Substrate Stiffness Affects Endothelial Cell Junctions and MAPK Signaling Pathway

Huanli Wang,Xiaomei Han,Yue Zhou,Jin Xu,Chao Zhang

doi:10.4236/jbm.2022.102012

Abstract

Vascular homeostasis is critical for maintaining normal vascular structure and function. Aging is an irreversible trigger of vascular sclerosis, which causes structural and functional damage to blood vessels, leading to severe atherosclerosis. Endothelial cells (ECs) can respond to mechanical stimuli from the extracellular matrix, causing disruption of endothelial barrier function and activating signaling pathways to regulate cellular behavior under pathological conditions. In this paper, we investigated the effect of substrate stiffness on endothelial cell junctions, and the activation of mitogen-activated protein kinase (MAPK) signaling pathways. An in vitro stiffness model was established using polyacrylamide hydrogels of 1 kPa, 20 kPa and 100 kPa. By transcriptome analysis, we found that the cell-cell junction, cadherin binding, cytoskeleton and classical signaling pathways such as MAPK and Rho GTPase of endothelial cells were regulated by substrate stiffness. The expression of cell junction-related molecules TJP1, TJP2, JAM3 and JCAD was also found to be reduced at higher stiffness. The MAPK signaling pathway-related molecules MAP2K3, MAP2K7, MAP3K3, MAP3K6, MAPK3, MAPK7 were upregulated with increased stiffness. qRT-PCR analysis showed that the gene expression of JCAD was reduced with increased stiffness. Immunofluorescence staining of VE-cadherin indicated that the total fluorescence level of VE-cadherin decreased significantly with increased stiffness, and stiffness impaired the cell-cell junction with increased punctuation and discontinuity. Western blotting analysis confirmed that the protein expression ratio of pp38MAPK/p38MAPK increased with stiffness. Our research suggested that substrate stiffness played an important role in regulating endothelial cell integrity and MAPK signaling pathway.

Highlights

The vasculature forms an interface between blood circulation and different organ environments
The results indicated that cell-cell junction assembly was the major biological process affected
Blood vessels exhibit a remarkable ability to adapt throughout life that depends upon genetic programming and well-orchestrated biochemical processes

Summary

Introduction

The vasculature forms an interface between blood circulation and different organ environments. As early as the 1850s, studies in anatomic pathology began to explore the consequences caused by endothelial dysfunction They observed that lipid accumulation occurs at the site of early lesions, and local endothelial permeability was altered [5]. Atherosclerosis and coronary artery disease lead to vascular dysfunction, causing an imbalance in the ratio between vasodilator and vasoconstrictor factors, promoting thrombosis and atherosclerosis as evidenced by vasoconstriction, leukocyte adhesion, platelet activation, vascular inflammation [9] [10]. Hypertension and hypercholesterolemia are involved in vascular inflammatory processes, and under pathological conditions ROS can stimulate NF-κB to activate various pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), increase the expression of cell adhesion molecules, accelerate endothelial dysfunction, and recruit monocytes into the intima, leading to chronic inflammation of the vessel wall [11] [12]

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