Cellular and Molecular Life Sciences
(2022) 79:372
https://doi.org/10.1007/s00018-022-04403-1
Cellular and Molecular Life Sciences
REVIEW
Historical and current perspectives on blood endothelial cell
heterogeneity in the brain
Ryota L. Matsuoka1,2
· Luke D. Buck1,2 · Keerti P. Vajrala1,2,3 · Rachael E. Quick1,2 · Olivia A. Card1,2
Received: 8 April 2022 / Revised: 18 May 2022 / Accepted: 25 May 2022
© The Author(s) 2022
Abstract
Dynamic brain activity requires timely communications between the brain parenchyma and circulating blood. Brain–blood
communication is facilitated by intricate networks of brain vasculature, which display striking heterogeneity in structure
and function. This vascular cell heterogeneity in the brain is fundamental to mediating diverse brain functions and has long
been recognized. However, the molecular basis of this biological phenomenon has only recently begun to be elucidated.
Over the past century, various animal species and in vitro systems have contributed to the accumulation of our fundamental
and phylogenetic knowledge about brain vasculature, collectively advancing this research field. Historically, dye tracer and
microscopic observations have provided valuable insights into the anatomical and functional properties of vasculature across
the brain, and these techniques remain an important approach. Additionally, recent advances in molecular genetics and omics
technologies have revealed significant molecular heterogeneity within brain endothelial and perivascular cell types. The
combination of these conventional and modern approaches has enabled us to identify phenotypic differences between healthy
and abnormal conditions at the single-cell level. Accordingly, our understanding of brain vascular cell states during physiological, pathological, and aging processes has rapidly expanded. In this review, we summarize major historical advances and
current knowledge on blood endothelial cell heterogeneity in the brain, and discuss important unsolved questions in the field.
Keywords Brain vascularization · Angiogenesis · Cell diversity · Blood–brain barrier · Fenestrations · Neurological
diseases · Vascular therapy
Abbreviations
AD
Alzheimer’s disease
ALPM
Anterior lateral plate mesoderm
ALS
Amyotrophic lateral sclerosis
AP
Area postrema
APC
Activated protein C
BBB
Blood–brain barrier
CNS
Central nervous system
CPs
Choroid plexuses
CVOs
Circumventricular organs
Cyp26
Cytochrome P450 family 26 enzymes
* Ryota L. Matsuoka
matsuor@ccf.org
1
Department of Neurosciences, Lerner Research Institute,
Cleveland Clinic, Cleveland, OH 44195, USA
2
Department of Molecular Medicine, Cleveland Clinic Lerner
College of Medicine, Case Western Reserve University,
Cleveland, OH 44195, USA
3
Present Address: Kansas City University College
of Osteopathic Medicine, Kansas City, MO 64106, USA
EMPs
ME
MS
NH
NVU
OVLT
PDGF-B
PDGF-C
PG
PLVAP
RA
SCO
SFO
TGF-β
TFRC
vECs
VEGFs
Erythro-myeloid progenitors
Median eminence
Multiple sclerosis
Neurohypophysis
Neurovascular unit
Organum vasculosum of the lamina terminalis
Platelet-derived growth factor B
Platelet-derived growth factor C
Pineal gland
Plasmalemma vesicle-associated protein
Retinoic acid
Subcommissural organ
Subfornical organ
Transforming growth factor beta
Transferrin receptor
Vascular endothelial cells
Vascular endothelial growth factors
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Introduction
Throughout the body, blood vessels deliver oxygen and
nutrients while removing metabolic wastes. In the brain,
continuous and coordinated blood supply and waste clearance ensure the constant engagement of brain cells in processing enormous amounts of environmental stimuli and
executing commands. To meet the high energy demands of
the brain, an elaborate network of blood vessels is formed
during development and maintained afterwards. Brain vasculature is organized in a specialized manner to support
the diverse functions of the brain while protecting it from
harmful blood-borne factors [1, 2]. Similar to peripheral
blood vessels, vascular endothelial cells (vECs), the innermost layer of blood vessels, are covered by perivascular
cells, such as smooth muscle cells and pericytes, to build
functional brain vasculature. A notable difference in the
brain as compared to other tissues is that vascular cells
form close associations with neurons and glial cells to
develop the neurovascular unit (NVU)—a brain–vascular
interface where the blood–brain barrier (BBB) forms to
limit cellular and molecular transport into the brain parenchyma [3, 4]. Most blood vessels in the brain establish this
semi-permeable barrier, while others lack BBB properties
and instead develop more permeable (fenestrated) phenotypes [5, 6]. Regardless of this heterogeneity in barrier
properties, blood vECs in the brain are broadly classified
into three types based on structural, molecular, and functional features: arterial, venous, and capillary endothelium
[7–9]. Arterial and venous ECs share multiple conserved
markers between the brain and other organs, while more
prominent transcriptional heterogeneity is noted for capillary ECs [10]. A greater extent of organotypic features at
the level of capillaries along the arterial–capillary–venous
axis raises the question of how endothelial phenotypes are
uniquely specified along this axis to meet organ-specific
needs. However, the mechanisms by which brain vECs are
specified into distinct subtypes within an interconnected
brain vascular network have been understudied, and therefore remain unclear.
Across species, brain barriers are critical interfaces
between the brain parenchyma and circulating blood
[11–15]. Thus, disruptions in barrier properties of brain
vasculature can be detrimental to brain health. For
instance, current evidence has illuminated age-related
changes in brain vascular structure and function. Declines
in capillary density, reduced angiogenic potentials,
decreased blood flow, impaired barrier properties, and
vascular hypoperfusion are all hallmarks of aging brains
[16–18]. These age-induced vascular declines, along with
increased BBB permeability, can trigger neuroinflammation, which may lead to neurodegeneration and subsequent
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neurological deficits [19, 20]. A recent study suggests that
counteracting the insufficiency of vascular endothelial
growth factor (VEGF) signaling that occurs as animals age
may prevent these vascular declines, thereby promoting
healthy aging and extended life spans [21]. Other studies
have proposed that age-induced shifts in endothelial transcellular transport machinery [22] and increased senescence
in brain vECs [23] are potential underlying mechanisms of
the endothelial dysfunction that causes BBB breakdown
[22, 23]. However, it remains unknown whether aging has
a specific or global impact on distinct subtypes of brain
vECs, and which structural and functional changes individual vEC subtypes experience with aging.
BBB breakdown is not only observed during brain aging,
but it has also been documented during the progression of
numerous neurological diseases, including Alzheimer’s disease (AD) and multiple sclerosis (MS) [24, 25]. Hence, therapeutic interventions which could mitigate BBB dysfunction
have been pursued as promising treatment options to prevent
the progression of these disorders [26–28]. For example, a
recent study demonstrates that genetically engineered Wnt7
ligands, which are crucial for BBB integrity, can prevent
BBB breakdown and disease progression in several neurological disease models, illuminating their pharmaceutical
potential to protect BBB function in both aged and neurological disease states [26]. However, in light of studies showing
that forced activation of canonical Wnt/β-catenin signaling
in fenestrated brain vECs can partially convert them into a
BBB state [29, 30], it remains unclear how BBB restoration
therapies may affect physiological function of fenestrated
brain vECs. A deeper understanding of brain vEC subtype
dysfunction and restoration will help develop vEC typespecific vascular therapies for neurological disorders.
