Identification of Basic Fibroblast Growth Factor as the Dominant Protector of Laminar Shear Medium from the Modified Shear Device in Tumor Necrosis Factor-α Induced Endothelial Dysfunction.

Huang-Joe Wang,Wan-Yu Lo

doi:10.3389/fphys.2017.01095

Abstract

Background and Aims: Endothelial dysfunction is a hallmark of cardiovascular diseases. The straight region of an artery is protected from atherosclerosis via its laminar blood flow and high shear stress. This study investigated the cytoprotective effects of a new laminar shear medium (LSM) derived from a modified cone-and-plate shear device and identified basic fibroblast growth factor (bFGF) secreted by human aortic endothelial cells (HAECs) as the dominant protective factor in the LSM.Methods: Based on a modified cone-and-plate shear device system, HAECs were exposed to laminar shear (15 dynes/cm2) and static control for 24 h to produce a new supernatant LSM and static medium (SM). Evaluation of the protective effects of LSM and SM on endothelial dysfunction induced by tumor necrosis factor (TNF)-α (10 ng/mL), which leads to production of reactive oxygen species (ROS), inflammatory monocyte adhesion, and tissue factor activity. ROS induction-, inflammation-, and thrombosis-related genes and protein expression were evaluated by quantitative-PCR and western blotting. To identify the cytokines that played a key role in the cytoprotective action of the LSM, we used cytokine antibody arrays, selected an abundant marker cytokine, bFGF, and validated the different cytoprotective effects of recombinant bFGF (rbFGF) and neutralization by monoclonal antibody (rbFGF+Ab) co-treatment. Aortic and lung tissues from different groups of C57BL/6J mice were examined by immunohistochemistry. SB203580 (specific inhibitor of p38) and BIX02189 (specific inhibitor of MEK5) were used to identify bFGF as the main cytoprotective factor acting via p38/MAPK and MEK5-KLF2 pathways.Results: Compared with traditional LSM, the new LSM not only significantly decreased TNF-α-induced intracellular adhesion molecule 1 and plasminogen activator inhibitor type 1 gene expression, but also significantly increased heme oxygenase 1 gene expression. The new LSM and bFGF attenuated TNF-α-induced ROS induction, inflammation, and tissue factor activity and inhibited the inflammatory- and thrombosis-related gene/protein overexpression both in vitro and in vivo. Mechanistically, the cytoprotective action of bFGF was mediated via the p38/MAPK and MEK5-KLF2 pathways.Conclusion: bFGF was identified as the critical factor mediating the cytoprotective effects of LSM derived from the modified laminar shear system.

Highlights

Endothelial cells are constantly exposed to blood flow; shear stress for blood flow within a vessel is defined as τ = 32μQ/πd3, where Q is the mean volumetric flow rate, μ is the mean velocity, and d is the vessel diameter (Papaioannou and Stefanadis, 2005)
Other human aortic endothelial cells (HAECs) were incubated in fresh media mixed with 2, 20, and 60% laminar shear medium (LSM) for 24 h, but there was no significant induction of cell death (Figure 1E)
The data indicated that the gene levels in the underlying HAECs exposed to LSS for 24 h were as follows: under-expressed cytokines (EGFR, granulocyte-macrophage colony-stimulating factor (GM-CSF), monokine induced by gamma interferon (MIG), and monocyte chemo-attractant protein-1 (MCP-1)) were 0.63, 0.64, 0.54, and 0.4-fold lower than the static control (Figure 4B) and over-expressed cytokines (HGF, granulocyte-colony stimulating factor (G-CSF), IL-17, and basic fibroblast growth factor (bFGF)) were 1.89, 1.7, 1.57, and 1.45-fold higher than the static control (Figure 4C)

Summary

Introduction

The original cone-and-plate system has been modified by numerous groups, including Dr Hanjoong Jo, whose modification of the design included a 10-cm tissue culture disc on which human umbilical vein endothelial cells (HUVECs) were exposed to laminar or oscillating shear stress from a rotating cone, and the angular separation between the cone surface and culture disc was 0.5◦. Using this traditional shear device (15 dynes/cm laminar shear stress, LSS), we observed that human aortic endothelial cells (HAECs) in the center of the culture disc, compared with those at the periphery, were detached on the culture disc and did not show the typical high shear stress-induced alignment of endothelial cell shape, likely because of the non-uniform shear stress levels of this device (Rezvan et al, 2011). This study investigated the cytoprotective effects of a new laminar shear medium (LSM) derived from a modified cone-and-plate shear device and identified basic fibroblast growth factor (bFGF) secreted by human aortic endothelial cells (HAECs) as the dominant protective factor in the LSM

Methods

Results

Conclusion