A team of scientists at the Wyss Institute at Harvard University and Boston University has created a 3D blood-vessel-on-a-chip model to investigate endothelial barrier failure, and found that inflammation disrupts the connections between endothelial cells and mural cells, causing the mural cells to retract or even detach from their usual position surrounding blood vessels and leading to further leakage.
“Most of the studies out there have thought that these different cell types communicate via the diffusion of growth factor molecules, but we’ve found that the physical connections, or junctions, between them are just as important for proper barrier function,” says co-first author Stella Alimperti, Ph.D., a Postdoctoral Researcher at the Wyss Institute and Boston University. The research is published in PNAS.
All of the blood in the human body circulates within its vast network of arteries, veins, and capillaries, separated from organs and tissues by an inner layer of endothelial cells and an outer layer of vessel-supporting mural cells and extracellular matrix that very selectively regulate what can diffuse in and out. When the endothelial cell barrier is disrupted, as in the case of injury and disease, blood leaks out of the vasculature and triggers inflammation and clotting, which can cause acute organ failure, tissue damage, and other problems.
While mural cells are known to be crucial to healthy blood vessel function, their response to inflammation has not been well studied. The blood-vessel-on-a-chip consists of a 3D matrix made of collagen through which passes a hollow tube that is seeded with endothelial cells on the inner surface and primary human bone marrow stromal cells (hBMSCs), a type of mural cell, on the outer surface. The researchers then perfused known barrier-disrupting molecules into the vessel-mimicking tube to evaluate how the cells respond: lipopolysaccharides (LPS; a toxin produced by some types of bacteria), thrombin (THBN; an enzyme that promotes blood clotting), and tumor necrosis factor alpha (TNFα; an inflammatory molecule produced by the body’s immune system). After one hour of treatment, they observed a dramatic increase in the permeability of the endothelial barrier and, interestingly, that the mural cells either retracted or detached from the vessels altogether. These results were confirmed by similar observations of openings forming in skin blood vessels in vivo following exposure to LPS.
Holes and breakages in the endothelial barrier are more likely to occur at the junctions between endothelial cells than via the cells themselves being punctured or breaking apart. The researchers found that all three of the inflammatory molecules increased the activity of a protein called RhoA, which is known to disrupt the integrity of those junctions, in both the mural and endothelial cells. When they selectively activated RhoA within the cells, they observed the same results as those caused by exposure to the inflammatory molecules (mural cell detachment and increased barrier permeability). Furthermore, activation of Rac1, a protein known to counteract RhoA, restored the integrity of the endothelial barrier and coverage of the mural cells, confirming that inflammation increases RhoA activity and decreases Rac1 activity, causing vascular leakage.
Because the mural cells physically detached from the endothelial cells when exposed to inflammation, the team hypothesized that the junctions between those different cell types were also disrupted. They focused their investigation on the protein N-cadherin, the primary molecule that mediates interactions between the two cell types. Antibody staining of N-cadherin revealed that it is highly concentrated at the junctions between healthy cells but more diffuse when the cells are either treated with inflammatory molecules or induced to overproduce RhoA, and the permeability of the blood vessel increases dramatically when production of N-cadherin in mural cells is genetically blocked.
Once mural cells detach from blood vessels they can migrate to other chronically injured parts of the body and contribute to fibrosis, or the accumulation of excess connective tissue that disrupts normal healing and organ function, which is implicated in a slew of diseases from cystic fibrosis to Crohn’s disease to liver cirrhosis. “Our study suggests that inflammatory molecules, not cell signaling proteins, are what drive mural cells into the bloodstream, and that the interaction between mural cells and endothelial cells plays a much larger role in fibrosis than previously thought,” says Chris Chen, M.D., Ph.D., Associate Faculty Member at the Wyss Institute and Founding Director of the Biological Design Center and Distinguished Professor of Biomedical Engineering at Boston University, who is the corresponding author of the paper.
“Because it is embedded within a full extracellular matrix, this blood-vessel-on-a-chip captures the essential features of the vasculature and allows different cell types to behave as they would in the body, an advance that enables discoveries that would be impossible using simple in vitromodels,” says Wyss Founding Director Don Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, as well as a Professor of Bioengineering at Harvard SEAS. “This device could become a platform that is used to understand how vascular permeability is regulated and how this influences the progression of the many fibrosis-related diseases.”
Other authors of the paper include Teodelinda Mirabella, Ph.D., Postdoctoral Associate at the Wyss Institute and Boston University; Varnica Bajaj, Student and Researcher at Boston University; William Polachek, Ph.D., Postdoctoral Fellow at the Wyss Institute and Visiting Scholar at Boston University; Dana Pirone Ward, Ph.D., Associate Professor at Mount St. Mary’s University; Jeremy Duffield, M.D., Ph.D., Associate Professor at the University of Washington; Jeroen Eyckmans, Ph.D., Postdoctoral Fellow at the Wyss Institute and Group Leader at Boston University; and Richard Assoian, Ph.D., Professor of Pharmacology at the University of Pennsylvania.
This research was funded by grants from the NIH, the Biological Design Center at Boston University, the Undergraduate Research Scholars Award, and the Ruth L. Kirschstein Institutional National Research Service Award. Additional support was provided by the Wyss Institute at Harvard University.