The brain is a ravenous organ.
A three-pound adult human brain consumes about a fifth of the body’s energy, yet it cannot store energy on its own and requires constant nourishment from the cardiovascular system.
The organ’s energy needs fluctuate greatly depending on neural activity, and sufficient blood must be delivered to the right place at precisely the right time to ensure healthy brain function.
To meet these ever-shifting demands, a process known as neurovascular coupling rapidly increases blood flow to areas of heightened neural activity.
This process is impaired in conditions such as hypertension, diabetes and Alzheimer’s disease, and also serves as the foundation for imaging technologies such as fMRI, which uses blood flow as a readout for brain activity.
Despite its importance, it was unclear how the brain and blood vessels communicate to enable neurovascular coupling.
Now, in a study published in Nature on Feb. 19, Harvard Medical School neuroscientists report the discovery of a control mechanism in the brains of mice that ensures adequate blood flow to areas of heightened neural activity in a rapid and precise manner.
Their experiments reveal that arteries in the brain actively regulate neurovascular coupling in response to neural activity, and that the protein Mfsd2a, previously implicated as a key regulator of the protective blood-brain barrier, is critical for this process.
The findings shed light on mechanisms that enable new avenues of study into the role of neurovascular coupling in neurological diseases.
“We now have a firm handle on the mechanisms involved in neurovascular coupling, including its molecular, cellular and subcellular components, which we’ve never had before,” said senior study author Chenghua Gu, professor of neurobiology in the Blavatnik Institute at HMS and a Howard Hughes Medical Institute faculty scholar.
“This puts us in a position to dissect this process and determine, for example, whether the neurovascular coupling impairments that we see in diseases like Alzheimer’s are the result of a pathology or the cause,” Gu said.
In previous studies, Gu and colleagues demonstrated that the protective integrity of the blood-brain barrier is ensured by the protein Mfsd2a, which suppresses the formation of caveolae–small lipid bubbles containing signaling molecules–from capillaries in the brains of mice.
To their surprise, they found that arteries, which carry nutrient-rich blood from the lungs and account for around five percent of the blood vessels in the brain, had the opposite characteristics as capillaries.
Arteries lacked Mfsd2a and exhibited high amounts of caveolae.
In the current study, the team investigated this observation. Spearheaded by co-first authors Brian Chow and Vicente Nunez, HMS research fellows in neurobiology, the researchers stimulated the whiskers of awake, healthy mice and simultaneously live-imaged the animals’ brain activity using a powerful technique known as 2-photon microscopy.
In response to whisker stimulation, normal mice showed increased neural activity, arterial diameter and blood flow in the corresponding sensory area of the brain.
However, mice genetically engineered to lack caveolae had the same neural activity but significantly reduced blood flow and arterial dilation, indicating deficits in neurovascular coupling.
The team specifically blocked endothelial cells that make up the lining of arteries from forming caveolae, by forcing these cells to express the normally absent Mfsd2a.
This again resulted in significant impairments to neurovascular coupling, demonstrating the importance of caveolae in the arteries.
Additional experiments demonstrated the unique role of arterial endothelial cells. It was previously known that neural activity relaxes the smooth-muscle cells that surround arteries, which leads to vessel dilation and increased blood flow.
In contrast, the results of the current study revealed a different mechanism in which caveolae in arterial endothelial cells enable neurovascular coupling by relaying the signal to relax from neurons to smooth-muscle cells.
“For over a century, we’ve known that this phenomenon exists, where neural activity rapidly increases blood flow in a very local and temporally precise manner,” Chow said. “But the mechanisms for how the nervous system talks to the vascular system to coordinate this event were largely unknown, and it was extremely surprising to find that arterial endothelial cells play such an active role in the process.”
The team also discovered that caveolae functioned independently of nitric oxide signaling–an important systemic pathway that regulates blood vessel dilation, famously targeted by medications like nitroglycerine for heart failure or sildenafil (Viagra) for erectile dysfunction.
When both caveolae and nitric oxide signaling were blocked, the team saw a complete absence of neurovascular coupling. Each mechanism appears to play an equally important but independent role in regulating blood flow in response to neural activity.
This finding suggests that caveolae in arteries may be responsible for more precisely targeted increases in blood flow, whereas nitric oxide acts more broadly, the authors said.
Arteries in a mouse brain. Image is credited to Gu Lab/Havard Medical School.
Gu and colleagues are now investigating the exact composition of the signaling molecules contained within caveolae to better understand this process.
The researchers said they hope that the newly revealed mechanistic underpinnings of neurovascular coupling will enable new experimental approaches to study the biology of this process and how it goes awry in disease.
“We’ve established a very powerful set of genetic tools that allow us to not only identify but manipulate the molecular mechanisms at the heart of neurovascular coupling,” Gu said.