The purpose of this review is to provide an overview
of the major advances in the field of brain vascular biology, thereby clarifying our current knowledge and setting
the ground for future research. This review focuses on covering topics related to the heterogeneous nature of brain
blood vECs and does not describe in great detail topics that
have been thoroughly reviewed elsewhere, including BBB
cell biology, physiology, and pathology [6, 25, 31–35].
Heterogeneous permeability, anatomy,
and transcriptional profiling of brain
endothelial cells: from historical
observations to current perspectives
The first evidence of brain barrier properties came from
dye injection studies in embryos [36–39]. Vital dyes such
as trypan red, Evans blue, methylene blue, and trypan blue
were injected into embryos of different species, including
rodents, rabbits, dogs, cats, guinea pigs, and chicks [36, 37,
Historical and current perspectives on blood endothelial cell heterogeneity in the brain
39, 40]. These dyes stained almost all tissues throughout the
body, but they were excluded from the cerebrospinal fluid
and most parts of the central nervous system (CNS). Later
studies discovered that the lack of staining in the brain was
due to the barrier properties of brain vECs, marking the
identification of the endothelial BBB [41–43]. Conversely,
there are small regions of the brain localized in close proximity to the midline brain ventricular systems that were
stained after vital dye injections [38, 39, 44–46]. These
stained brain regions were the choroid plexuses (CPs) and
circumventricular organs (CVOs), named by Helmut Hofer
in 1958 [47].
The CVOs include the subcommissural organ (SCO),
organum vasculosum of the lamina terminalis (OVLT),
subfornical organ (SFO), median eminence (ME), area postrema (AP), pineal gland (PG), and neurohypophysis (NH)
[5, 48]. All CVOs contain neural tissues responsible for
neuroendocrine function. In contrast, the CPs do not contain a neural tissue, thus they are not typically classified as
CVOs. In both the CPs and CVOs, capillary networks lack
the BBB and tight junctions that create a paracellular barrier in vECs, permitting high vascular permeability. These
structural and functional features are in stark contrast to the
limited permeability of those that form the BBB in the rest
of the brain. The exception to this high vascular permeability
is the SCO which does not form fenestrated capillaries [49].
Previous studies noted vascular permeability differences
between the sensory and secretory CVOs in the adult mouse
brain [50, 51]. In these studies, the authors used tracers of
various molecular masses and observed different degrees of
extravascular leakage and diffusion of these tracers across
and within the CVOs, indicating that vasculature in each
CVO displays unique barrier properties [50, 51]. Tight junction protein ZO-1 immunolabeling displays discrete protein
localization patterns associated with vECs across the CVOs,
providing additional evidence of heterogeneous vEC barrier
properties in the CVOs [52].
Ultrastructural analyses using electron microscopy have
been the gold standard to date for characterization of vEC
anatomical structures. Electron microscopic studies in the
1960s revealed the presence of specialized tight junctions
in vECs of the cerebral cortex that form the BBB [41, 42,
53]. In the late 1900s, studies identified the presence of
endothelial fenestrae in the NH [54, 55], the AP [56, 57],
the PG [58, 59], the SFO [60, 61], the OVLT [62, 63], the
ME [54, 64, 65], and the CPs [66, 67]. Additionally, electron
microscopic studies in some of these CVOs suggest that the
number of endothelial fenestrae increases during development following vessel formation [54, 55, 65, 67], indicating
that the induction of endothelial fenestrations takes place
progressively after vessel formation.
The evolution of DNA sequencing and genome editing
technologies since the late 1900s has facilitated molecular
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genetic studies in a variety of model organisms. Besides
mammals, zebrafish are an excellent example of an emerging model organism that exhibits barrier and non-barrier
properties across the brain, similar to mammals [13, 68, 69].
Moreover, the well-conserved anatomical features of resident cell types and endothelial fenestrations in the CVOs and
CPs have been documented between mammals and zebrafish
[48, 70–73]. The fruit fly, Drosophila melanogaster, has also
recently emerged as a genetic model to study BBB biology
[12, 74], although it is unclear whether or not this organism possesses fenestrated brain vECs. The use of a range
of model organisms has compensated for the limitations of
each individual organism, and allows insights into evolutionarily conserved mechanisms of brain barrier operations.
Comparative anatomy and function of BBB and fenestrated
vECs between the human, mouse, and zebrafish brain are
illustrated in Fig. 1.
Most recently, cutting-edge next-generation sequencing
platforms have offered genome-wide gene expression profiling at a wide range of sample scales and cellular resolutions.
For instance, bulk transcriptomic analyses of vECs isolated
from different organs using microarray and RNA-seq technologies have identified organ-specific, endothelial transcriptional signatures, including those unique to the brain
[75–79]. Furthermore, single-cell RNA sequencing (scRNAseq) of adult mouse and human brain vECs revealed gradual
transcriptional changes along the artery–capillary–vein axis,
a phenomenon known as zonation (Fig. 2) [9, 10, 80–82].
Even greater transcriptional differences were noted between
BBB and fenestrated vECs [10]. Unsupervised clustering of
the scRNA-seq data obtained from male and female adult
mouse brain vECs revealed sex differences in brain endothelial cell transcriptomes [83, 84], indicating that sex is a crucial factor influencing transcriptional heterogeneity in brain
vECs. These technologies have begun to be applied to the
generation of brain vEC transcriptomes in many different
contexts, which are discussed in later sections.
Embryonic origins of brain endothelial cells
The initial steps of vascular development take place during gastrulation as mesodermal progenitors commit to an
endothelial fate [85, 86]. vECs have been shown to derive
from both the lateral plate mesoderm and the paraxial
mesoderm (also known as the presomitic mesoderm) in
zebrafish [87–89], chicks [90–92], and mice [93–95]. It
was long believed that after the de novo formation of a
primitive vascular network, new blood vessels arise exclusively by sprouting from pre-existing endothelial cells via
angiogenesis [96, 97]. However, a recent study identified
yolk sac-derived erythro-myeloid progenitors (EMPs) as
another source of brain vECs in mice [98], challenging the
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A
B
C
Fig. 1 Comparative anatomy and function of BBB and fenestrated
vECs between human, mouse, and zebrafish. A Schematic diagrams of the midline sagittal views of the brain in human, mouse,
and zebrafish, indicating the locations of the CPs (blue) and CVOs
(green). B Representative anatomical and functional features of BBB
and fenestrated endothelium. C Schematic diagrams of the unique
anatomical and functional features of BBB (left two panels) and
fenestrated (right two panels) endothelium listed in B. AJs adherens
junctions, AP area postrema, BBB blood–brain barrier, CB cerebellum, CC cerebral cortex, CP choroid plexus, CVOs circumventricular organs, ME median eminence, NH neurohypophysis, OB olfactory
bulb, OT optic tectum, OVLT organum vasculosum of the lamina terminalis, PG pineal gland, SCO subcommissural organ, SFO subfornical organ, TC telencephalon, vECs vascular endothelial cells
current dogma that embryonic vessels expand solely by
the proliferation of pre-existing endothelial cells. These
findings have become controversial after a separate study
found no evidence for the contribution of EMPs to brain
vasculature using similar murine genetic tools [99]. It
is presently unclear whether ECs derived from different
embryonic origins are functionally distinct, or preferentially contribute to different brain vascular beds.
