Endothelial cells and astrocytes: a concerto en duo in ischemic pathophysiology.
ABSTRACT The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions. We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke.
- SourceAvailable from: stroke.ahajournals.org
Article: Time is brain--quantified.[show abstract] [hide abstract]
ABSTRACT: The phrase "time is brain" emphasizes that human nervous tissue is rapidly lost as stroke progresses and emergent evaluation and therapy are required. Recent advances in quantitative neurostereology and stroke neuroimaging permit calculation of just how much brain is lost per unit time in acute ischemic stroke. Systematic literature-review identified consensus estimates of number of neurons, synapses, and myelinated fibers in the human forebrain; volume of large vessel, supratentorial ischemic stroke; and interval from onset to completion of large vessel, supratentorial ischemic stroke. The typical final volume of large vessel, supratentorial ischemic stroke is 54 mL (varied in sensitivity analysis from 19 to 100 mL). The average duration of nonlacunar stroke evolution is 10 hours (range 6 to 18 hours), and the average number of neurons in the human forebrain is 22 billion. In patients experiencing a typical large vessel acute ischemic stroke, 120 million neurons, 830 billion synapses, and 714 km (447 miles) of myelinated fibers are lost each hour. In each minute, 1.9 million neurons, 14 billion synapses, and 12 km (7.5 miles) of myelinated fibers are destroyed. Compared with the normal rate of neuron loss in brain aging, the ischemic brain ages 3.6 years each hour without treatment. Altering single input variables in sensitivity analyses modestly affected the estimated point values but not order of magnitude. Quantitative estimates of the pace of neural circuitry loss in human ischemic stroke emphasize the time urgency of stroke care. The typical patient loses 1.9 million neurons each minute in which stroke is untreated.Stroke 02/2006; 37(1):263-6. · 6.16 Impact Factor
- Journal of Vascular Surgery. 12/2008; 48(6):1634–1635.
- [show abstract] [hide abstract]
ABSTRACT: Microvessels and neurons respond rapidly and simultaneously in focal regions of ischaemic injury in such a way as to suggest that the responses could be coordinated. The ability of neurons to modulate cerebral blood flow in regions of activation results from neurovascular coupling. But little is known about the microvessel-to-neuron direction of the relationship. The presence and participation of intervening glial cells implies the association of microvessels, glia, and neurons in a 'neurovascular unit'. The interdependent functions of the cellular and matrix components of this theoretical unit have not been rigorously explored, except under conditions of injury where, for the most part, only single components or tissue samples have been studied. Whereas maintenance or timely re-establishment of flow reduces tissue and neuron injury in both humans and animal models, protection of neuron function in humans has not prevented the evolution of injury despite the inherent mechanisms of neurovascular coupling. However, occlusion of flow to the brain rapidly identifies regions of neuron-vascular vulnerability within the vascular territory-at-risk. These coalesce to become the mature ischaemic lesion. The failure, so far, of clinical trials of neuron protectant agents to achieve detectable tissue salvage could be explained by the vulnerability (and lack of protection) of essential components of the 'unit'. This presentation summarizes evidence and thoughts on this topic. These support the need to understand component interactions within the neurovascular unit.Journal of Internal Medicine 02/2010; 267(2):156-71. · 6.46 Impact Factor
Hindawi Publishing Corporation
International Journal of Cell Biology
Volume 2012, Article ID 176287, 16 pages
Vincent Berezowski,1,2,3Andrew M.Fukuda,4Rom´ eo Cecchelli,1,2,3andJ´ erˆ ome Badaut4,5
1Universit´ e Lille Nord de France, 59000 Lille, France
2UArtois, LBHE, EA 2465, 62300 Lens, France
3IMPRT-IFR114, 59000 Lille, France
4Departments of Physiology, Loma Linda University School of Medicine, Loma Linda, CA 92354, USA
5Departments of Pediatrics, Loma Linda University School of Medicine, Loma Linda, CA 92354, USA
Correspondence should be addressed to J´ erˆ ome Badaut, email@example.com
Received 2 March 2012; Accepted 30 April 2012
Academic Editor: Carola F¨ orster
Copyright © 2012 Vincent Berezowski et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the
cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge
of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions.
We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the
only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side
effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator-
activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-
brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved
clinical outcomes after stroke.
In the United States, stroke is the number one cause of
chronic disability and the fourth leading cause of death,
with approximately 7 million adults affected . Annually
there are approximately 800,000 strokes in the US, of which
87% are ischemic strokes, 10% are primary hemorrhages,
and 3% are subarachnoid hemorrhages . Together they
cause the country a financial burden of approximately 62.7
billion dollars . Cerebral ischemic stroke is caused by an
occlusion of a cerebral blood vessel, typically by a thrombus,
which causes a decrease in cerebral blood flow and thus
limits the supply of oxygen and nutrients globally (in global
ischemia) or to certain regions of the brain (in focal brain
neuronal death in addition to damaging the vascular tree;
the vascular tree is usually made more fragile during the
ischemic period and damaged during reperfusion. Time is
an important parameter in the evolution of brain injury.
In 2006, Saver et al. have estimated the impact of stroke
on the brain tissue  to be immense; the brain may lose
up to 120 million neurons, 830 billion synapses and 714km
of myelinated fibers for each hour after stroke onset .
Ischemic stroke seems to accelerate aging of the brain at
a rate of 3.6 years each time when the symptoms are
not treated . Therefore, the clinical goal of acute stroke
treatment is to reduce brain damage by limiting the time of
ischemia through thrombectomy (mechanical endovascular
approach) or thrombolytic therapy, which consists of in
lysing the blood clot in order to restore cerebral blood flow.
