Background
There is currently a total of 13 types of Aquaporins in mammals, each with its expression
in different body tissues such as red blood cells, lungs, pancreas, kidneys and nervous
tissue. All AQPs form tetramers in membranes in which monomers, each of ∼30kd molecular
size, contain six transmembrane helical domains and two helical segments surrounding
cytoplasmic and extra-cellular vestibules. The vestibules are connected by a narrow
aqueous pore allowing single-file water transport in which water selectivity is coferred
by electrostatic and steric factors.[1] They are proteins that regulate the flow of water into and out of the cell. However,
some of the AQPs such as AQP3 and AQP8 also allow the passage of glycerol and ammonia,
called aquaglyceroproteins.[2]
[3] In the central nervous system there is an increase in AQP 1 and AQP 4 in astrocytes
submitted to neoplasms and trauma.[4] The structure, function and location of AQPs in the various organs are known. However,
there is still to be discovered about the relationship and interaction that these
proteins play in the various pathophysiological processes involving such organs. These
findings would allow the genesis of new ways to approach pathologies such as neoplasia,
especially of the CNS, in which AQP 4 has a great influence, not yet fully known.
Gliomas are the most common central nervous system tumors. Glioblastoma multiforme
(GBM) is the most common and malignant brain tumor with high mortality and poor prognosis.
The vast majority of patients develop symptoms within ∼3 months and die within 8 to
18 months of diagnosis. Less than half live more than 6 months and only 3% have a
2-year survival. Malignant gliomas have a very aggressive and relapsing behavior,
giving the patient a very poor prognosis. For this reason, new therapies are needed
to change this scenario.[3]
Therefore, the aim of this review is to address the literature on the structure and
location of this protein in the CNS, as well as its function and expression in the
healthy CNS and brain gliomas. Finally, this study will address the relationship between
this protein and peritumoral edema that is present in the literature and the new therapeutic
approaches in research.
Results
Structure and Location of AQP4 in Brain and Tumors
Aquaporins are a family of integral membrane proteins that regulate the osmotic permeability
of the plasma membrane, allowing water to pass through the membrane while blocking
entry of ions or charged solutes.[2] It contains 6 transmembrane α-helices, with 2 asparagine-proline and alanine (NPA)
loops, each with its cytoplasmic B and extracellular E portion oriented 180 degrees
apart.[2] There are a total of 13 types of aquaporins in mammals.[5] In the Brain tissue there are three types of aquaporins: AQP 1, AQP 4 and AQP 9.[6]
AQP4 is usually expressed at the end of astrocyte perivascular cytoplasmic protrusions
([Fig. 1]), ependimoglia and glia limitans, in the black and gray substance where there is
10 to 15 times more molecular AQP4 than in the rest of brain tissue.[2]
[4]
[7]
[8]
[9]
[10] AQP4 is also found in the hippocampal dentate gyrus, medial habenular nucleus, cerebellum,
neocortex, and supra-optic and suprachiasmatic nuclei of the hypothalamus.[2] The polarized expression of AQP4 coincides with the location of potassium channels,
however, in gliomas, the location is lost and the water channels disperse throughout
the cell surface.[11] The protein comes in two different isoforms, M1 and M23, with 22 fewer amino acids.[4] Both isoforms have the same water permeability, but different aggregation properties.[12] With the mixture of the two isoforms and the agrin and dehydroglycan proteins, the
so-called Orthogonal Arrays of Particles (OAPs) of different sizes are formed, which
are present mainly at the extremities of the astrocyte protrusions[4]
[13] ([Fig. 2]). OAPs rich with the presence of the M23 isoform promote the formation of large
OAPs with little mobility, standing stationary at the end of the astrocyte perivascular
process.[12] M1isoform rich OAPs are small and diffuse freely into the cell membrane, mainly
for astrocyte processes, to aid in cell migration[12] ([Fig. 2]). The M1 isoform can exist alone in the cell, also moving freely through the membrane.[4]
[12] The isoform M23, when it does not agglomerate with the isoform M1, does so among
themselves, forming larger and less mobile clusters.[4]
[12] Agrine is an extracellular matrix proteoglycan and destroglycan is a component of
the dystrophin-destroglycan complex. The agrina connects to the dystrophin-dehydroglycan
complex, which in turn connects to OAPs. There complexes also contain the inwardly
rectifying potassium channel Kir4.1, a protein involved in spatial buffering of K+ ions released, because of synaptic activity, into the extracellular space.[14] Noel et al. Showed that this chain of molecules undergoes glioma alterations.[4] The hypothesis that Agrina and destroglycan are responsible for the polarization
of AQPs in the vascular extremities of astrocytes has been tested in vitro
[15] and in vivo
[4]
[16]. In vitro it has been shown that rats lacking agrin undergo alteration of membrane distribution
of AQP4.[3] In rats with no dehydroglycan, there was loss of OAPs at the vascular end of the
astrocytes, which correlated with down-regulation of AQP4 in the region.[3]
[4]
Fig. 1 Extracellular structure and astrocytic interaction with the normal brain blood-brain
barrier.