Previous studies in zebrafish indicated that vECs in
the head derive from the anterior lateral plate mesoderm
(ALPM) while those in the trunk and tail originate from the
posterior lateral plate mesoderm [100–102]. A recent study
employing retrospective cell-lineage tracing via light-sheet
fluorescence microscopy implied that the dorsal–anterior
side of the gastrula, early embryonic cell populations present
even before the ALPM forms, is the major origin of vECs in
the zebrafish head [102].
Despite these advances, how distinct brain vEC subtypes
are specified and how they develop lineages from embryonic
origins remain unanswered. Single-cell resolution fate mapping of whole brain vECs using a combination of lineage
tracing, clonal analysis, time-lapse cell tracking, and omics
approaches will help reveal vEC lineage trajectories from
embryonic origins, filling this knowledge gap.
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Molecular and structural signatures of brain
endothelial cell types
The BBB represents an evolutionarily conserved, highly
selective separation at the interface between the circulatory
system and brain parenchyma. To limit paracellular and
transcellular molecular transport, BBB vECs establish a
unique physical barrier characterized by the presence of tight
Historical and current perspectives on blood endothelial cell heterogeneity in the brain
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Fig. 2 Flowchart summary of scRNA-seq analysis steps of isolated single vascular cells. This schematic flowchart provides a snapshot of the
experimental workflow for scRNA-seq analysis. ECs endothelial cells
junctions, an extensive transport machinery, and restricted
transcytosis [25, 33, 34]. Additionally, the expression of leukocyte adhesion molecules in BBB vECs is maintained at
low levels to limit the entry of immune cells into the brain
parenchyma [25, 34]. Various isoforms of tight junction
proteins belonging to the claudin family are expressed in
brain vECs [103]; however, Claudin-5 is the most abundant
claudin [103, 104], and its localization at BBB endothelial
membranes and cell junctions is conserved across vertebrates [103, 105–107]. Conversely, Claudin-5 expression
is undetectable in fenestrated brain vECs [29, 30, 73, 106,
108]. Thus, Claudin-5 has been used as a specific marker
for BBB vECs. Other well-established specific markers
for BBB vECs include the glucose transporter GLUT1,
the docosahexaenoic acid transporter MFSD2A, the transcription factor LEF1, the tight junction proteins Occludin
and ZO1, and the multi-drug resistance pump MDR1 [29].
The BBB vEC-specific expression of these genes relative
to fenestrated brain vECs is also supported by recent transcriptomic data [29].
Plasmalemma vesicle-associated protein (PLVAP) is
an endothelial cell-specific protein, and the only known
molecular component of fenestral diaphragms and stomatal diaphragms of caveolae and trans-endothelial channels [109–111]. Fenestrae and trans-endothelial channels
are transcellular pores that allow rapid exchange of molecules [111], while caveolae are spherical invaginations of
the plasma membrane that play a role in transcytosis, the
vesicular transcellular transport of macromolecules [112,
113]. Once vasculature becomes mature, PLVAP expression is restricted to fenestrated vECs in the brain, making
it a unique molecular signature for these cells [10, 29, 72,
73]. PLVAP has also been implicated in controlling angiogenesis and immune cell trafficking in addition to its functions as a regulator of trans-endothelial molecular transport
and vascular permeability via the formation of fenestral and
stomatal diaphragms [72, 110, 111]. Inactivation and forced
activation of endothelial-specific β-catenin in mice suggest
a model whereby active β-catenin signaling suppresses
PLVAP expression in brain vECs to limit their permeability,
thereby establishing the BBB [29, 30, 114].
Over the past decades, studies have revealed that eliminating the unique molecular component(s) underlying BBB or
fenestrated vEC properties leads to impaired vascular development and/or integrity. For example, Claudin-5 deficiency
in mice resulted in vascular leakage of small molecular tracers in the brain, while endothelial tight junctions at the BBB
were largely preserved [115]. Additionally, recent studies
showed that haploinsufficiency or endothelial-specific deletion of GLUT1 in mice leads to impaired angiogenesis and
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diminished vascular complexity in the thalamus [116] and
the cerebral cortex [117], in addition to reduced pericyte
coverage resulting in increased BBB leakage [118, 119].
Lastly, in mice and zebrafish deficient for PLVAP or its
zebrafish ortholog, impaired formation of fenestral and stomatal diaphragms led to excessive or accelerated transcellular transport of blood-borne proteins through fenestrated
vasculature [72, 120, 121].
Cellular and molecular control of brain
endothelial acquisition of unique properties
Over the last two decades, substantial progress has been
made with regards to our understanding of the molecular
mechanisms governing brain vascularization and endothelial
cells’ acquisition of BBB and fenestrated properties. Brain
vascularization is initiated by angiogenic sprouting from the
peri-neural vascular plexus (PNVP), a primitive vascular
network that covers the entire surface of the neural tube, into
the brain parenchyma [122–124]. Paracrine VEGF-A signaling derived from the developing CNS is critical for PNVP
formation and subsequent blood vessel invasion, branching,
and density in neural tubes across multiple vertebrate species [125–130]. Genetic loss of heparin-binding VEGF-A
isoforms (VEGF120/120 mice that are genetically engineered
to produce solely the soluble isoform VEGF-A120) led to a
significant reduction in vessel branch points and complexity
in the brain [131], demonstrating VEGF isoform-specific
control of brain vascularization.
Classical chick-quail transplantation experiments suggested that BBB barrier properties are not intrinsic to brain
vECs, but rather, they are induced and maintained by neural environmental signals [132, 133]. These studies showed
that neural tissues transplanted into the coelomic cavities
are sufficient to induce BBB characteristics in mesenteric
vessels, whereas brain vessels do not display BBB properties
when invading into somite tissues transplanted into the brain
ventricles [132, 133]. Isolated brain vECs in culture exhibit
a rapid loss of BBB-specific transcripts and chromatin features [134], supporting the notion that neural environmental
signals are necessary for vECs to maintain BBB properties.
Development and maintenance
of endothelial BBB properties
Wnt/β-catenin signaling Canonical Wnt/β-catenin signaling has been well established as a central regulator of brain
angiogenesis, as well as of the induction and maintenance
of endothelial BBB properties. Transgenic Wnt/β-catenin
reporter mice demonstrated the specific activation of Wnt/
β-catenin signaling in CNS vECs [114, 135, 136], but not
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in those of other organs [135]. Recent work revealed that
β-catenin activities in fenestrated vECs of the CPs and
CVOs are maintained at much lower levels than in those
forming the BBB in both mouse and zebrafish models [29,
30]. Mechanistically, endothelial cell-specific deletion of
β-catenin in developing mice results in a drastic reduction
of BBB-specific proteins, including Claudin-5 and GLUT1,
while leading to increased expression of the fenestrated
vEC marker PLVAP [114, 135, 136]. Conversely, endothelial cell-specific β-catenin stabilization leads to the opposite
outcomes in these gene/protein expressions [114]. Moreover,
β-catenin stabilization in fenestrated vECs of the CPs and
CVOs is sufficient to partially convert them into BBB-like
states in vivo [29, 30], although it is unable to restore the
loss of the BBB transcriptional and chromatin landscapes in
primary brain vECs after short-term in vitro culture [134].