Recombinant tissue plasminogen activator (rtPA) is cur-
rently the only thrombolytic molecule administered during
acute cerebral infarction that provides a clinical benefit in
terms of survival and neurological outcome . The rtPA
administration must be within the first 4 hours 30 minutes
after stroke onset to maintain the beneficial effects without
substantially raising the side effects/risk [5, 6], which limits
2International Journal of Cell Biology
of stroke patients are eligible for this therapy in this narrow
time window, which leaves the remaining 95% of patients
without any beneficial treatment available. The major risk of
rtPA is the extension of the damage due to potential bleeding
. The need for drug development to prevent the neuronal
save viable neurons located in the ischemic penumbra area.
However, all of the proposed neuroprotective treatments
specifically targeting neurons that showed promise on the
bench have failed in clinical trials .
In 2000, the neurovascular unit (NVU) was proposed as
a physiological unit composed by neurons, astrocytes, and
endothelial cells ; there is a growing interest in studying
the changes of the NVU after stroke. In addition to cell
death, ischemic stroke is characterized by changes in the
properties of the blood-brain barrier (BBB) with physical
disruption of the tight junctions contributing to aggravation
of cerebral edema and consequently neuronal death. The
new strategy for drug development is to have molecules with
a broader spectrum targeting not just the neurons but the
NVU as a whole entity. In the present paper, we will focus on
some molecular and cellular mechanisms of astrocytes and
endothelial cells. We will look specifically at: (1) the ways
astrocytes and endothelial cells work in concert in stroke
pathophysiology such as BBB disruption and edema forma-
(3) new drug developments in the future.
2.Definition of the Neurovascular/
Several groups have proposed the NVU as a physiological
unit composed of not only endothelial cells, astrocytes,
and neurons but also pericytes, smooth muscle cells, and
the interacting circulating peripheral immune cells [10–12].
The term “gliovascular” emphasizes the importance of the
interactions between astrocytes and cerebral blood vessels
within the NVU , which are critical in cerebral blood
flow regulation , brain energy metabolism , and also
the maintenance of the BBB properties .
with the presence of tight junctions and adherens junctions
between the cells (Figure 1) that prevent paracellular diffu-
sion and act as a unit to regulate ions and other molecules
between peripheral blood flow and brain parenchyma. Tight
junctions are composed of several protein families: trans-
membrane proteins (claudins and occludins), cytoplasmic
proteins, and zona occludens proteins. They bind the afore
mentioned proteins with structural cytoskeletal proteins
such as actin. Adherens junctions are formed by proteins
and vascular endothelial-cadherin, which contribute to the
close physical contact between endothelial cells and facilitate
the formation of tight junctions.
The brain endothelial cells of the BBB also present spe-
membranes for nutrients, ions, and toxins to cross the endo-
example, energy molecules are transported by specific solute
carriers such as glucose transporter 1 (GLUT 1) and mono-
carboxylate transporters 1 and 2 (MCT1, MCT2). Large
molecular weight solutes (e.g., large proteins and peptides)
are able to cross the BBB and enter the intact CNS via endo-
cytotic mechanisms called receptor-mediated transcytosis,
such as with insulin, or adsorptive-mediated transcytosis,
exemplified by albumin. On the other hand, transport
can also be achieved by the ATP-binding protein (ABC)
family, which consumes ATP to effectively transport a wide
range of lipid-soluble compounds from the brain endothe-
lium. In the BBB examples of ABC transporters for efflux
transport are P-glycoprotein (P-gp), multidrug resistance-
associated protein (MRP), and breast cancer resistance pro-
tein (BCRP) . These efflux transporters are understood
as gatekeepers of the brain because they keep tight control
over which substances are allowed to enter the CNS through
the endothelial cell barrier (Figure 1). Endothelial cells also
present a metabolic barrier of the BBB, which functions
to inactivate molecules capable of penetrating cerebral
Quite recently it has been proposed that the primary
barrier of the BBB may extend to the basal lamina, thus
preventing the entry of immune cells into the parenchyma
under normal brain conditions . Historically the brain
was thought to be an immune cell deficient organ, and the
BBB was thought to prevent passage of any immune cells
into the brain. However, peripheral immune cells from the
blood have been observed to enter and be present in the
 and in normal physiological conditions in adults .
organ has recently started to be reexamined and revised.
Engelhardt and collaborators elegantly compare the perivas-
cular space as a castle moat with perivascular antigen pre-
senting cells floating as guards, confined by the inner and
outer wall, which is the basement membrane of the astro-
cytic endfeet and the endothelial cell, respectively .
Endothelial cells and other cells, such as the astrocytes, may
also contribute to the tight regulation of the movement of
immune cells between the peripheral blood stream and the
brain. However, the exact mechanisms by which peripheral
rather than the BBB being a rigid wall, it provides a dynamic
interface between the brain and the rest of the body.
As mentioned previously, the presence and the mainte-
nance of these barrier properties are important for brain
homeostasis and for neuronal functioning . In fact,
disruption of tight junctions leads to BBB disruption and
extravasation of blood components and water, which con-
tribute to vasogenic edema formation. We will cover these in
more detail in the following section.