“This is important given how many aspects of neurovascular coupling are still unclear.”
“For example, even if increased local blood supply is impaired, the brain still has blood flow and oxygen. What is the impact of this on neurons?
How does this affect brain function?
And does it contribute to conditions like neurovascular dementia?” Gu said.
“We are now in the position to perform rigorous science that could allow us to answer questions like these.”
Funding: The study was supported by the National Institutes of Health (K99 NS102429, R37 NS046579, DP1 NS092473, P30 NS072030), a Quan Fellowship, the Jane Coffin Childs Memorial Fund, Fidelity Biosciences Research Initiative and a faculty scholar grant from the Howard Hughes Medical Institute.
Additional authors include Luke Kaplan, Adam J. Granger, Karina Bistrong, Hannah L. Zucker, Payal Kumar and Bernardo L. Sabatini
Pericytes are multi-functional cells embedded within the walls of capillaries throughout the body, including the brain. Pericytes were first identified in the 1870s, but little attention was paid to them during the following century.
More recently, numerous vascular functions of pericytes have been identified including regulation of cerebral blood flow, maintenance of the blood-brain barrier (BBB), and control of vascular development and angiogenesis.
Pericytes can also facilitate neuroinflammatory processes and possess stem cell-like properties. Pericytes form part of the neurovascular unit (NVU), a collection of cells that control interactions between neurons and the cerebral vasculature to meet the energy demands of the brain.
Pericyte structure, expression profile, and function in the brain differ depending on their location along the vascular bed. Until recently, it has been difficult to accurately define the sub-types of pericytes, or to specifically target pericytes with pharmaceutical agents, but emerging techniques both in vitro and in vivo will improve investigation of pericytes and allow for the identification of their possible roles in diseases.
Pericyte dysfunction is increasingly recognized as a contributor to the progression of vascular diseases such as stroke and neurodegenerative diseases such as Alzheimer’s disease. The therapeutic potential of pericytes to repair cerebral blood vessels and promote angiogenesis due to their ability to behave like stem cells has recently been brought to light.
Here, we review the history of pericyte research, the present techniques used to study pericytes in the brain, and current research advancements to characterize and therapeutically target pericytes in the future.
History of Pericyte Research
Pericytes were first identified in the late 19th century by Eberth, then Rouget, as spatially isolated cells associated with the capillary wall (Attwell et al., 2016). They were found embedded within the basement membrane, both on straight sections and at branch points of capillaries, with projections extending from the soma to wrap around the underlying vessel.
In the 1920s, Zimmerman named these cells pericytes, stating that he included various cell morphologies under the definition of pericyte, including their transitional forms to vascular smooth muscle cells (VSMC) at the arteriolar end (Zimmermann, 1923).
This definition is used broadly in research investigating pericyte function and has led to debate over what constitutes a pericyte (Hill et al., 2015; Attwell et al., 2016). Throughout the 20th century, there was relatively little research into pericytes.
With advancing technologies and more accurate techniques to identify and study pericytes, research into pericyte function has increased exponentially over the last 15 years (Figure 1A).
Numerous studies have reported novel functions for pericytes in different organs of the body, including the heart (Avolio and Madeddu, 2016) and the brain (Sweeney et al., 2016), with 25% of all papers on pericytes focused on the brain (Figure 1A).
Current research largely focuses on characterizing pericyte mechanisms of function, defining pericyte sub-classes (Figure 1B), their roles in health and disease, and their potential as a therapeutic target to treat disease.
Current State of Knowledge on Pericytes in the Brain
Pericytes in the Neurovascular Unit
The brain is one of the most energy-demanding organs in the body. Despite only accounting for 2% of the body’s mass, over 20% of cardiac output is distributed to the brain to meet oxygen and glucose requirements at rest (Xing et al., 2017).
To deliver blood flow to neuronal and glial cells, the mammalian brain has evolved the neurovascular unit (NVU), a complex unit of cells that connects the brain parenchyma to the cerebral vasculature.
The NVU consists of brain parenchymal cells including excitatory neurons, inhibitory interneurons, astrocytes, and microglia interacting with vascular cells including pericytes (on capillaries), VSMC (on arterioles and arteries), and endothelial cells (EC) (McConnell et al., 2017; Figure 2).
The NVU plays important roles in maintaining brain function, particularly the regulation of cerebral blood flow (CBF) and the formation of the blood-brain barrier (BBB).
Pericytes are central to NVU function as they are located at the interface between the brain parenchyma and the blood vessels, and so can act as chemical sensors to enable communication between the two groupings of cells.
Functions of Pericytes
Cerebral Blood Flow
The mammalian brain has evolved a mechanism for regional control of CBF known as neurovascular coupling, which ensures a rapid increase in the amount of CBF directed to active neurons (Attwell et al., 2010). Neurovascular coupling is controlled by the cells within the NVU, which includes pericytes.
