The blood-brain barrier (BBB) plays an essential role in protecting the central nervous system (CNS) from harmful bacteria, toxins, and other blood-borne pathogens. However, what exactly controls the permeability of the blood-brain barrier is a question that has long bedeviled scientists. Building on their previous work, a team led by Chenghua Gu, PhD, professor of neurobiology at the Blavatnik Institute at Harvard Medical School (HMS) and a Howard Hughes Medical Institute investigator, has now revealed new insights into the regulation of a cellular trafficking system known as transcytosis, which plays a key role in controlling blood-brain barrier permeability by determining how easily molecules can be transported across it.

Their studies in mice, reported in Neuron, describe a mechanism by which cells in the surrounding microenvironment, or extracellular matrix (ECM), signal to cells that make up the BBB. They found that this intercellular communication inhibits transcytosis to keep the barrier less permeable and ensure that molecules cannot easily pass through.

“Our work opens the door to better understand how and why the microenvironment is important for maintaining the blood-brain barrier,” Gu said, which could inform the development of better laboratory models to study it.

Identifying the mechanism offers a potential avenue for manipulating the barrier to make it more or less permeable, the researchers suggested. If their findings are replicated in further animal studies and then in humans, they could point to new ways of treating certain diseases, or strategies for delivering medicines into the brain.

Gu et al., described their findings in a paper titled, “Pericyte-to-endothelial cell signaling via vitronectin-integrin regulates blood-CNS barrier,” in which they concluded, “ … our findings reveal new molecular targets and pathways within the CNS for the development of novel therapeutics that could aid CNS drug delivery.”

The CNS requires an optimal and tightly regulated microenvironment for efficient synaptic transmission, the authors explained. “This is achieved by blood-CNS barriers that regulate substance flux to maintain tissue homeostasis.” Composed of a tight row of endothelial cells, the BBB is semipermeable and highly selective. It allows small molecules and nutrients to pass from the blood into the central nervous system, while blocking substances that might cause infection, inflammation, and otherwise disrupt the system’s delicate balance. Moreover, the investigators pointed out, “Barrier properties are not intrinsic to these cells; rather they are induced and maintained by CNS microenvironment.”

Contrary to what its name might suggest, the blood-brain barrier is not simply a wall that physically blocks harmful molecules from crossing into the central nervous system. Rather, it acts more like a self-regulating filtration system, and its permeability changes based on the properties of the endothelial cells.

How the permeability of the BBB is controlled is a question that Gu has been studying for almost a decade. In 2014, Gu and her team at HMS established a role for transcytosis, the cellular trafficking process by which molecules cross the BBB in vesicles that are formed in endothelial cells. The team identified a gene called Mfsd2a, which suppresses transcytosis. This suppression maintains the integrity of the barrier by ensuring that molecules aren’t transported across endothelial cells. Building on this work, in 2017 Gu’s team revealed that transcytosis may be the main mechanism that controls the permeability of the blood-brain barrier. “The restrictive permeability of CNS endothelial cells that constitute these barriers is a result of specialized tight junctions and low rates of transcytosis, which limit substance exchange between blood and the CNS tissue,” the authors noted.

However, the investigators suspected that there was more to the story. They began to wonder how the permeability of the BBB is affected by the surrounding microenvironment, which is composed of cells that are physically close to the blood vessels in the central nervous system. More specifically, they became interested in adjacent neural cells called pericytes that wrap around these blood vessels. “Pericytes ensheathe capillary endothelial cells and share the same basement membrane with them allowing for elaborate cell-cell signaling between these two cells,” the team noted.

“We started off asking what are the cells in the microenvironment that could be conferring these barrier properties to endothelial cells,” explained lead author Swathi Ayloo, PhD, who conducted the research as a postdoctoral fellow at HMS and is now a senior scientist at Sanofi. “And a lot of history and other work prompted us to look at pericytes.” Similar to the blood-brain barrier, the blood-retina barrier (BRB) separates the blood from the retina. And “intriguingly” the authors noted, “the brain and retina have the highest pericyte-to-endothelial cell ratio compared with that in other tissue.” However, they acknowledged, “how pericytes signal to endothelial cells to maintain barrier integrity is unknown.”