3.EdemaProcess afterStroke: Endothelium
andAstrocyte, Concerto en Duo
3.1. BBB Disruption and Edema Formation. Cerebral edema
has been traditionally divided into 2 major classes: cytotoxic
and vasogenic  for cerebrovascular diseases and other
International Journal of Cell Biology3
Glucose transporter 1 (GLUT 1)
(ZO1, 2, 3)
Figure 1: (a) Schematic drawing of the neurovascular unit (NVU) in the capillary bed composed by the neuron, astrocyte endfoot, basal
the tight junction composes the physical barrier and the movement of substrates is controlled by several transporters. The astrocyte endfeet
are linked with the gap junction, allowing movement of several solutes in the astrocyte network. The basal lamina is composed of several
proteins such as agrin, dystroglycan, and perlecan. (b) A close-up schematic drawing of the endothelial cells and astrocyte endfeet with some
of the proteins involved in edema formation and resolution.
accumulation of water coming from the extracellular space
without BBB disruption. Vasogenic edema appears after
BBB disruption, leading to a diffusion of proteins from the
blood to the tissue followed by water accumulation in the
extracellular space . However, this division alone does
not explain fully the diversity and the complexity of the
edema process in brain ischemia as well as in the other brain
injuries and disorders. Based on several recent advances in
the understanding of the molecular mechanisms of edema
formation and BBB properties, a third subtype of edematous
processes was named ionic edema and described as a contin-
uum between the cytotoxic to vasogenic edema in the cere-
brovascular diseases [19, 20]. In fact, cytotoxic, or anoxic,
edema occurs within the first few minutes after cerebral
blood flow stoppage and is characterized as swelling of the
astrocytes and neuronal dendrites [20, 21]. The cellular
swelling within the first 10 minutes is a result of oxygen and
4International Journal of Cell Biology
glucose deprivation followed by a slow rise in extracellular
[K+] . The absence of oxygen and energy nutrients
induces a disruption of the cellular ionic gradients and leads
to entry of ions into cells. Water follows this ionic gradient
into the cells and induces cellular swelling. Cytotoxic/anoxic
edema may evolve quickly to become ionic edema because
the absence of oxygen and nutrients further alters the
energy balance in endothelial cells and the ionic gradients,
including transcapillary flux of Na+in these cells [19, 23].
The endothelial cells also require a large amount of ATP
production, characterized by the high density of mito-
chondria, which are important for the regular homeostatic
BBB functions such as maintenance of ionic gradients and
membrane transporters [24, 25]. The absence of energy
Reperfusion induces overpressure accompanied by shear
stress on the nonperfused vascular tree that results in early
transient leakage of the BBB [26, 27]. This leakage results in
further entry of water through the endothelial cells resulting
inbrain swellingwithin 30 minutesafterreperfusion[26,27]
of the BBB has also been described clinically in humans
and is frequently associated with hemorrhagic transforma-
tion . Early reperfusion probably mitigates the BBB
alterations, but if it is delayed, reperfusion will exacerbate
the amount of endothelial injury [30–32]. The final step
is the development of vasogenic edema, in which there
is disruption of cerebrovascular endothelial tight junctions
leading to increased permeability to albumin and other
plasma proteins . Another contributing factor of brain
edema formation in addition to tight junction disruption is
brain endothelial transcytosis . BBB disruption is usually
coupled with the inflammatory response and activation of
matrix metalloproteinases (MMP) [34, 35]. In fact, vaso-
genic edema development is aggravated by MMP-9, which
degrades basal lamina, the connection between astrocytic
endfeet and endothelial cells .
In the clinic, diffusion-weighted imaging (DWI) and
T2-weighted imaging (T2WI) magnetic resonance imaging
(MRI) modalities are used extensively to assess postischemic
edema [20, 37, 38]. T2 values represent water content and
apparent diffusion coefficient (ADC) values derived from
DWI images represent water mobility in the tissue [20, 37].
ADC values decrease rapidly after stroke onset, indicating
restricting water movement, and are interpreted as evidence
of ionic edema with the characteristic swelling of the brain
cells causing a decrease in extracellular space as proposed
in our classification mentioned before. T2 values increase at
later time points, which are associated with vasogenic edema
The molecular mechanisms and temporal development
of edema after stroke have been well studied. However, the
cellular and molecular mechanisms involved in edema
resolution are not well understood in stroke and other brain
diseases. The healing of the endothelial cells with stabiliza-
tion of the tight junctions may be a critical step to limit
the entry of blood components into the brain. Thus, stabiliz-
ing the NVU may be an essential component of controlling
edema formation and BBB breakdown after stroke.
Postischemic BBB disruption has been commonly
believed to be biphasic , but recent work suggests that
the BBB disruption may be continuous for up to 5 weeks
after ischemia in rats . BBB leakage was demonstrated
using gadolinium and magnetic resonance imaging (MRI) at
25min; 2, 4, 6, 12, 18, 24, 36, 48, and 72 hours; and 1, 2, 3, 4,
and 5 weeks after ischemia . Similarly, albumin leakage
through the BBB, especially in the hippocampus, has also
been observed in spontaneously hypertensive stroke prone
rats long term . Although these data do not completely
rule out the possibility of a biphasic pattern in the opening
of the BBB, the long-term leakage of the BBB is important to
disruption could account for a prolonged vasogenic edema.
3.2. “Concerto en Duo”: Astrocyte Network in Edema Forma-
in contact to the blood vessels are well known for to swell
after stroke [42–44]. The recent knowledge on the trans-
porters and channels in this astrocyte subdomain gives new
perspectives on the understanding of astrocyte swelling. In
channel proteins, is proposed to have an important role in
edema formation [20, 45]. AQP4 is the most abundant water
channel in the brain, in part due to its high concentration on
astrocytic endfeet which are in contact with all the cerebral
blood vessels [46, 47]. More recently, AQP1 has also been
described in a subpopulation of astrocytes within the non-
human primate but not in rodents, suggesting interspecies
differences and a possible role in brain water homeostasis
endothelia and primary rat brain endothelial cell cultures
. Interestingly, Dolman and collaborators observed that
mRNA AQP1 levels were lower in cultured brain endothelial
cells when cocultured with astrocytes , suggesting an
inhibition effect of the astrocytes on the AQP expression in
endothelia. In fact, there are publications reporting a low
level of AQP in endothelial cells in vivo , although AQP
is more abundant in astrocytes [49, 51–54].
