Using 3D Model to Study Effect of Curvature on Endothelial Cells

Three-dimension image of a purple-hued blood vessel
Seen here from a top-down view, engineered spiral blood vessels twist and curve through three-dimensional space (endothelial cells are shown in magenta).

Picture the route a car might take through a city. To reach the other side of town, it must navigate a complex network of wide, straight highways, busy streets and avenues, and side roads that bend and curve in unpredictable patterns. Civic engineers and traffic safety experts hoping to understand how differences in this varied environment influence human behavior might use sophisticated models to study whether each road type made drivers more or less likely to slow down, speed up, change lanes, or collide with one another.

On a much smaller scale, our blood vessels also come in many shapes and sizes. And researchers who study the human body are just as curious about the traffic patterns in blood vessels.  One long-standing area of inquiry involves the relationship between the dynamics of blood flow through a vessel and the response of cells of the vessel wall, known as endothelial cells.

To date, most research on this question has focused on the factors like diameter and flow rate in the larger vessels. This scenario, in which blood cells move in orderly fashion through a straight passage, is referred to as laminar flow. Blood often moves like this in our bodies, but not always. Disturbed flow occurs in the aortic arches that help pump blood throughout the body.

How endothelial cells respond to conditions when vessels narrow and twist and turn has remained a relatively open question.

Side-by-side image of scientists
Ying Zheng PhD (L) and Christian Mandrycky (R)

New research led by Ying Zheng, Associate Professor of Bioengineering and a faculty member in the Institute for Stem Cell and Regenerative Medicine (ISCRM) sheds new light on the interplay of vessel geometry and the behavior of endothelial cells. The findings of the study, authored by PhD student Christian Mandrycky, were published this week in the journal Science Advances.

In a clever feat of engineering, the research team developed a synthetic three-dimensional, spiral-shaped platform that allowed them to introduce a new factor that they suspected influenced endothelial cells in important ways. That factor, which has been prohibitively difficult to reproduce, is known as curvature in three-dimensional space.

“The vascular system is full twists and turns,” says Mandrycky. “As you branch off and go into the organs, blood flow becomes more complicated. Our goal was to generate 3D engineered vessels that reproduce this environment and then see how curvature affects the endothelial cells.”

Side-by-side images of a straight vessel and spiral vessel shown stained red and blue
When vessels are simultaneously perfused with two fluorescent bead solutions (red and blue), rotation and mixing occur only in spiral vessels

The ability to explore the complex dynamics at work in our blood vessels has important real-world implications.  Our blood vessels – from the larger aorta to the tiniest capillaries – carry oxygen and nutrients throughout our body. They are constantly growing, adapting, and repairing to meet changing needs over the course of a lifetime. Exploring the factors that regulate these changes can tell researchers a great deal about the mechanisms of regeneration and about the origins of vascular diseases.

According to Mandrycky, the curvature in the spiral platform causes a rotational mixing effect. Where molecules moving through the vascular equivalent of a straight highway tend to stay in their proverbial lanes, curves in the vessels cause them to become jumbled, cut each other off, and even jump over one another.

To analyze how the endothelial cells responded to this manufactured chaos, Mandrycky and Zheng partnered with Brandon Hadland MD, PhD, Assistant Professor, Pediatric Oncology and Stem Cell and Gene Therapy Programs at Fred Hutchinson Cancer Research Center. Together, the researchers used bulk and single-cell RNA sequencing methods.  “The most fundamental thing we showed is that cells indeed behaved differently,” explains Mandrycky.  “Specifically, we saw surprising transcriptional patterns, meaning that the curvature was leading to changes in gene expression.”

Mandrycky and Zheng believe the study provides important insights about why our bodies have so much heterogeneity in terms of vascular response – why some vessels grow more and some grow less. From an engineering standpoint, it also gives researchers new design principles for growing 3D tissue in the lab and new ideas about how to potentially treat people with heart disease or other vascular conditions.

Engineered tumor models
Proof-of-concept spiral tissues included an engineered tumor model (left, vascular cells in green, tumor cells in red) as well as an vascularized heart chamber (right, vascular cells in green, heart cells in red).

In a proof-of-concept aspect of the investigation, the researchers used the 3D system to engineer a tumor, which they placed in the middle of the spiral so that it was surrounded by – but not connected to – the blood vessels also present in the model. In this manmade setup, the tumor pulled healthy vessels toward it just as it would in the body. To further demonstrate that the platform could be used to model complex tissue functions, Zheng and Mandrycky also created a vascularized heart chamber in which stem cell-derived heart muscle contracted in the spiral vessels, just as they might in their natural environment.

Looking ahead, the researchers are digging deeper into the actual mechanisms causing behavior changes in the endothelial cells.  “We want to know more about the biophysical dynamics at work, what exactly the cells are sensing, and the functional consequences for the cells,” says Zheng.  “We want to learn more about the engineering nature of our blood vessels and to build better models to combat diseases.”

Acknowledgements:

This work is supported by National Institute of Health: R01HL141570, 815 UG3TR002158, and UH2/UH3 DK107343 (to YZ), NHLBI K08HL140143 (to BH), and 816 Gree Foundation (to CM).