The critical role of β-catenin in BBB maintenance was further demonstrated via its deletion in endothelial cells of
postnatal or adult mice [137, 138]. Endothelial β-catenin
signaling was also shown to regulate vascular pericyte coverage in the mouse brain via modulation of Pdgfb expression
[139]. Altogether, these findings support a model in which
endothelial β-catenin signaling is central to the induction
and maintenance of BBB properties in vECs and inhibits
the expression of the fenestration marker PLVAP to limit
vascular permeability.
Two classes of β-catenin activators (Wnt7a/Wnt7b and
Norrin) have been well characterized in mice, which redundantly direct brain angiogenesis and BBB formation/maintenance in a brain region-specific manner. Wnt7a and Wnt7b
are expressed largely in overlapping domains, and the combined, but not individual, loss of these two genes results in
severe brain angiogenesis defects in mice [135, 136]. Wnt7a
overexpression in the neural tube in vivo, or in primary cultures of mouse brain endothelial cells in vitro, is sufficient
to enhance GLUT-1 expression in brain endothelial cells
[135, 136]. Functional redundancy between Wnt7a and Norrin in BBB maintenance was identified in the cerebellum
where these double mutant mice displayed increased BBB
permeability and elevated PLVAP expression in vECs [140].
The key components of Wnt7s/β-catenin signaling are the
receptor complexes consisting of Frizzled, Lrps, Gpr124,
and Reck [138, 141–144], while Norrin/β-catenin signaling requires Frizzled-4 [145, 146] and another co-receptor
component called Tspan12 [140, 147]. A recent study has
reported that the guidance cue Netrin-1 signaling through
its Unc5B receptor is critical for activating endothelial
β-catenin pathways to maintain BBB integrity in mice [148].
Similarly, the crucial roles of Wnt7/β-catenin signaling
through Gpr124/Reck receptors in regulating brain angiogenesis and BBB formation have been well documented in
zebrafish [26, 149–151]. Additionally, a study in zebrafish
showed that brain angiogenesis and transcriptional induction
Historical and current perspectives on blood endothelial cell heterogeneity in the brain
of endothelial BBB differentiation occur simultaneously during development [152]. Whether brain vECs establish BBB
or fenestrated functional properties in a simultaneous or progressive manner in relation to vessel formation remains an
important question.
Retinoic acid (RA) signaling RA signaling is indicated
to be another important inducer of BBB properties in
brain vECs, although its role in vivo is not entirely clear.
High concentrations of RA treatments in vitro can induce
BBB properties in cultured murine brain endothelial cells
[153]. Pharmacological inhibition of RA receptor signaling in pregnant mice at the stage of brain angiogenesis and
BBB formation (E10.5–16.5) leads to increased vascular
leakage of tracers in the brain, in addition to resulting in
significantly reduced expression of several BBB marker
genes [154]. Endothelial RA signaling acts upstream of the
Wnt/β-catenin pathway and suppress β-catenin expression
in vECs via transcriptional suppression and phosphorylation-dependent protein degradation [139]. In mice globally
lacking the RA-biosynthetic enzyme Rdh10, reduced levels of RA production occur. Subsequently, Wnt signaling
activation and Wnt-responsive gene expression were significantly diminished, while gene expression of endogenous
Wnt inhibitors (Dkk1 and Sfrps) was upregulated [155]. In
zebrafish, pharmacological inhibition of the RA degrading enzymes, cytochrome P450 family 26 (Cyp26), led to
increased expression of Claudin-5 in fenestrated hypophyseal vessels [71], indicating that increased levels of RA can
induce BBB properties in these fenestrated vessels and that
Cyp26-mediated RA degradation represses the induction of
the BBB properties. Further investigations into the signaling
crosstalk between RA and Wnt/β-catenin pathways in vivo
will clarify the epistatic and redundant relationships between
these two pathways in BBB function.
Suppressed transcytosis Brain vECs with BBB properties
display low rates of transcytosis, the transcellular vesicular
transport of macromolecules from one side of vECs to the
other [156, 157]. Recent studies have revealed molecular
components that actively suppress transcytosis specifically
in brain vECs. Selective expression of the lipid transporter
Mfsd2a was identified in BBB vECs [158, 159], and its
genetic deletion results in impaired BBB function in mice
[158] and zebrafish [160]. This BBB endothelial-specific
expression of Mfsd2a is important to establish a unique
lipid environment that inhibits caveolae-mediated transcytosis in these ECs, thereby maintaining BBB integrity
[161]. Endothelial cell-specific deletion of β-catenin in mice
results in significant reduction of Mfsd2a expression in the
brain [162], while EC-specific β-catenin stabilization leads
to the upregulation of Mfsd2a in fenestrated vECs of the
brain [29]. A recent study has identified the extracellularmatrix protein Vitronectin secreted from brain pericytes as a
crucial ligand that restricts endothelial transcytosis in a cell
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non-autonomous manner [163], illuminating a cellular mechanism by which transcytosis in BBB vECs is suppressed.
Pericytes Pericytes are an important cell type that induces
and maintains endothelial BBB properties [164–166]. A
recent scRNA-seq study investigated endothelial cell transcriptional changes in response to pericyte deficiency
(70–80% brain pericyte loss) in adult brains of Pdgfbret/ret
mice [167]. This study showed that pericyte deficiency led
to a significant reduction of BBB endothelial gene expression, notably transporters such as Mfsd2a, while inducing
the upregulation of Plvap and leukocyte adhesion molecule
expression, resulting in BBB disruptions. Expression of
many BBB markers (e.g., Claudin-5 and Glut1) remained
unchanged in this pericyte deficiency model, indicating a
specific role for pericytes in regulating BBB function. These
gene expression changes in the adult brains are consistent
with the earlier study that identified the critical role of pericytes in BBB induction during embryogenesis and described
similar gene expression changes in embryonic brains of Pdgfrb mutant mice deficient for pericytes [164]. In this pericytedeficient mouse model, 2 distinct modes of BBB disruptions
were observed: widespread increase in vesicular transcytosis
across brain vECs [166, 167] and focal disruption of tight
junctions causing hotspot leakage [167]. Reduced endothelial
expression of Angiopoietin 2 triggers the latter mode of BBB
disruptions in the absence of brain pericytes [167].
Studies among several taxa revealed multiple embryonic
origins of brain pericytes [168], including avians [169, 170],
mice [171–173], and zebrafish [174]. A recent study suggests the existence of differences in morphology and distribution pattern of pericytes along capillary vessels across
brain regions [175], indicating that pericytes from different
developmental origins exhibit morphological and functional
differences in regulating specific types and/or function of
brain vasculature. The well-conserved role of PDGF-B signaling through its cognate PDGFR-β receptor in brain pericyte
development was documented between mice [176–179] and
zebrafish [174, 180]. CD146 is indicated to function as a coreceptor of PDGFR-β to mediate pericyte recruitment to cerebrovascular ECs and promote BBB maturation [181]. Notch3
expression in pericytes was shown to have a conserved role in
maintaining BBB integrity and mural cell coverage of brain
vasculature between mice [182] and zebrafish [183]. A recent
study identified lactate, a metabolite produced through glucose metabolism in brain vECs, as a crucial energy source
for pericytes in maintaining BBB function [119]. Emerging
scRNA-seq data have identified potential new specific markers for brain pericytes in humans [81, 184, 185], mice [80,
186], and zebrafish [187], accelerating the study on brain
pericyte heterogeneity in structure and function.