Currently, AQP4 is considered as a key player in the
edema process by its location on the astrocyte endfeet [20,
55]. AQP4 is assembled in homotetramers where each indi-
vidual aquaporin represents a water channel . Interest-
ingly, AQP4 is also organized in the astrocyte endfeet mem-
brane in a larger geometric structure known as an orthog-
onal array of particles (OAPs), which has been described
with freeze-fracture techniques and electron microscopy
studies (Figure 2) . OAPs are present in all astrocyte
endfeet in contact with the blood vessels as well as the glia
limitans. OAPs are formed with two isoforms of AQP4:
long (AQP4-M1) and short splice variants (AQP4-M23).
The ratio of AQP4-M1 to AQP4-M23 determines the size of
these OAPs  in contact with the basal lamina of brain
vessels (Figure 1). Experiments in oocytes showed that the
AQP4-M23 isoform stabilizes the OAP structure [57, 58].
However, the exact functional roles of the OAPs remain
unknown in normal and pathological conditions. Recently,
after stroke onset, while AQP4-m23 remained the same.
International Journal of Cell Biology5
AQP4 in OAPs
disruption of OAPs
Figure 2: (a) Schematic drawing of the aquaporin homotetramer assembly within the lipid membrane: the central pore is proposed to
be permeable to cations and gases (green arrows). Each individual aquaporin facilitates bidirectional water movement depending on the
osmotic gradient (blue arrows). (b) AQP4 homotetramer is assembled in a higher structure named orthogonal array of particles (OAPs).
Two isoforms of AQP4, AQP4-M1 (purple circles) and AQP4-M23 (blue circles) isoforms, contribute to the formation of OAPs. In vitro
experiment showed that higher expression of AQP4-M23 contributes to the formation of larger OAPs. (c) Increase of AQP4-M1 induced
disruption of OAPs. Recent knowledge on AQP leads us to hypothesize that the large OAPs contribute to gas and cation diffusion in the
astrocyte membranes through central pores (green arrows).
The increase of AQP4-m1 early after ischemia could favor
a shift toward M1 in the M1/M23 balance, which is known
to favor small size OAPs . In accordance with this work,
previous studies have shown that early disorganization of
OAPs on the astrocyte endfeet after global cerebral ischemia
preceded astrocyte swelling . Although a direct effect of
the modification in the ratio of AQP4-M1 to AQP4-M23
on water permeability has not yet been directly investigated
in vivo, in a preconditioning model, a strong increase in
AQP4 expression and increase of AQP4-M1 were correlated
with reduced edema and less water in the tissue, suggesting
increased water diffusibility which resulted in the removal
of excess liquid from the brain tissue . Interestingly, it
was recently proposed that the assemblage of 4 aquaporin
molecules forms a central pore, through which water, ions,
and gases may flow depending on the AQP subtype. For
example, the central pore is permeable for O2, CO2, and
possibly nitric oxide for AQP1, 4, and 5 [56, 60]. Thus, the
disruption of the OAPs may also affect the diffusion of ions
and gas through the central pore.
Due to its location in the astrocyte endfeet in contact
with the blood vessels, AQP4 has been proposed to be linked
with BBB integrity [52, 61, 62] and cell adhesion . In
the epithelial cells of the eye lens AQP0 is present in the
OAPs and participates in epithelial cells linkage; however it
does not facilitate water flux . In this case, the presence
of AQP4 in the astrocyte endfeet membrane was dependent
on the presence of proteins in the basal lamina such as
agrin, α-dystroglycan, and laminin [65, 66] in addition to
syntrophin and dystrophin protein complexes [67, 68]. The
connection of AQP4 to proteins in the basal lamina may
explain the ability of astrocytes to maintain the integrity of
the blood-brain barrier, suggesting a possible role for AQP4
as a structural molecule within the perivascular space .
However, reports using AQP4 knock out (AQP4-KO) mice
BBB structure suggesting that AQP4 may not be integral to
the BBB structure [61, 69]. Similarly in our siRNA silencing
studies, BBB permeability was not significantly changed at
distance from the site of injection after injection of siRNA
against AQP4, even though AQP4 expression was decreased
. We also showed that the upregulation of AQP4 in a
preconditioning model did not prevent the early opening of
the BBB after stroke .
Heparan sulfate proteoglycan is a large family of proteins
with agrin and perlecan, involved in the basal lamina
composition located between the astrocyte endfeet and
endothelial cells [54, 70]. Agrin and dystroglycan seem to
play an integral role in the maintenance of astrocyte polarity
by the interaction with AQP4 in the astrocyte endfeet .
Specifically, agrin KO mice showed a significantly decreased
density of OAP in the astrocyte endfeet when compared
to wildype but overall immunoreactivity of AQP4 did not
differ significantly . Dysfunctions in the basal lamina
are related to increase of the BBB disruption, promoting
edema formation. In fact, a family of endopeptidases, matrix
metalloproteinases (MMPs), has been shown to degrade the
proteins of the basal lamina and contribute to vasogenic
cerebral edema . In the human brain, MMPs are usually
very low in concentration under nonpathological conditions
. However, after injuries such as ischemic stroke, certain
MMPs such as MMP-2, -3, and -7 and especially MMP-9
have been shown to be upregulated in the brain (reviewed
in ). This layer between astrocytes and endothelial cells
is a potential future target for the NVU protection. Recently,
Dr. Bix and collaborators have shown that administration
6International Journal of Cell Biology
of perlecan domain V, which is the c-terminal fragment,
administered 24 hours after ischemic stroke has beneficial
effects by interacting with integrins . Perlecan domain
V increased expression of vascular endothelial growth factor
(VEGF), thus promoting angiogenesis, and interestingly did
not lead to increased BBB permeability  even though
VEGF is known to increase BBB permeability after ischemia
astrogliosis through interaction with dystroglycans and
integrins in the astrocytes .