Fig. 2 OAP components and their location in the astrocyte membrane.
In same study, Noel et al showed that astrocytes at the extremities of glioblastomas
present the classic star shape with the presence of AQP4 at the vascular end of astrocytic
protrusions, the same pattern found in non-tumor astrocytes.[4] In the primary glioblastoma cell membrane, the density of OAPs is small even if
there is contact with the basal lamina ([Fig. 3]). In the relapsing Glioblastoma cell membrane, the occurrence of regular OAPs is
also small, and of non-polarized distribution, but with density similar to the parenchyma
and membrane area outside the astrocyte protrusion end. This shows the loss of cell
polarization of astrocytes during glioma transformation[8] in both primary and recurrent tumors, and in the latter, the density of OAPs is
closer to the normal pattern.[4] In addition, tumor regions with greater presence of small blood vessels showed greater
immunoreactivity to AQP4 than regions with larger blood vessels[4] ([Fig. 4]). It is possible to observe the redistribution of AQP4 already in the tumor infiltration
zone around the tumor, where the neurovascular functional structure is normal.
Fig. 3 Astrocyte extracellular structure in Glioblastoma and cellular localization of OAPs
- AQP 4.
Fig. 4 Effect of neoangiogenesis on AQP4 expression in small OAPs in Glioblastoma.
Reinforcing the above, Ndoum et al., In 2013,[17] through MRI analysis and immunohistochemistry, showed that low-grade gliomas preserve
their astrocyte processes, vascular structure and blood-brain barrier, and there is
no significant angiogenesis in the lesion. ([Fig. 5]). The author explained this fact by the tumor cell's ability to invade healthy tissue
and receive nutrients through the phenotypically normal astrocyte structure. The opposite
occurs in high grade gliomas. Astrocyte structure and basement membrane are lost around
the tumor vasculature. There is diffuse reactivity of AQP4 by the tumor and loss of
uniformity in AQP4 reactivity where the blood brain barrier is still intact[10] ([Fig. 3]).
Fig. 5 Astrocyte extracellular structure in astrocytomas and cellular localization of OAPs
- AQP 4.
AQP4 Function in Brain and Gliomas
The main function of AQP 4 are to regulate the exchange of intracellular and extracellular
water molecules, provide a transportation route for the rapid movement of water, participate
in water regulation in vivo, and maintain the water balance in vivo.[18]
AQP4 is known to participate in the formation of cerebral edema following trauma or
other brain diseases.[18] The distribution of AQP4 in the astrocyte cytoplasm suggests that this protein has
the function of controlling the flow of water into and out of the brain parenchyma.[2]
[4]
[10]
[19]
[20] As a bidirectional water channel, AQP 4 facilitates brain water accumulation in
cytotoxic edema and clearance of excess brain water in vasogenic and interstitial
edema.[1] Moreover, astrocytes are known to express AQP4 in OAPs primarily in cell protrusions.