Astrocytes Astrocytes are another important cellular constitute of the BBB in mammals. Generation of astrocytes in
the mammalian brain occurs postnatally after the functional
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BBB is established [158, 164], eliminating the possibility of
an astrocyte role in endothelial acquisition of BBB properties during embryonic development. However, after passing through postnatal developmental stages, astrocytes can
be critical for BBB maintenance and repair. A recent study
indicated that mature astrocyte ablation from the mouse
brain using the inducible astrocyte glutamate transporter
Glast-CreERT line led to increased leakage of fluorescently
labeled small molecule Cadaverine (< 1 kDa) into the brain
parenchyma, suggesting BBB dysfunction following brain
astrocyte ablation [188]. However, several studies using similar astrocyte-targeted genetic ablation systems with larger
molecular tracers in the spinal cord did not report significantly increased BBB permeability [189, 190].
In the developing mouse brain, Reelin-induced activation
of endothelial Dab1 plays an instructive role in directing
astrocyte end-feet attachment to cerebral blood vessels for
functional BBB development, as genetic inactivation of this
signaling axis led to insufficient astrocyte end-feet coverage of these vessels, resulting in defective barrier properties
[191]. This Reelin-endothelial Dab signaling is not critical for
pericyte coverage and maintenance of the BBB [191], suggesting its developmental role in the assembly of the NVU.
In adulthood, astrocyte-specific deletion of Netrin-1 or Neogenin in mice resulted in increased BBB leakage and reduced
pericyte vascular coverage [192], indicating a crucial role for
astrocyte-derived Netrin-1 signaling in maintaining BBB and
NVU integrity. Similarly, genetic inhibition of Wnt secretions from astrocytes led to impaired astrocyte end-feet morphology, reduced pericyte coverage, and increased levels of
BBB permeability and endothelial transcytosis [193]. Thus,
astrocyte-derived Wnts maintain BBB and NVU integrity
after developmental stages by sustaining adequate levels of
Wnt/β-catenin activity in brain vECs and astrocytes. Future
studies will be needed to identify the specific Wnt ligand(s)
secreted from astrocytes and their underlying cellular mechanisms responsible for maintaining the NVU integrity.
Development and maintenance
of endothelial fenestrations and permeable
properties
VEGF signaling has been demonstrated as a key regulator
of endothelial fenestrations in both in vitro [194] and in vivo
[195]. VEGF-A signaling downregulates the expression of the
tight junction proteins Claudin-5 and Occludin at the mRNA
and protein levels [196], while it upregulates the mRNA and
protein expression of PLVAP [197]. Across the CVOs, higher
levels of Vegf-A mRNA expression were observed than in
adjacent brain regions in adult mice [198]. Distinct cell types
have been shown to regulate endothelial fenestrations across
the CPs and CVOs.
13
R. L. Matsuoka et al.
In the ME, specialized ependymal cells, tanycytes, display
increased Vegf-A expression in response to fasting, which
results in increased levels of endothelial PLVAP expression,
density, and fenestrations [199]. Recent work indicated that
Melanin-concentrating hormone-expressing neurons, which
extend axonal projections to the ME in close proximity to
fenestrated capillaries, directly controls endothelial density
and fenestrations via activity-dependent Vegf-A release from
their axon terminals [200]. These results are consistent with
a previous study that reported Vegf signaling-dependent continuous endothelial proliferation and angiogenesis in the ME
of adult mice [201].
In the NH, Vegf-A signaling derived from pituicytes, glial
cells of the posterior pituitary, controls continuous endothelial proliferation and angiogenesis in adult mice [202]. In the
developing zebrafish NH, pituicyte-derived Vegf and TGF-β
signaling induces plvap expression and high vascular permeability in its fenestrated capillaries [71]. In the same system,
defective fenestrated vasculature forms in the pituitary in the
absence of both the hypothalamic–hypophyseal axon tract and
signaling evoked by the neuropeptide oxytocin secreted from
its axon terminals [203].
In the CPs, several studies suggest a role for Vegf-A and
TGF-β signaling derived from the ependymal epithelium in
maintaining the integrity and fenestrations of capillaries in
adult mice [195, 204]. Our recent study identified a unique
combination of Vegf ligands required for driving fenestrated
vascular development in the zebrafish hindbrain CP, while
these ligands have little impact on the formation of neighboring BBB brain vasculature [73].
In the AP, OVLT, and SFO, Vegf-A expression in neurons
and/or astrocytes is indicated to sustain continuous endothelial
proliferation and vascular permeability of fenestrated capillaries in adult mice [198]. Additionally, in the AP, Wnt Inhibitory Factor-1 knockout mice displayed an elevated level of the
GLUT1 BBB marker [29], suggesting that locally expressed
endogenous inhibitors of Wnt/β-catenin signaling suppress
vEC’s β-catenin activities to prevent BBB formation in the
fenestrated vascular beds of the brain. Melatonin, a hormone
secreted by the PG, has been implied in the regulation of angiogenesis [205], especially inhibition of multiple tumor angiogenesis; however, its role in fenestrated capillary development
or maintenance in the PG remains unclear.
Perivascular cell diversity and their
emerging roles in controlling endothelial
properties
Recent scRNA-seq data have revealed transcriptionally
diverse subtypes of brain perivascular cells, including pericytes [80, 81, 184], fibroblasts [80, 82, 206, 207], smooth
muscle cells [80, 186, 208], as well as macrophages and
Historical and current perspectives on blood endothelial cell heterogeneity in the brain
microglia [209–212]. An increasing number of scRNA-seq
datasets that contain brain perivascular cell populations
have been reported under physiological and pathological
conditions across species, developmental stages, and brain
regions. However, except pericytes, the functional roles of
each perivascular cell type in regulating brain vEC properties remain largely unknown.
A recent study showed that perivascular fibroblasts lie
adjacent to smooth muscle cells in arterioles and largediameter venules, but are absent in capillaries, in the
adult mouse brain [213]. scRNA-seq results of embryonic
mouse brain meninges identified transcriptionally distinct
fibroblast subtypes derived from different embryonic origins in brain meningeal compartments [206]. Our recent
work identified mesoderm-derived meningeal fibroblasts
as important sources of Vegf ligands critical for directing fenestrated vessel formation in the zebrafish CP [73].
Future studies will be needed to uncover how fibroblast
subtypes, including perivascular fibroblasts, contribute to
the establishment of brain vascularization and the endothelial acquisition of unique properties across the brain.
Similarly, recent scRNA-seq data have indicated the
unique transcriptional signatures of perivascular macrophages, which are distinguishable from those of microglia, in the mouse cortex [212]. This and other emerging
scRNA-seq results have provided valuable information to
identify and establish reliable cell type-specific markers
for perivascular cells. Given the unique distributions of
perivascular cell types along vEC types and across brain
regions [175, 213, 214], future studies on dissecting the
functional roles of each cell population and subtype in
regulating vEC properties across the brain will expand
our understanding of dynamic and heterogeneous brain
vEC properties.