Astrocytic AQP4 is not only linked with the matrix pro-
teins but also with several other channels present in higher
concentration in the astrocyte endfeet such as potassium
inner rectifying channel 4.1 (KIR4.1), connexins (Cx), and
also chloride channel 2 (CIC-2) [76, 77]. Colocalization
of AQP4 and KIR4.1 suggests that AQP4 may have a role
in potassium homeostasis by facilitating water diffusion
along the potassium gradient and AQP4-KO mice display
a delay in potassium reuptake during electrical activity
. The decrease of AQP4 expression using siRNA showed
an associative decrease of connexin 43 (Cx43), a protein
involved in gap junction formation, and a decrease of CIC-
2, involved in the regulatory volume decrease function of
the astrocytes. Interestingly, gap junctions and AQP4 are
morphologically closely associated  with the astrocyte
endfeet. The gap junctions in the astrocyte contribute to
the formation of a complex network named the astroglial
network . Intercellular and intracellular communication
that facilitate the movement of second messengers, amino
acids, nucleotides, energy metabolites, and small peptides
[79–82] in astrocyte processes occur through gap junctions,
which are made up of a family of channel proteins called
connexins [83, 84]. In astrocytes, Cx30 and Cx43 are
predominant [83–85]. However, it is also important to note
that Cx43, along with Cx37, Cx40 [86, 87], and Cx45 ,
is also expressed in brain endothelial cells. The protein
level of Cx40 and Cx45 was shown to increase in cerebral
arteries, but no change in protein or mRNA was observed
for brain endothelial Cx43 and Cx37 after a model of brain
injury causing cerebral vascular dysfunction . The effect
of astrocytic Cx43 upregulation or downregulation after
as to what provides beneficial effects . However, in
humans, there are reports that show that Cx43 protein
levels were increased in the penumbra . And because
Cx43 and Cx30 knockouts have been observed to be more
edema prone , it is possible that the increase in Cx43
after ischemia may be a physiological response to decrease
edema. The induction of Cx43 may be facilitating water flow
throughout the astrocyte network to diversify and dissipate
the accumulation of fluid from just one region. From these
data we hypothesize that gap junction proteins, specifically
Cx43 on astrocytes, are working with AQP4. Evidence for
this also comes from a significant decrease of Cx43 observed
in mouse astrocyte cell cultures after administration of
small interference RNA against AQP4 . Although direct
functional data are still lacking, one possibility is that AQP4
and Cx43 is working together to direct water flow between
astrocytes and could be controlling astrocytic swelling.
The role of AQP4 in cerebral edema formation and
resolution has been studied in several models. However
the precise role of AQP4 remains unclear and depends on
the pathological model used [92, 93]. Indeed, the absence
of AQP4 was shown to prevent the formation of edema
in a permanent ischemia model in AQP4-KO mice .
Similarly, edema formation is prevented in α-syntrophin
knockout mice at 24h after stroke . This decrease of
brain swelling was correlated with the loss of the perivascular
AQP4 domain in α-syntrophin-KO mice . These results
suggest that perivascular AQP4 has an important role in
edema formation. However, the absence of AQP4 in AQP4-
KO mice also prevents water clearance in an experiment of
intrastriatal infusion of a saline solution, showing that AQP4
is critical for water removal from tissue . Conversely, in a
preconditioning stroke model, a higher induction of AQP4
was correlated with edema reduction . However, this
reduction of edema may be referring to vasogenic edema,
in which case, AQP4 is said to aid in edema resolution
by actively pumping out water from the cerebral tissue to
peripheral blood . The redistribution of the water in the
also be possible for the CSF compartments. This hypothesis
ependymal cells in the border of the ventricles in a traumatic
brain injury model .
To summarize, the exact mechanism causing decreased
edema formation is not yet fully understood, but AQP4 and
the astrocyte network with the gap-junction proteins may
certainly be contributing. Osmotic gradients can also play
an important role, and recently, high AQP4 expression was
observed in hypersaline treatment after stroke correlating
with decreased edema formation at 48 hours .
4.rtPA: AUniqueDrugfor Stroke Treatment
withAversiveEffects on the NVU
4.1. Clinical Evidence (from Bed to the Bench, Neurotoxicity
of rtPA). As discussed in the introduction, recombinant
tissue plasminogen activator (rtPA) is currently the only
thrombolytic molecule FDA approved for treatment of
acute ischemic stroke . The intact BBB is usually an
obstacle for most neuropharmacological agents in healthy
patients. The dysfunction of the BBB after ischemia could
cause problems for the therapeutic function of rtPA. This
protease targets fibrin-bound plasminogens and converts
them into plasmins, which then cut the fibrin clot and lyse it.
Intravenously infused at a dose of 0.9mg/kg over one hour,
rtPA provides increased survival and better neurological
outcomes . To be beneficial for the patient, rtPA must be
administered within the first 4h 30min after stroke onset
[5, 6]. Despite the organization of emergency care, only 5%
of stroke patients are eligible for this therapy. In fact, late
administration of rtPA translated to a higher risk of bleeding
and extension of the lesion . Higher doses of rtPA do
not bind only the fibrin clot but also activate the circulating
plasminogen activator (tPA). This activation contributes
to a generalized fibrinolysis and fibrinogenolysis, which is
suspected to be a cause of bleeding. But the mechanisms of
International Journal of Cell Biology7
the hemorrhagic transformation after rtPA treatment seem
to be more complex than can be accounted for by the
affinity of rtPA for fibrin alone. In fact, the enhanced fibrin
specificity of tenecteplase and reteplase, two rtPA derivatives,
resulted in no significant difference in terms of cerebral
hemorrhage [98, 99].