Some of these protrusions involve the basal lamina of the cerebral vessels, fundamentally
participating in the maintenance of the blood-brain barrier.[3] The deletion of AQP4 can reduce water permeability through the cell plasma membrane
in the brain.[6] In addition to controlling the flow of water into and out of the cell, it has been
postulated that AQP 4 actively participates in the cell migration process.[3]
[9]
[19] By expelling water, the cell can easily change its morphology. As in gliomas, the
expression of aquaporin 4 is increased, the tumor cell has a great capacity for migration
and tissue invasion. This hypothesis was tested by Zhao et al.[9] using LN229 glioblastoma cells, in which there is low expression of AQP4, showing
low capacity for migration and tissue invasion in vitro. LN229 cell chemotaxis, compared with the control group, was lower. In addition to
chemotaxis, chemokinesis, which is independent of concentration gradient, was also
lower in the group with lower AQP4 expression.[19] Lack of AQP4 seems to change cell morphology. The cell body of LN229 cells became
thin and elongated. Reduction of AQP4 decreases the cell's ability to adhere to the
substrate due to reduced actin-F protein polymerization. Moreover, these cells showed
higher cell-cell adhesion capacity and lower invasion capacity according to the Matrigel
Boyden chamber test (in vitro). In vivo, the same pattern is repeated. Mice injected with glioblastoma cells without AQP4
showed less cellular invasion than the control group.[19]
Increasing AQP4 expression to some extent may facilitate the reabsorption of water
accumulated in the extracellular space, whereas a large increase in AQP4 expression
may cause water accumulation in glial cells leading to cell death.[9]
Studies by Ding et al., 2013, showed that AQP4 directly participates in the processes
of glioblastoma cell proliferation and apoptosis. By reducing the amount of AQP4 using
RNA inhibitor, human glioblastoma cells LN229 and U87 suffered apoptosis and had their
replication cycle shortened.[10]
AQP4 Expression in Gliomas
Mou et al.,[21] showed that AQP 4 expression is higher in the peritumoral region than in the tumor
itself and in normal brain tissue, besides increasing according to the histological
grade of the tumor. In addition, a positive correlation was observed between AQP4
expression with VEGF and HIF-1α. This result differs from that found in older literature,
which showed higher protein expression in the center of the tumor.[3] Tan et al.[22] compared MRI images and AQP4 mRNA expression between high-grade and low-grade gliomas.
The author found higher mRNA values in high-grade tumors than in low-grade solid tumors,
which is consistent with most of the literature.
Isoardo et al. compared AQP-4 expression in Glioblastoma multiforme between patients
with and without seizures.[23] In his study, the average tumor size among patients with and without seizures was
no different, as well as cortical involvement and MRI bleeding. Regarding the expression
of AQP-4 by immunohistochemistry, a greater increase in expression was detected in
patients with seizures than in those without seizures. The presence of AQP-4 was detected
in the GBM cell membrane. In patients with epileptic seizures the distribution of
AQP-4 was diffuse or perivascular, while in patients without seizures the immunohistochemistry
was undetectable or showed a diffuse pattern of expression.[23] However, by PCR there was no difference in expression between the 2 groups and both
showed increased expression of AQP-4.[23] Michael DeLay et al,[24] in 2012, showed that anti-VEGF antibody resistant glioblastomas show higher AQP4
expression. Through electron microscopy, Noel et al., In 2012, confirmed the greater
amount of AQP4 in glioblastoma than in control tissue.[4] In the same study, Western blotting results showed that both M1 and M23 isoforms
increased expression in the primary tumor. However, in relapsing glioblastoma tissues,
AQP4 expression is lower, comparable to control tissue, and with M23 isoform more
expressed than M1. In addition, tumor regions with greater presence of small blood
vessels showed greater immunoreactivity to AQP4 than regions with larger blood vessels[4] ([Fig. 3]). While AQP4 expression is increased in glioblastomas, the concentration of OAPs
