Novel findings on extracellular matrix stiffness effects on endothelial cells and role in atherosclerosis at a single-cell level

by Roxanna Van Norman
November 4, 2022

A recent study led by Stanford School of Medicine researchers suggests that extracellular matrix effects on the phenotypic modulation of endothelial cells play a critical role in atherosclerosis, a buildup of plaque in the artery walls that can narrow arteries and block blood flow.

"This is a project that has been several years in the making, and for us as cardiovascular engineers, it addresses a very important question of how the extracellular matrix directly impacts cell behavior," said Ngan F. Huang, PhD, Associate Professor at the Stanford Department of Cardiothoracic Surgery and corresponding author of the study.

The extracellular matrix is a three-dimensional network of proteins and other components that helps cells bind together and regulate several cellular functions; it plays a critical part in disease development. Stiffening of the extracellular matrix, which can alter the phenotypes of endothelial cells and other vascular cell types, is associated with atherosclerosis.

Drawing on the multidisciplinary expertise of collaboration with researchers from biomedical engineering, mechanical engineering, molecular biology and genetics, and cardiovascular medicine, Huang and her colleagues used single-cell RNA sequencing to analyze thousands of cells to further their understanding of the extracellular matrix stiffness effects and cellular mechanisms that lead to endothelial cells de-differentiation.

They worked closely with the study’s co-author and computational biology collaborator, Patrick Cahan, PhD, and his group from John Hopkins University to analyze the single-cell RNA sequencing data.

The study was published in an early-view research article in September of Advanced Functional Materials.

Cell behaviors

Arterial stiffening is often linked to atherosclerosis, as well as other characteristics of cardiovascular diseases. Understanding the cellular mechanisms in the development of atherosclerosis is critical for identifying disease progression, the study highlighted.

"Studies are showing that endothelial cells play a role in giving rise to cells of atherosclerotic plaques, namely through the complex process of endothelial-mesenchymal transition (EndMT) in which they change their identity," said Huang.

She and her team investigated the effects of extracellular matrix stiffness on EndMT, a complex process in which endothelial cells lose their endothelial identity and take on mesenchymal phenotypic markers.

While studies have shown many contributing factors to atherosclerosis, there was no definitive way in vivo to conclusively show that stiffness was a defining parameter that induces the EndMT process, Huang explained.

To address this, the researchers developed an in vitro model using cultured human coronary artery endothelial cells on constructed hydrogels substrates of physiological or pathological stiffness.

"In an in vitro model, we can carefully control the system so we can show a direct relationship and study how this exactly changes or induces EndMT in endothelial cells," said Huang. "This was a simple model where we could show the direct effects of stiffness changes on the EndMT process."

Single-cell level 

The researchers applied single-cell RNA sequencing to examine the phenotype modulation change in endothelial cells in response to substrate stiffness. They found that the pathological substrate stiffness promoted EndMT, whereas physiological stiffness hydrogels blunted it. 

Huang further noted there was a great degree of heterogeneity in the cell populations in how the cells responded to different substrate stiffness.

"We observed a great deal of heterogeneity, so we used the single-cell RNA sequencing to look at the phenotypes of single cells to see how their biology changed with respect to other cells," said Huang, who collaborated with Cahan’s group to help with analyzing the cells at the single-cell level.

Even though the researchers studied the process using in vitro models, they discovered that the de-differentiation characteristics of endothelial populations from the pathological stiffness environment bore a resemblance to the de-differentiation found in human atherosclerotic plaque endothelial cells.

The study showed that endothelial cells were more protected from losing their healthy phenotype on substrates with physiological stiffness, supporting the study’s hypothesis that the physiological stiffness protected cells from taking on pathological features associated with EndMT and atherosclerosis.

Looking ahead

The study highlights novel findings on the effects of extracellular matrix on endothelial cells and the association with atherosclerosis via single-cell RNA sequencing.

Understanding how substrate stiffness impacts endothelial cell behaviors helps researchers to think about therapeutic approaches for addressing the progression of atherosclerosis and other disease developments.

According to Huang, future work will continue to look at the pathways regulated by these stiffness effects to see what specific pathways are activated. "The hope is that we can potentially find druggable targets that we can use to combat atherosclerosis," she said.

The paper's authors included Maedeh Zamani, PhD, Yu-Hao Cheng, Frank Charbonier, Vivek Kumar Gupta, Aaron T. Mayer, PhDAlexandro E. Trevino, PhDThomas Quertermous, MD, and Ovijit Chaudhuri, PhD.

Read more about the paper.

Dr. Ngan Huang