Within the brain, pericytes can actively relax or contract to change CBF in response to localized changes in neuronal activity (Hall et al., 2014; Mishra et al., 2016; Kisler et al., 2017b; Cai et al., 2018).
Pericytes possess contractile proteins including alpha-smooth muscle actin (α-SMA), tropomyosin, and myosin which give rise to their contractile ability (Rucker et al., 2000; Alarcon-Martinez et al., 2018).
However, it appears that only a subset of pericytes perform this role, namely the ensheathing pericytes at the arteriolar end of the capillary bed which express higher amounts of α-SMA compared to thin-strand pericytes in the middle and at the venous end of the capillary, though this remains controversial (Alarcon-Martinez et al., 2018).
Still, it is important to note that red blood cells deform the walls of capillaries when passing through (Jeong et al., 2006), so any change to tone or rigidity of mid-capillary or venule end pericytes may also affect CBF by altering the stiffness of the capillary wall, thereby changing capillary transit time (Attwell et al., 2016).
Vascular Development and Maintenance
Correct development of the cerebral microvasculature is essential for neuronal function and pericytes play a critical role in both development and maintenance of cerebral microcirculation.
In adult-viable pericyte-deficient mice, pericyte loss leads to vascular dysfunction via a reduction in brain microcirculation, diminished capillary perfusion, loss of blood flow responses to brain activation, and BBB breakdown associated with brain accumulation of neurotoxic serum molecules (Bell et al., 2010).
EC use growth factors such as angiopoietin 1, transforming growth factor beta (TGF-β) and platelet-derived growth factor-BB (PDGF-BB) to direct pericytes to migrate to new vessels in order to stabilize the vascular wall (Ribatti et al., 2011).
At the capillary level, pericytes appear to control the cell cycle of EC, as well as directly contribute to the formation of the basement membrane (Bergers and Song, 2005).
Pericytes also secrete angiogenic-promoting factors such as vascular endothelial growth factor and neurogenic locus notch homolog protein (NOTCH) 3 to activate angiogenic processes in the adult central nervous system (Ribatti et al., 2011).
The Blood-Brain Barrier
The BBB is a diffusion barrier vital for preventing toxic material from the circulation entering the brain. Function of the BBB is reliant on non-fenestrated EC that form the blood vessel wall and is supported by both pericytes and astrocytes.
Pericytes can modulate and maintain the BBB through the release of signaling factors to determine the number of EC tight junctions and direct the polarization of astrocyte endfeet (Armulik et al., 2010).
A reduction in pericyte numbers can cause a loss of tight junctions between EC, leading to increased BBB permeability (Sengillo et al., 2013).
In addition, pericytes can control the movement of substances between the blood stream and the brain parenchyma, including the vascular clearance of toxic species out of the brain (Ma et al., 2018).
Neuroinflammation is primarily driven by microglia, astrocytes, and infiltrating leukocytes, though pericytes are also capable of performing immune cell functions (Jansson et al., 2014). Pericytes can phagocytose other cells, respond to and express inflammatory molecules and cytokines, and present antigens to immune cells (Rustenhoven et al., 2017).
Exposure of pericytes to cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) triggers the release of inflammatory molecules and matrix metalloprotease 9 (MMP9), leading to BBB breakdown (Herland et al., 2016).
In addition, it has been shown that expression of apolipoprotein E, a major genetic risk factor for Alzheimer’s disease (Kim et al., 2009), or a lack of murine apolipoprotein E can lead to vascular dysfunction and BBB breakdown by activating the pro-inflammatory CypA-nuclear factor-κB-MMP9 pathway in pericytes (Bell et al., 2012).
As a result, pericytes are able to recruit immune cells and enable their extravasation into the brain (Rustenhoven et al., 2017). In transgenic pericyte-deficient mice there is reduced leukocyte trafficking across the microvasculature, suggesting that pericytes play a role in leukocyte recruitment into the brain (Wang et al., 2006).
Stem Cell Potential
Pericytes are often considered to be similar to mesenchymal stem cells and both in vitro and in vivo studies have shown they can differentiate into multiple cell types including angioblasts, neural progenitors, vascular cells, and microglia (Ozen et al., 2014; Nakagomi et al., 2015).
The exact mediators that control differentiation of pericytes are still under investigation but pericyte pluripotency can be utilized as a target for therapeutic intervention for a number of diseases.
An interesting example of pericyte pluripotency is from the dental pulp where pericytes could be differentiated into both glial and neuronal cell types (Farahani et al., 2019).
However, this example uses peripheral pericytes and there is increasing evidence at the single-cell transcriptomic level that there are molecular and functional differences between brain-derived pericytes and pericytes residing in the periphery (Vanlandewijck et al., 2018).