Current and emerging models for the fate
determination of brain endothelial cells
In light of classical studies that conducted chick-quail
transplantation experiments, neural environmental signals
or extrinsic factors are thought to be the key determinants
of brain vEC barrier properties [132, 133]. In support of
this finding, additional studies have established that canonical Wnt/β-catenin signaling is necessary [114, 135] and
sufficient [29, 30] to induce and maintain BBB properties
in brain vECs (Fig. 3A). However, it remains unknown
whether neural environmental signals are necessary and
sufficient to induce a fenestrated endothelial cell fate and
if so, what the molecular determinants of this cell fate are.
This incomplete knowledge has limited our current understanding of brain vEC fate specification across the brain,
Page 9 of 22
372
leaving open the question of whether BBB and fenestrated
vEC properties in the brain are determined entirely by
extrinsic signals.
There still remains the possibility that the distinct fates
of brain vECs derive from different embryonic origins.
Our recent results indicate that during brain vascularization in zebrafish, adjacent vECs exhibit individually distinct responses to local Vegf ligands, resulting in different
fates [73]. These findings suggest that brain vECs undergo
fate pre-determination prior to angiogenesis and that these
intrinsic properties facilitate migration of vECs to their destinations in response to environmental angiogenic factors
via “EC type-specific angiogenesis” (Fig. 3B). Thus, it is
possible that a combination of intrinsically programed predetermination and dynamic spatiotemporal presentation of
local morphogenetic cues direct fate decisions and heterogeneous network formation of brain vECs. This model is in
line with a recent study that demonstrated lineage history as
a critical determinant of EC specialization [95]. The authors
reported that the lymphatic lineage derived from the paraxial
mesoderm contributes to lymphatic endothelium in multiple
organs, but in an organ-restricted manner [95], indicating
that EC fate is imprinted before the established endothelial
genetic specification process is initiated. Identifying the key
developmental determinants of fenestrated brain vEC identities will accelerate our understanding of the mechanisms
underlying the generation of brain vEC heterogeneity.
Plasticity of brain endothelial fates
and states under physiological
and experimental conditions
Endothelial fates and states are plastic during development and in adulthood [215–217], resulting in heterogeneous permeability of this cell type. Neuronal activity has
been proposed as a crucial modulator of influx [218] and
efflux [219] transport activities in BBB vECs, thereby
controlling BBB permeability dynamics [220]. In addition, efflux transporter function in BBB vECs undergoes
circadian regulation [219, 221, 222], inducing circadian
changes in BBB permeability throughout the day. Since
the production of certain molecules and metal ions such
as hormones, neuropeptides, cytokines, and magnesium
has been reported to undergo circadian oscillations [223],
the rhythmic expression of these molecules and ions may
mediate circadian changes in BBB permeability. Sleep
loss also causes BBB permeability changes through downregulation of tight-junction protein expression [224] and
levels of endocytosis [225] in brain vECs. Other physiological factors that modulate BBB tightness include pregnancy, nutrition, body temperature, physical activity, gut
microbiota, and psychological stress [226].
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Page 10 of 22
R. L. Matsuoka et al.
A
B
Fig. 3 Current and emerging models for the fate determination of
brain endothelial cells. A In a prevailing model, brain-specific signaling induced by extrinsic factors, such as Wnt7s and Norrin, induces
the differentiation of primitive endothelial cells into a BBB EC type
as they migrate to target brain regions. B In an emerging model,
primitive endothelial cells first undergo pre-differentiation, which
allows them to migrate to target brain regions in response to specific
sets of angiogenic cues via EC type-specific angiogenesis. In the CPs
and CVOs where fenestrated ECs are generated and maintained, BBB
inducing extrinsic signals, such as Wnt7s, Norrin, and RA, are suppressed by Wnt inhibitory factors and/or RA degrading enzymes,
Cyp26. BBB blood–brain barrier, CPs choroid plexuses, CVOs circumventricular organs, Cyp26 cytochrome P450 family 26 enzymes,
ECs endothelial cells, RA retinoic acid, TGF-β transforming growth
factor beta, VEGFs vascular endothelial growth factors, WIFs Wnt
inhibitory factors
In contrast to our increasing knowledge of BBB permeability dynamics and their modulators, much less is known
about physiological states that modulate fenestrated vEC permeability in the brain. One study showed that fasting, or glucose deprivation, increases PLVAP expression and endothelial
fenestrations in fenestrated capillaries of the murine ME [199].
The activity of hypothalamic neurons involved in food intake,
locomotor activity, and sleep also controls fenestrated endothelial density and permeability in the ME [200], suggesting that
these physiological factors modulate vEC permeability states
in this brain region. Seasonal changes in the length of daylight
(i.e., photoperiod) lead to the oscillatory pattern of melatonin
secretion from the PG, which is indicated to affect fenestrated
vascular density in the sheep ME [227].
Recent studies in mice demonstrated that fenestrated brain
vECs respond differentially to forced activation of β-catenin
across the CVOs and CPs in terms of their phenotypic conversions into a BBB state [29, 30], suggesting that heterogeneity in the phenotypic plasticity of vECs exists across fenestrated vascular beds of the brain. Changes in fenestrated brain
vEC permeability can influence fluid balance, waste clearance, immune surveillance, and the efficiency of hormonal
secretion and reception, through this vessel type. However,
fenestrated vascular permeability dynamics have been understudied. Future studies on structural and functional changes
in fenestrated brain vasculature in response to physiological
and pathological stimuli will advance our knowledge of the
plasticity of this vEC type.
13
Pathological and age‑induced changes
in brain endothelial properties
and heterogeneity
Significant structural and functional changes of brain
capillaries have been reported with aging and in disease
states [2, 228–230]. For example, the brain of elderly
humans [231] and of aged mice [22] displays capillary
wall thinning and declines in capillary density, blood
flow, vascular perfusion, angiogenic potentials, and barrier properties [16–18]. Decreased pericyte coverage of
brain vasculature and diminished Mfsd2a expression were
also observed with aging [22]. Moreover, pericyte degeneration is associated with BBB disruptions in patients
Historical and current perspectives on blood endothelial cell heterogeneity in the brain
with neurological diseases such as AD and amyotrophic
lateral sclerosis (ALS) [232–235]. Since brain pericytes
regulate blood flow via capillary constriction [236, 237],
reduced cerebral blood flow as a consequence of pericyte
degeneration is another hallmark associated with aging
and age-related neurodegenerative diseases [238]. In support of these observations, forced pericyte ablation with
diphtheria toxin using an inducible pericyte-specific Cre
line in mice led to rapid BBB breakdown and neurodegeneration [239].
While brain pericyte deficiency clearly promotes BBB
disruptions, viable pericytes can also produce numerous proinflammatory mediators that are detrimental to
BBB function [240]. In aging human and rodent brains,
increased production of the pleiotropic cytokine TGF-β
was reported [241], which can upregulate a number of
inflammatory genes in pericytes in vitro [242]. Conditional loss of the transcription factor RBPJ in pericytes
of mouse brains triggers excessive production of TGF-β3
that leads to overexpression of inflammation-related genes,
suggesting a potential role for RBPJ in regulating a proinflammatory genetic program in pericytes [243]. Notably,
pharmacological inhibition of TGF-β signaling restored
cognitive impairments in aged mice [241] and ameliorated
autoimmune encephalomyelitis in a mouse model [244],
indicating that TGF-β signaling inhibition counteracts detrimental consequences of neuroinflammation.