is reduced. According to Noel et al., 2012, this could be explained by the increased
expression of the M1 isoform, which does not lead to the formation of dense OAPs ([Fig. 3]). However, the expression ratio between M1 and M23 forms did not change in glioblastomas.[4] Similarly, Becker et al., 2016, found similar results when performing real-time
PCR and electron microscopy to compare different expression of AQP4 isoforms in different
glioma grades. The author found a fall relationship in the presence of OAPs from low
to high grade gliomas.[15] However, there was no correlation between tumor malignancy and M1 isoform expression,
since this expression was uniform. In fact, there was an increase in M23 isoform expression
on average 1.5 higher than M1 isoform, being higher in grade IV gliomas. However,
the M23 / M1 ratio in a healthy brain is at least 3.[15] The author concludes, therefore, that the formation of OAPs depends on other mechanisms
besides the change of the M23 / M1 isoforms relation.[15]
Contrary to what is shown in the previously mentioned studies, Zhao et al., 2012,
observed a pattern of decreased AQP4 expression from grade II to IV gliomas. Their
conclusion was that AQP4 expression does not depend entirely on tumor malignancy,
but rather on tumor type. However, it has been shown that glioma cells with higher
migration capacity show higher AQP4 expression.[9] Moreover, the study also showed that in low-grade gliomas, the concentration of
AQP4 in astrocyte perivascular processes is higher than in high-grade gliomas.[9]
To relate radiological characteristics in MRI of gliomas and expression of AQP4, Tan
et al., In 2016, compared diffusion imaging of kurtosis (mean kurtosis, radial kurtosis
and axial kurtosis) and tension diffusion (mean diffusion) in High and low grade gliomas.
Their study showed that there is greater expression of AQP4 in the solid part of high
grade gliomas compared with low grade. Moreover, it showed that there is a directly
proportional relationship between the mean, radial and axial kurtosis with the expression
of AQP4, while the mean diffusion showed an inversely proportional relationship with
the expression of AQP4.[25] The study also showed that the mean kurtosis value in the peritumoral edema area
is higher in high grade gliomas than in low grade gliomas. This result may have been
due to a larger tumor infiltration of the high grade gliomas,[25] which may mean a tumor staging method through imaging.
Wang et al[26] proposed to research the expression of AQP 1 and 4 in the main pediatric brain tumors.
Regarding aquaporin 4 in astrocytomas, the author found that high-grade gliomas did
not express AQP 4, while there was high protein expression in pilocytic astrocytomas,
but with great variability. Houng et al[27] also demonstrated low AQP 4 expression in pilocytic astrocytoma samples, but high
expression in low-grade diffuse astrocytoma samples, mainly around the microvasculature
and with an intact blood-brain barrier.
AQP-4 Relationship with Edema
Brain edema is typically present in human brain cancers and affects both the course
and outcome of pathology, therefore can be considered a prognostic factor.[28] The appearance and effects of edema in clinical progression of brain cancers has
been known since long ago in clinical practice. Schoenegger et al. showed shorter
survival time after surgery in patients with major peritumoral edema (≥ 1 cm) in MRI.[28]
Cerebral edema is associated with various neurological disorders such as ischemia,
trauma and tumor; all leading to increased intracranial pressure and its comorbidities
such as herniation and death.[2] Under normal conditions, water moves in and out of the central nervous system, obeying
osmotic pressure.[2] There are 3 mechanisms of formation of cerebral edema: cytotoxic, vasogenic and
interstitial. Cytotoxic edema results from the disturbance of cellular metabolism,
increasing the movement of liquid into the intracellular space. Vasogenic edema occurs
due to alteration of the BBB, allowing greater passage of water and macromolecules,
accumulating fluid in the extracellular space. Interstitial edema occurs due to an
obstruction of the ventricular channels, causing hydrocephalus, in which the accumulated
fluid leaks through the periventricular walls.[2] Filippidis et al., 2016, reviews the relationship of aquaporins with cerebral edema.