Interestingly, the comparison with myocardial infarc-
tion shows a low incidence of cerebral hemorrhage after
rtPA administration  suggesting a direct link between
bleeding and the ischemic pathophysiology. Clinical studies
showed that 80% of bleeding after cerebral thrombolysis
occur preferentially in the ischemic territory .
To have a better understanding of the aversive effects of rtPA
its neurotoxic effects were examined. It is well known that
endogenous tPA is present in the blood stream, endothelial
cells, neurons, and microglial cells . In the brain
parenchyma, tPA activity was found to be pleiotropic and
associated with synaptic plasticity and cell death [102–104].
In fact, tPA interacts with several neuronal proteins such
as N-methyl-D-aspartate (NMDA) receptors, one subtype
related protein (LRP), and Annexin-II [101, 105, 106]. tPA
is synthesized in neurons, stored in presynaptic vesicles,
and released following depolarization in synergy with the
neurotransmitters. In the synaptic cleft, tPA binds and
cleaves the NR1 subunit of NMDA receptors that causes an
amplification of calcium influx in postsynaptic neurons and
an increase of the glutamatergic response in physiological
conditions. However, this physiological response becomes
excitotoxic after ischemia and is magnified after rtPA injec-
tion [101, 107, 108]. The injection of antibodies against
the NR1-subunit prevented these proexcitotoxic effects of
endogenous tPA and reduced brain infarction and BBB
receptor may be a protective drug target for the NVU after
stroke and may provide a potential extension of the rtPA
therapeutic window .
The presence of rtPA in the brain parenchyma has been
explained by its passage through the BBB in several in vitro
models with different proposed mechanisms.
(i) rtPA diffuses into the brain parenchyma through an
already opened BBB as a consequence of the ischemic
process. As we discussed previously, the kinetics of
the BBB opening is complex in the early stages
after stroke and it is difficult to observe this with
clinical imaging . Interestingly, in vitro endothe-
lial monolayer cultured with astrocytes enables us
to observe the ability of rtPA to cross the intact
BBB , which is increased under oxygen-glucose
deprivation (OGD) . Therefore, as rtPA poten-
tially diffuses through an open or closed BBB in
early time points after stroke onset, it may aggravate
neuronal cell death as described previously.
(ii) rtPA could cross the BBB by degrading the endothe-
lium via its own proteolytic activity, but it is not
a requirement in the intact BBB . The ability
of rtPA to cross the intact BBB at a thrombolytic
the endothelial cells before the BBB breakdown. In
fact, rtPA promotes breakdown of the BBB  by
stimulating the synthesis activity of MMP-9 [113–
116] and other MMP isoforms  exacerbating
the degradation of the basal lamina and subsequent
vasogenic edema formation and hemorrhage. The
thrombolytic products could exacerbate the pro-
posed mechanism .
(iii) Finally, LRP potentially contributes in trans-endo-
thelial transport of the exogenous rtPA [106, 119,
120] and then activates the astrocytic MMP-9 and
of inducible nitric oxide synthase (iNOS). This
increase of NO results in increased BBB permeability
With all these data together, Yepes and collaborators
have proposed the following potential cellular and molecular
(1) Circulating endogenous tPA and rtPA cross the BBB
(intact or damaged endothelial layer) and increase
MMP-9 activity in the basal lamina soon after stroke
onset which compromises the NVU integrity and
makes it fragile.
(2) Then tPA and rtPA bind to the astrocytic LRP,
inducing the loss of the extracellular domain of LRP
[122, 123] in the basal lamina, and release the intra-
cellular domain of LRP in the astrocytic cytoplasm
to activate NF-κB. This NF-κB activation increases
iNOS and MMP9 expression and overall function in
feet from the basal lamina. This is usually observed
at the later stages of BBB breakdown. However, it
is tempting to speculate that this cascade, which
involves the perivascular cells of the NVU, would be
use of rtPA. It is possible that rtPA and tPA may
also affect the phenotype of the astrocyte endfeet by
as AQP4 and also Cx43.
4.3. New Therapeutic Strategies for rtPA Treatment after
Stroke. The BBB is definitely not a barrier to rtPA in stroke
but the BBB does become a serious barrier to the effective
usage of this drug in clinic due to the neurotoxic effects
and the risk of hemorrhagic transformation. Interestingly,
tPA may be endogenously synthesized by the central nervous
system in neurons and endothelial cells . However, tPA
and rtPA have effects on the endothelial cells, astrocytes,
and neurons and possibly other glial cell types such as
oligodendrocytes and microglia. In order to prevent the
reperfusion, several new therapeutic strategies have been
examined to prevent the interaction of rtPA with the NMDA
receptor within the NVU . In fact, NMDA receptors are
8International Journal of Cell Biology
expressed not only in neurons but also in oligodendrocytes
and endothelial cells [125, 126]. One of these strategies uses
an LRP antagonist (RAP) to minimize the binding of rtPA
with LRP in the endothelial cells. A second strategy uses
the ATD-NR1 antibody to block rtPA binding of the NR1
subunit on neuronal NMDA receptors. The last one uses
a mutation of the rtPA to decrease its adverse effects on
the nervous tissue . An example of a natural drug,
desmoteplase, the vampire bat Desmodus Rotundus Salivary
Plasminogen Activator (DSPA), is a thrombolytic agent
under development. It shows little neurotoxicity and has the
ability to interact with the BBB endothelium through the
the clinical trial of DIAS-2 (Desmoteplase In Acute ischemic
Stroke) showed no benefit of the desmoteplase versus
placebo . Although the outcome of this clinical trial
investigated. In fact, Gleevec, a FDA approved drug for
treatment of chronic myelogenous leukemia, was recently
proposed to prevent the complications associated with rtPA
treatment . Gleevec inhibits the activation of platelet-
derived growth factoralpha receptor (PDGFR).It wasshown
that tPA increases BBB permeability through the indirect
activation of perivascular astrocytic PDGFR .