The researchers began by sifting through RNA databases for genes highly expressed in pericytes of the retina and the brain. They identified a gene in pericytes that makes a protein called vitronectin, found in the microenvironment of the central nervous system. Then, they turned to the retina to test the importance of vitronectin in maintaining the BBB. The retina is an ideal model system, Ayloo explained, because in the first few days after a mouse is born, the blood-retinal barrier is still permeable on the outer edge of the retina, but impermeable in the middle, setting up an easy comparison.

As it turns out, vitronectin was present in the microenvironment near the endothelial cells in the impermeable part of the barrier, but was missing at the outer edge of the barrier where the endothelial cells were permeable. Furthermore, when the team knocked out the gene that produces vitronectin, the barrier became leaky.

“That set the stage for us to say, OK, we know that vitronectin is important for barrier permeability, but why is it functioning like this, what is the mechanism,” Ayloo said.

Through a series of genetic experiments in mice, the researchers established that vitronectin binds to a receptor called integrin α5 in endothelial cells to form a signaling pathway. “Lack of vitronectin or mutating vitronectin to prevent integrin binding, as well as endothelial-specific integrin deletion—all of these genetic manipulations result in dysfunctional blood-CNS barriers, highlighting the role of ligand-receptor interactions of pericyte-to-endothelial signaling in barrier function,” they wrote. This signaling pathway inhibits transcytosis by telling endothelial cells in the blood-brain barrier to maintain the tension of their membranes, which prevents the formation of vesicles that can transport molecules across the barrier.

“The adhesive forces generated by the engagement of integrins have been shown in various cell types to maintain plasma membrane tension. Disengaging integrin receptors from vitronectin likely causes a reduction in membrane tension, and it is well known that decreased membrane tension promotes increased endocytosis,” the scientists added. “Thus, it is plausible that vitronectin binding to integrin α5 exerts adhesive forces to maintain the plasma membrane tension to ensure low rates of transcytosis in CNS endothelial cells.

“When we zoom out, it totally makes sense, because this mechanism is basically controlling the biophysical properties of the membrane, and that determines how easy or how difficult it is to form those vesicles,” Gu said. Ayloo added, “The big punch line is that there is this very active ligand-receptor signaling between pericytes and endothelial cells, and you need that active interaction for the maintenance of the barrier.”

Gu described the microenvironment as “a really mysterious thing” that is present around all tissues yet is exceedingly difficult to study. For her, the paper not only reveals a specific mechanism in this microenvironment that controls permeability of the BBB, but opens the door for more research on cellular signaling in the extracellular matrix.

In fact, the extracellular matrix is known to break down in neurodegenerative diseases such as multiple sclerosis. “That’s one of the hallmark features of neurodegenerative diseases, so I think we need to do more extracellular matrix research,” Ayloo said. She added that there are many protein-to-protein interactions between the microenvironment and the blood-brain barrier that need to be better understood, especially within the context of disease.

Understanding the microenvironment could also lead to better laboratory models to study the BBB. Currently, Gu explained, these models typically include only endothelial cells, and don’t incorporate the microenvironment. “You cannot just put endothelial cells together in an in vitro model and claim that this is the blood-brain barrier,” Gu said. “Billions of dollars are being spent on inaccurate models.” Once researchers identify the full collection of proteins in the microenvironment that influence the permeability of the blood-brain barrier, she added, “we can re-create those factors in a model to more accurately mimic the barrier.”

If the newly reported findings are affirmed in further experiments and, eventually, in humans, the specific interaction between vitronectin and integrin α5 may offer a new molecular target for manipulating the permeability of the blood-brain barrier.

Gu estimates that around 90% of retinal diseases are related to barrier leakage, necessitating treatments that make the barrier less permeable—something that also appears to be true for certain neurodegenerative diseases. On the flip side, in some conditions, the barrier needs to become temporarily more permeable so that medicines can be delivered into the brain. “By identifying this basic molecular mechanism, now maybe we can find agonists that target this mechanism to tighten the barrier, and inhibitors that target this mechanism to open it,” Gu said.

“Not surprisingly, ECM breakdown is a hallmark feature associated with many diseases and disorders of the CNS,” the scientists pointed out. “Together, our results indicate that ligand-receptor interactions between pericyte-derived vitronectin and its integrin receptor expressed in CNS endothelial cells are critical for barrier integrity and may provide novel therapeutic opportunities for CNS drug delivery.”

Perhaps most importantly, the work highlights the fundamental role of the microenvironment in controlling the permeability of the blood-brain barrier and brings researchers another step closer to a complete understanding of how the blood-brain barrier works.

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