The author points out several studies on the subject such as the reduction of intracranial
pressure due to cytotoxic edema in rats without AQP4 expression. Fazzina et al., 2010,
demonstrated that treatment with protein kinase C irreversible activator reduces cerebral
edema by decreasing AQP4 expression.[29] Besides, AQP 4 deletion in mice reduces cytotoxic brain edema.[30] Yang et al., in 2008, showed that AQP4-overexpressing mice had an accelerated progression
of cytotoxic brain swelling on acute water intoxication produced by intraperitoneal
water injection. This lad to higher intracranial pressures (ICP). In contrast, ICP
was lower in AQP4 knockout mice.[30]
Both benign and malignant tumors produce cerebral edema, which may be due to BBB defect
and increased tumor angiogenesis (Papadopoulos et al., 2003). Saaduon et al., 2002,
demonstrated that rats without AQP4 with edematous brain tumor showed higher intracranial
pressure values and more neurological complications when compared with rats with AQP4,
showing a possible protective effect of AQP4.[1] This information contrasts with the study of the same author, 2005, in which AQP4
promotes tumor metastasis by facilitating cell migration and angiogenesis,[1] thus promoting the formation of vasogenic edema.
Isoardo et al. compared the edema rate among patients with GBM who had epileptic seizures
and who had no seizures. There was no difference in edema between the two groups and
there was no difference between patients with positive and negative immunohistochemical
results. The author explains these results by choosing patients with lesions of similar
dimensions since the aim of the study was to determine if AQP-4 is related to the
onset of epileptic seizures regardless of edema.[23]
The work of Yang et al., 2012, observed the relationship between VEGF and AQP4 in
edema formation. By associating AQP4 expression with glioma cells expressing different
amounts of VEGF, the author found that there is no difference in AQP4 expression by
different amounts of VEGF.[31] However, there is increased expression of AQP4 in tumor tissue with increased VEGF,
vascular permeability and water content. The author concludes that VEGF does not directly
affect AQP4 expression, but that AQP4 redistribution in glioblastoma cells is a reaction
to VEGF-induced vasogenic edema to facilitate resorption of excess fluid.[31]
Mou et al., 2010[21] found that in edema associated with gliomas, AQP4 was also regulated by local osmotic
pressure and hypoxia. The degree of peritumoral edema could only be directly related
to AQP4 expression in the peritumoral region of the samples.
The findings in the study by Nduom et al. In 2013 have clinical implications in that
the breakdown of the BBB and astrocyte structure may lead to ultrafiltrate leakage
through endothelial vessels and cause peritumoral vasogenic edema.[17]
Henker et al., In 2016, related 10 different types of polymorphisms with the preoperative
volumetric characteristics of multiform glioblastomas. Using MRI and PCR, the study
shows a strong relationship between 4–31G and 131G Aquaporin polymorphism and the
ratio of tumor volume to peritumoral edema. The presence of this polymorphism determines
lower measured peritumoral edema compared with tumor volume.[32] In situations where there is no polymorphism, the ratio of peritumoral edema to
volume showed that the edema was on average twice as large as the tumor volume, and
the necrosis area was one quarter of the total tumor volume.[32] AQP4–131G> A is the promoter area of the AQP4 gene. Thus, a change in this area
may lead to alteration of AQP4 expression and consequent decrease of water accumulation
and reduction of peritumoral edema.[32]
Discussion
Frequently, cell lines or primary cells cultures from glioblastoma are used to measure
the cell volume regulation, but many glioma cell lines do not express the water channel
proteins. Besides, the majority of freshly isolated glioma cells do not express any
of these water channels in vitro in primary cell cultures. However, in glioma tissue,
from which the cells were isolate, AQP 4 were detected.[4]
[13]
Could the occurrence of AQP4 redistribution in the astrocyte plasma membrane already
in the tumor infiltration zone suggest an early factor to predict transformation to
Glioblastoma? The tumor infiltration zone still has normal neurovascular structure
and can be compared with normal tissue.[8] Moreover, the results shown by Nduom et al. 2013, could support the hypothesis that