MMP inhibition is a good strategy based on reports
of easy monitoring of MMP blood levels, defining them
as potential biomarkers of brain damage [131, 132]. But
because endogenous MMPs are also key mediators in stroke
recovery by contributing to inflammatory and remodeling
responses, pharmacological targeting must be accurately
applied for acute stroke phases so; their beneficial effects are
not compromised [133, 134]. Despite efforts to understand
the complex link between BBB integrity and the hemorrhage
risk , a better definition and understanding of NVU
kinetics and the mechanisms underlying their dysfunction
is still needed to better define eligibility criteria for rtPA
treatment. Thus, alternative approaches other than MMP
inhibition as mentioned before in some recent developments
will offer interesting treatment strategies after stroke.
5.NVU ProtectionMay Be the Futureinsteadof
Neuroprotection inStroke Treatment
5.1. Preconditioning for Future Development of New Drugs.
Given the small number of patients eligible for thrombolysis,
many pharmaceutical compounds have been developed to
limit the progression of brain injury by targeting differ-
ent mechanisms leading to neuronal death . Despite
promising protective effects observed in preclinical studies,
no compound to date has demonstrated benefit against
stroke-induced neuronal death after facing the rigorous wall
of clinical trials .
As mentioned in Section 1, research on brain diseases
has focused on neuronal damage, as it was thought to be
the major cause of cognitive deficits. However, ischemic
stroke is a complex brain disease characterized by sudden
onset of disabilities related to brain damage with a vascular
origin. Because the development of many neuroprotective
molecules for treatment over the last twenty years has been
unsuccessful, researchers have switched gears towards inves-
tigating the natural endogenous neuroprotection of ischemic
tolerance . The purpose of the ischemic tolerance pre-
conditioning is to induce endogenous defense mechanisms
prior to the ischemic event that will attenuate the even-
tual consequences of ischemia. This resistance to ischemic
damage can be achieved experimentally by several stimuli
including ischemic preconditioning . The concept and
protocols were adapted from previous studies done in
myocardial infarction. In fact, a short duration of coronary
occlusion is unable to cause myocyte necrosis. However,
when carried out before a prolonged occlusion, a short
myocardium . This initial nonharmful ischemic insult
triggered endogenous mechanisms that made the organ
more resistant to the next attack for up to two periods of
ischemic tolerance . The first period of ischemic toler-
ance resulted from posttranscriptional responses and began
minutes after preconditioning. The second, longer period,
began 24 hours after preconditioning and lasted up to 7 days
with maximal protection found at 3 days.
As with the cardiac preconditioning, ischemic tolerance
in the brain also has delayed mechanisms leading to neuro-
protection .However,themechanisms arecomplexand
not well understood. The induction of ischemic tolerance
likely depends on the coordinated responses at the genomic,
molecular, cellular, and tissue levels [141–143], which sug-
gests the importance of the interactions between the astro-
cyte and endothelial cells in the NVU. Regarding neurovas-
cular events in stroke pathophysiology, there has been a
growing interest in vascular approaches to the precondition-
ing mechanisms. Protective effects of preconditioning were
observed in vivo, demonstrating that endothelium function
is preserved by improving cerebral blood flow during reper-
fusion in areas surrounding the lesion , and that BBB
integrity is maintained with a reduction in edema formation
. The induced protection was again correlated not
only with a decreased expression of MMP-9  but also
with a reduced neutrophil adhesion to endothelial cells
through a decreased expression of ICAM-1 [147, 148]. These
results were confirmed by in vitro studies that report a
protective effect via preservation of BBB integrity, by both a
decreased expression of the inflammatory molecules ICAM-
1 and VCAM-1 [149, 150] and maintenance of tight junction
structure . Moreover, preconditioning also facilitates
the increase of AQP4 expression at early time-points after
stroke onset, which is associated with a decrease of the
edema formation . A recent study also reported the
protective role of glial tissue preconditioning in severe stroke
. These recent observations suggest that future drug
development must focus on drugs affecting the entire NVU
instead of one cell type as was proposed in the 1990s with
the development of calcium channel and NMDA inhibitors.
Recently, some compounds like edaravone, an antioxidant,
showed benefits in preclinical and clinical studies by protec-
tion of the NVU [152, 153]. But further trials are needed to
confirm these promising preliminary results .
International Journal of Cell Biology9
5.2. Protection of the NVU: Focus on PPARs. Preventive neu-
roprotection also involves management of risk factors, which
is supported by studies showing that physical exercise 
or lipid-lowering treatment reduces the occurrence and
severity of stroke [156–158]. In this context, the involvement
of pharmacological agents that are activators of nuclear
receptors like peroxisome proliferator-activated receptors
(PPARs) could be a promising study. Present in three iso-
forms, α, β/δ, and γ, these receptors exhibit pleiotropic
activity in the sense that they can activate or repress the tran-
scription of many genes involved in lipid and carbohydrate
metabolism in addition to inflammation [159, 160]. PPARs
are expressed in neurons, endothelial cells, and glial cells
. Activation of the PPARs has long-term effects lasting
from hours to days, which correspond to an activation of
gene transcription (named transactivation) as has been seen
in lipid and carbohydrate metabolism. However, activation
of the PPARs induce a cellular response within minutes to
hours and this corresponds to an inhibition of gene tran-
scription named transrepression . The latter mecha-
nism does not require binding to DNA, but rather protein-
protein interaction involving other transcription factors like
NF-κB of STAT-3 and AP-1, to inhibit their activity as
reported for inflammatory genes .
carried out in mice subjected to transient ischemia with pre-
WY-14643, and resveratrol (a polyphenol present in grapes)
[164–166]. The observed protection is the result of an anti-
inflammatory mechanism, which decreases the expression
of adhesion molecules, ICAM-1 and VCAM-1, in brain
endothelial cells. Effects of antioxidants were also observed.