There is a growing body of transcriptomic datasets that
offer enriched information on gene expression changes of
brain vECs over the course of development, and also in
response to disease conditions. A recently established transcriptome database of isolated mouse brain vECs under
healthy and various neurological disease states identified
a common, core pathway leading to BBB dysfunction
regardless of BBB disruption triggers [79]. This common
BBB dysfunction module among diseases includes upregulation of genes that are enriched in vECs of peripheral
organs under normal physiological conditions, indicating a
shift of the BBB vEC identity toward peripheral non-BBB
cell states [79]. Moreover, single-cell atlases of human
brain vasculature from individuals with AD [245], Huntington’s disease [185], or arteriovenous malformations
[81] have provided valuable resources to understand vascular cell-type-specific perturbations of gene expression
under these disease states compared to healthy individuals.
In contrast to BBB dysfunction linked to aging and neurological disorders, very little is known about structural
and functional alterations of fenestrated brain capillaries
under these conditions. Given that defective brain–blood
communications via fenestrated vasculature can lead to
impaired metabolic sensing and hormonal release into
the bloodstream, it is likely that structural and functional
changes in this vEC type abrogate neuroendocrine control
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and action. Indeed, alterations of neuroendocrine function
with aging have been documented [246, 247], which include
imbalanced hormone production [248] and reduced signaling reception and sensitivity for secreted hormones [249].
Considering age-related VEGF signaling declines [21] and
the crucial role of this signaling in maintaining endothelial
fenestrations in the CPs and CVOs [71, 195, 204], it is likely
that endothelial fenestration numbers, sizes, and permeability in these brain regions decline with aging and that these
age-related changes affect neuroendocrine function. Future
investigations into age-related changes in the number, size,
and/or permeability of endothelial fenestrations across the
CPs and CVOs will help understand the malfunction and
disease associated with these brain regions.
Development of potential therapeutics
targeting unique brain endothelial
properties
Pharmacological inhibition of the PDGF-C/PDGFR-α
signaling axis has been pursued as a powerful approach to
restore BBB dysfunction in neurological disorders [27].
Previous studies showed that the intracerebroventricular
injection of either PDGF-C or tissue plasminogen activator was sufficient to increase BBB permeability and induce
BBB dysfunction [250, 251]. Conversely, inhibition of the
PDGF-CC signaling by neutralizing antibodies or inhibition of PDGFR-α with imatinib reduced BBB dysfunction
in mouse models of ischemic stroke [250, 252], MS [253,
254], seizure [255], and traumatic brain injury [256]. Thus,
preserving BBB integrity by targeting this signaling axis is
a promising therapeutic approach for a wide range of neurodegenerative and neuroinflammatory diseases associated
with BBB breakdown.
Strong neuroprotective effects of activated protein C
(APC) on both acute brain injury and chronic neurodegenerative conditions make it another potential therapeutic target [28]. Intravenous injections of APC were shown to exert
beneficial therapeutic effects in mouse models of ischemic
stroke [257], MS [258], ALS [259], and AD [260]. It has
been proposed that APC functions by eliciting anti-inflammatory effects and neuroprotective actions within the NVU
to prevent BBB breakdown [28].
Another therapeutic strategy is the repurposing of a key
developmental BBB induction signal as a BBB protective
agent for neurological pathology [26]. This successful translation of developmental biology knowledge into potential
therapies opens a new avenue for developing therapeutics
for a variety of neurological and cerebrovascular diseases,
as well as for age-related vascular declines in the brain. By
conducting a screen of the numerous Wnt7a variants generated via single-residue substitutions, the authors identified
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R. L. Matsuoka et al.
Page 12 of 22
genetically engineered Wnt7a ligands that can preferentially
activate the Gpr124/Reck co-receptor complex while dramatically reducing activation of its other cognate Frizzled
receptors [26, 144]. This study further demonstrated that
these Gpr124/Reck-specific activators acted as BBB repair
agents and mitigated the progression of neurological disease
in mouse models, including glioblastoma and stroke. This
finding is consistent with a separate study where endothelialspecific β-catenin stabilization improves BBB function in
conditional Gpr124-deficient mice under pathological neurological conditions [261].
Emerging therapeutic targets include the Netrin1-Unc5B
signaling axis that is indicated to act upstream of canonical β-catenin pathways in the brain [148]. Current evidence
shows that Netrin-1 global knockouts [262] or endothelialspecific deletion of the Unc5B receptor in mice [148] led
to the down-regulation of BBB markers in vECs, while
upregulating their PLVAP expression [148]. In contrast,
Netrin-1 treatments can enhance BBB marker expression
both in vitro and in vivo [148, 192, 262] and reduce BBB
leakage in mouse models of MS [262] and BBB breakdown
A
B
Fig. 4 Expected therapeutic effects of a BBB repair agent(s) on brain
disease/injury associated with BBB dysfunction. (A) In a healthy
brain, no neuroinflammation and pathological BBB breakdown are
detected. (B) In a diseased/injured brain without any treatment, severe
neuroinflammation and BBB breakdown cause serious brain damage.
Brain disease/injury elicits a series of neuroimmune responses via
activation of microglia, astrocytes, and pericytes into proinflammatory states [240] that lead to their release of inflammatory mediators
involved in BBB disruptions. This permits blood-circulating immune
13
[192], suggesting BBB protective effects of Netrin-1-Unc5B
signaling.
The four strategies discussed above all help mitigate
BBB dysfunction, thereby preventing the progression and
worsening of neuroinflammation and BBB breakdown that
are the hallmarks of a broad range of neurological diseases
(Fig. 4). Future studies may seek to test whether treatment
approaches targeting different signaling pathways could have
additive or synergistic therapeutic effects on these diseases.
Furthermore, our increasing knowledge of brain vEC’s transcriptomic changes under a variety of disease conditions and
their shared BBB dysfunction module [79, 245] can help
identify potential new therapeutic options for BBB repairs
that may stand alone or boost the effects of the existing
approaches.
In many pathological and developmental conditions
where endothelial BBB integrity and identities are disrupted, upregulation of PLVAP expression in vECs has been
reported. For example, PLVAP upregulation is detected in
microvasculature associated with brain tumors and ischemia
[263–265] where neuroinflammation and BBB breakdown
C
cell infiltration into the damaged brain region. These neuroinflammatory events induce neurological damage. (C) In a diseased/injured
brain subjected to treatment with a BBB repair agent, mitigated neuroinflammation and BBB breakdown cause only limited brain damage. As compared to the scenario described in (B), timely administration of a BBB repair agent, such as PDGF-C signaling inhibitor
or engineered Wnt7a ligand, can ameliorate the damaging effects of
neuroinflammation by limiting BBB disruptions. BBB blood–brain
barrier, PDGF-C platelet-derived growth factor C
Historical and current perspectives on blood endothelial cell heterogeneity in the brain
occur, or in vasculature where reduced levels of endothelial β-catenin signaling are observed [114, 138, 140, 148].