the finding of AQP4 redistribution by astrocytes could be considered as a malignancy
factor for the tumor, since low grade tumors preserve their macrostructure more similar
to normal tissue. High-grade tumors, on the other hand, do not preserve the blood-brain
barrier, as well as diffuse expression of AQP4 throughout the cell membrane, even
near vascularization.[17]
Increasing AQP4 expression to some extent may facilitate the reabsorption of water
accumulated in the extracellular space, whereas a large increase in AQP4 expression
may cause water accumulation in glial cells leading to cell death.[9] The expression level of AQP4 correlates with the level of cerebral edema. One of
the factors that contribute to the maintenance of the blood-brain barrier is the concentration
of AQP4 in perivascular astrocyte processes.[9]
[20] Electron microscopy has shown that gap junction opens in the high-grade astrocytoma
microsvasculature. This, added to the loss of polarization of AQP4 in astrocytes can
lead to increased edema.[9] Conversely, as a result of the breakdown of the blood-brain barrier, the redistribution
of AQP4 into glioblastoma cells may act as a countermeasure to vasogenic edema. This
reinforces the hypothesis that AQP may be involved in the dynamics of edema formation
or resolution. The fact is that there is more about brain edema formation than just
AQP4. It has been shown that Na+-K+-Cl- cotransportes 1, matrix-metalloproteinase 9, thrombin, substance P and chemokine
receptors are also involved in the process.[33]
Increasing the amount of AQP4 expression in higher VEGF tumor tissue[31] could explain the greater amount of AQP4 found in tumor cells near small vessels
compared with large vessels,[4] since VEGF is the main modulator of angiogenesis. The author himself concluded that
there is no statistical relationship between VEGF value and AQP4 expression, but these
two factors could be physiopathologically involved.
The process of cellular invasion of malignant gliomas is complex and multifactorial.
In addition to increased cellular mobility, other mechanisms such as reduced substrate
and neighbor cell adhesion and extracellular matrix degradation are also involved.
The role of aquaporin 4 in these mechanisms is not yet fully understood. The same
could also be seen in the systematic review by Lan et al.[3]
Although the biomolecular behavior of brain gliomas has not been clarified, treatment
methods are a target of research. Nico et al., 2009, found that the combination of
chemotherapy and radiotherapy reduces AQP4 expression[4]
[8] and restores cell polarization.[8] It has also been shown that LN229 glioblastoma cells show lower AQP4 expression.
As a result, these cells showed lower capacity for migration and tissue invasion.[8] Thus, the use of AQP4 inhibitory therapies for the treatment of cerebral gliomas
would be possible. Ding et al., 2013 showed this possibility by using AQP4 inhibitory
RNA in human glioblastoma cells causing apoptosis of these cells.[10] Another proposed idea is the use of AQP4-specific antibody linked with toxin to
selectively damage AQP 4-expressing glioblastoma cells.[6] The use of curcumin to attenuate brain edema is also proposed, as one of its effects
is the reduction of expression of AQP 4 and 1.[34]
In addition to molecular therapies, noninvasive evaluation methods for gliomas are
being studied. The study of Tan et al.[22] correlates mean diffusion coefficient values obtained by serial MRI images between
high and low grade gliomas and AQP4 mRNA expression. The study shows higher AQP4 expression
in high grade tumors and a directly proportional relationship between AQP4 expression
and mean diffusion values in solid parts of the tumor. However, there was no relationship
between the diffusion coefficients for the peritumoral edema region. The author believes
that the redistribution of AQP4 in the cell surface of high grade gliomas is responsible
for the high values of mean diffusion coefficient, showing greater water flow in the
solid region of these tumors. Such imaging study could serve as a new form of assessment
of staging and expression of AQP in astrocytomas.
No studies were found in the literature associating the expression value of AQP4 with
patient follow-up or survival after surgery. Such studies could show some relationship
from which there was a predictive value of patient survival after possible surgery.
Moreover, most of the laboratory studies had the limitation of a small sample space,
impairing the impact or the statistical weight of the results.