However, a study using a BBB in vitro model combining
endothelial cells with glial cells from wild-type or PPAR-α
knockout mice has demonstrated not only that the observed
protection against OGD-induced hyperpermeability was
dependent on this nuclear receptor activation but also that
the ligand targeted specifically the endothelial cells without
modulation of the classical PPAR-α target genes associated
with inflammation or metabolism . Moreover, pro-
tective effects of PPAR-γ were not only reported through
similar mechanisms  but also via an inhibition of NFκB
cells activation, thus preventing cytokine production .
One study also suggests that PPAR-γ agonists could inhibit
excitotoxicity-induced neuronal death .
Statins are HMG-CoA reductase inhibitors. This enzyme
catalyzes the conversion of HMG-CoA (3-hydroxy-3-meth-
ylglutaryl coenzyme A) to mevalonate, a precursor of choles-
terol. As lipid lowering agents statins also exert pleiotropic
effects at the vascular level . In addition to protection
against excitotoxicity in cultured neurons , statins have
against glutamate excitotoxic challenge in vitro .
These compounds also enabled the reduction of MMP-9
synthesis in rtPA-activated astrocytes . The effects of
statins may involve nuclear receptors, through an increase
in both expression and activity of PPAR-α [177–179]. More
recently, brain endothelial PPAR-δ activation has proven to
be protective against ischemia-induced cell death through
inhibition of the miR-15a microRNA, thus strengthening the
therapeutic concept based on activation of PPARs for the
treatment of stroke-related microvascular dysfunction .
5.3. Inhibition of JNK Activation and NVU Protection. The
c-Jun N-terminal kinases (JNKs) belong to the mitogen
activated protein kinase (MAPK) family; the two other
members being p38 and ERK [181, 182]. The isoforms
JNK1 and JNK2 are ubiquitously distributed, while JNK3 is
primarily expressed in the heart, brain, endocrine pancreas
and testis . JNKs are activated by phosphorylation,
which is catalyzed by upstream kinases—MKK 4 and 7 [182–
184]. JNK activation is essential for normal brain devel-
opment and organogenesis during embryonic development
. However, the activation of JNKs plays several roles
ranging from regulation of cell survival and apoptosis to
cell proliferation [183, 185–187]. They are activated under
pathological conditions both in the brain [188, 189] and
in the periphery [190, 191]. In fact, JNK phosphorylation
initially decreases after stroke and then starts to increase at
1.5 hours with a maximum at 9 hours after onset .
Phosphorylation of c-Jun, a JNK substrate, follows the same
temporal pattern, peaking at 8 hours post-stroke [192, 193].
The development of the peptide named DJNKi, a
competitive inhibitor of the JNK signaling pathway, has
been shown to reduce lesion volume of mice with transient
MCAO by 90% even when induced 6 hours after injury.
This lesion volume decrease was accompanied by behavior
improvements as well , suggesting an increase of the
therapeutic time window almost 2 times longer than tPA.
This positive outcome was also observed in a more severe
model with a permanent occlusion model . Moreover,
DJNKi has been shown to be compatible for treatment
of ischemic stroke even in the presence of rtPA and was
neurobehavior scores and decreased hemispheric swelling
after a model of intra-cerebral hemorrhage . Thus,
DJNKi could possibly attenuate the highly probable side
effect of hemorrhagic transformation caused by rtPA. Inter-
estingly, in this model of intracerebral hemorrhage, DJNKi
administration significantly increased AQP4 expression 48
hours after injury. This increase in AQP4 expression nega-
tively correlated with decreased hemispheric swelling, thus
pointing towards a possible role of DJNKi controlling edema
as well. In fact, activation of the JNK pathway is present
not only in the neurons but also in glial cells  and
brain endothelial cells . Such activation in nonneuronal
cells may negatively impact neuronal cell death and function
. In the context of broad effects of this drug, Benakis
et al.  showed that DJNKI-1, injected peripherally, is
As discussed previously, the development of a drug targeting
several cells such as in the NVU may help to move towards
success in the clinic.
10International Journal of Cell Biology
In summary, the data found in the literature suggest that the
failure of agents in protecting the brain against stroke may
come from the fact that each developed compound targeted
only one mechanism and one cell type of stroke pathophys-
iology. Ischemic preconditioning appears to be an attrac-
tive experimental strategy that would identify endogenous
of such protective mechanisms supports a complex action
on cells of the NVU, underlining the importance of the
interactions between endothelial cells and astrocytes in the
pathophysiology after stroke. As our knowledge of the NVU
increases, molecules with pleiotropic activity will become
increasing useful in the development of post-ischemic treat-
ments in the clinics.
Conflict of Interest
The authors declare that they have no conflict of interests.
French Soci´ et´ e de Biologie” for permitting the translation
of some topics previously published in the French journal
“Biologie Aujourd’hui. This paper was supported in part by
the NIH R01HD061946 (to J. Badaut), the Swiss Science
Foundation (FN 31003A-122166 to J. Badaut), and the Euro-
pean Union’s Seventh Framework Programme (FP7/2007-
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