each level of Steady WSS. p∗<0.05, p∗∗<0.01, and p∗∗∗<0.001 versus static control.

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