These observations are consistent with the current model
that active β-catenin signaling suppresses PLVAP expression in brain vECs to establish the BBB [29, 30, 114]. Since
anti-PLVAP therapy has been explored as a potential antiangiogenesis and anti-edema therapeutic option for cerebral
edema caused by ischemic stroke and brain tumors [110,
111], this option may be pursued to limit vascular leakage
and BBB breakdown in conjunction with the other BBB
repair strategies. However, further investigations will be
needed to define the cellular and molecular mechanisms
underlying PLVAP upregulation and its contributions to
vascular leakage and BBB breakdown during aging and
neuroinflammation.
Beyond BBB-targeted therapies, efficient drug delivery
across the BBB has remained a significant challenge in treating or preventing the progression of neurological deficits.
Efforts have been made to exploit endogenous receptormediated transcytosis pathways to enhance uptake of large
molecules and therapeutics in the brain [266, 267]. Transferrin/transferrin receptor (TFRC) pathway is one of the major
transport pathways specific to the brain and BBB [268–270].
Since TFRC is highly expressed in BBB vECs [268, 269]
and brain tumors such as glioblastomas [271–273], nanoparticles targeted with TFRC-binding peptides have been
explored as diagnostic probes for imaging brain tumors [274,
275] and for boosting brain uptake of therapeutic antibodies
[276–278]. Another strategy for promoting drug delivery
across the BBB includes the use of the monoclonal antibodies that can block specific Netrin-1-Unc5B interactions or
induce Unc5B internalization [148]. These Unc5B blocking
agents were suggested to enhance size-selective BBB permeability within a limited time window [148], indicating their
potential application to transient BBB opening for therapeutic uptakes in the brain.
In contrast to the promising BBB repair approaches,
current molecular targets for repairing fenestrated brain
vECs are substantially limited. PLVAP is a well-established
marker for fenestrated brain vECs and currently known to
be the only structural components of fenestral and stomatal diaphragms [109–111]. In conditions where high vascular permeability in fenestrated brain vECs declines due
to reduced numbers of endothelial fenestrations with aging
and/or disease, restorations of fenestrations can be possibly achieved by targeting PLVAP itself, or VEGF and/or
TGF-β signaling, which are indicated to be required for
induction of PLVAP expression during development [71]
and maintenance of endothelial fenestrations in adults [195,
204]. However, systemic upregulation of PLVAP expression
can be problematic because this may result in a significant
increase in overall brain vascular permeability or unwanted
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angiogenesis due to the potential role of PLVAP in developmental and pathological angiogenesis [72, 263].
Recent bulk and single-cell transcriptomes have indicated
many other genes that are highly expressed in fenestrated
brain vECs [10, 29, 279], including Plpp1, Plpp3, Igfbp3,
Cd24a, and Ldb2. Future studies on these and other candidate genes in the development and/or maintenance of fenestrated brain vEC will shed new light on potential new targets
for repairing this cell type.
Future perspectives
Brain barriers act as the important boundaries that separate
the brain parenchyma from the periphery across species.
Multiple blood vEC types present in the brain play a central
role in establishing and maintaining some of these barriers, thereby ensuring an optimal brain microenvironment.
In addition to the widely recognized BBB properties that
a majority of brain vECs exhibit, fenestrated vECs possess
unique barrier properties across the CVOs and CPs. Numerous factors can affect the states of brain vECs globally or
locally, thereby generating the heterogeneity and highly
dynamic changes in their vascular permeability. The evolution of technology has enabled us to explore more diverse
scientific approaches and research directions than in the past.
Here, we discuss some emerging approaches untouched in
earlier sections that will help address unsolved questions in
the field.
Recently, there have been significant advances in in vitro
model systems. These include the vascularization models
of human brain organoids-on-a-chip [280, 281], self-assembling multicellular BBB spheroids [282, 283], and improved
endothelial BBB differentiation protocols using human
pluripotent, or induced pluripotent, stem cells [284, 285].
Human organoid and cell reprogramming technologies have
been rapidly expanding to mimic human brain development
and disease modeling with the construction of a functional
vasculature. These in vitro models will become powerful
tools for studying human-specific vascular traits absent
in animal models, drug delivery across the BBB, and as a
drug-screening platform for ameliorating BBB dysfunction
in neurological diseases.
Emerging large-scale biological resources from multiomics data have increased the demand for an efficient in vivo
platform to screen numerous candidate genes emerging from
big data analytics. Given the high conservation of the molecular mechanisms underlying brain vascularization across vertebrates, one effective approach includes the use of the zebrafish
system, which provides technical advantages such as rapid ex
utero development and facile 3D visualization of brain vasculature. Recently, our group, along with others, reported highly
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Page 14 of 22
efficient and scalable CRISPR/Cas9-based mutagenesis protocols to generate F0 zebrafish knockouts [73, 286, 287]. These
technical advantages will make the zebrafish a valuable model
to perform phenotype-based F0 screens of many candidate
genes individually, or in combinations, in vivo.
The identification of brain regional differences in BBB or
fenestrated vEC barrier properties has indicated brain areaspecific regulations of brain vEC characteristics mediated by
their surrounding cellular compositions (e.g., perivascular,
glial, and/or neuronal types). Cutting-edge technologies,
such as spatial transcriptomics [288], combined with an
in situ sequencing method [289] will enable the visualization
and analysis of many transcripts with single-cell resolution
[290–292]. These approaches will compensate for the lack
of positional information on emerging single-cell and bulk
transcriptome data of brain vECs and perivascular cell types
in health and disease, allowing for spatial mapping of gene
expression profiles in situ.
The discovery of novel regulators of BBB permeability in
recent years has illuminated potential new targets for delivering therapeutics across the BBB. Endothelial transcytosis
pathways have been a major target of non-invasive brain delivery approaches [293]. Several recently identified molecules
(Mfsd2a, Vitronectin, and Integrin α5) critical for suppression
of transcytosis in BBB vECs [158–161, 163] have become
attractive targets for non-invasive drug transport into the
brain parenchyma. For example, drugs that inhibit MFSD2A
activity may release the suppression of transcytosis in BBB
vECs, thereby increasing endothelial molecular transport via
transcytosis into the brain parenchyma. Recent advances in
structural biology and protein design methods [294–298] will
facilitate in silico structure-based molecular designs that can
enhance the efficiency of drug delivery across the BBB.
The approaches listed here represent only a few examples of emerging directions in the field that have evolved
with technological advances. Cutting-edge multi-omics
platforms have offered the opportunity to simultaneously
profile RNA and DNA/protein at the single-cell level and
with spatial resolution, becoming increasingly powerful
tools to study the heterogeneous identities and states of
brain vascular cell types in health and disease. Future
investigations into the complex physiology and pathology
of brain–blood interfaces using multifaceted approaches
will reveal new therapeutic horizons for treatments of cerebrovascular and neurological diseases.
Acknowledgements We apologize to colleagues whose work could
not be cited in this review due to space limitations. We thank Dr. Olga
Cherepanova for helpful comments on this manuscript. All Figures
were created with BioRender.com.
Author contributions RLM wrote the manuscript; LDB, KPV, REQ,
and OAC collected literature information and edited the manuscript.
All authors reviewed and commented on the manuscript.
13
Funding This work was supported by funding from the National Institutes of Health (R01 NS117510) and start-up funds from the Cleveland
Clinic Foundation to R.L.M.
Availability of data and materials Not applicable.
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
Ethics approval and consent to participate Not applicable.
Consent for publication All authors have approved the manuscript for
publication at Cellular and Molecular Life Sciences.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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