**2. Development and structure of the blood-brain barrier**

Blood-brain barrier (BBB) separates in a selective manner the nerve tissue of the central nervous system (CNS) from the blood. It is present throughout the CNS, except in the circumventriculars organs, where the capillaries are fenestrated as occurs in choroid plexuses. Indeed, such an anatomical membrane can selectively transport, through the capillary wall, large (>500 Da) or water soluble (hydrophilic) substances, whereas, small, lipid-soluble (hydrophobic) substances can freely pass the endothelium by passive diffusion [4, 5].

Classically, BBB was recognized as the structural base of the so-called *immune privilege* of the brain. Thereby, antigens would be sequestered within the brain and would be invisible to the immune system [6].

tissue from blood. TJs are elaborated and sealed, transcytosis decreases, leukocyte adhesion

**Figure 1.** Blood-brain barrier components and their interactions (figure partially modified from Hawkins BT and Davis

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Vascular endothelial growth factor (VEGF), VEGFR-2 and its ligand, play a pivotal role in embryonic angiogenesis and vasculogenesis [8]. In the developing CNS, embryonic brain cells of the subventricular neuroectoderm synthetize VEGF that directs angiogenesis via a concentration gradient through the angiogenic sprouting from vessel networks outside the CNS, in particular, the perineural vascular plexus (PNVP). Within the brain, blood vessels furtherly, then, generate huge networks as the neural tissue grows and concomitantly remodel into a vascular tree with arterial and venous hierarchy [9, 10]. Such a process, when VEGF is reduced or absent, develops in an incorrect way generating decreased blood vessel branching and density in the cortex [11].

Vasculogenesis and angiogenesis have been extensively studied in experimental setting in mouse retina and hindbrain and in zebrafish with the development of the "tip-stalk" model of angiogenic sprouting, which describes the different precursor of endothelial cells within

**1.** Tip cells drive the sprouting, migrating and extending filopodia that scan the environment

**2.** Behind the tip cells, another group of elements, termed stalk cells, proliferate and form the

Once mature connections and blood flow have been established, the proliferation and the migration of "activate endothelium" ceases. Tip and stalk cells display differential gene expression profiles [12]. The Notch signaling pathway is a very important tool in the regulation

molecules are downregulated and efflux transporter expression increases [7].

newly formed vascular sprouts:

TP [105]).

nascent vascular lumen.

for signals that can act as guides for vascular growth.

This view has been challenged in recent years, due to the discovery of the so-called "glymphatic system" (GS) and of meningeal lymphatics (MLs) of dura mater. GS allow that cerebrospinal fluid flows into brain within periarterial spaces and interstitial fluid and solute clear via perivenous spaces. Furtherly, MLs follow dural blood vessels and cranial nerves and exit the cranium via the foramina together with the venous sinuses, arteries, and cranial nerves in order to join cervical lymph nodes. The relationship of this system with the equilibrium of the bloodbrain barrier is unknown and its role in human pathology has yet to be clarified (**Figure 1**) [6].

Tight junctions (TJs) between endothelial cells (ECs) are the main component of BBB. TJs are occluding cell junctions, which act to seal off the intercellular space and consist of transmembrane proteins such as occludins (Oclns) and claudins (Cldns), connected intracellularly to the actin filaments, forming strands in the plasma membrane.

TJs blocks the intercellular pathway and forms a barrier between the arterial blood and the nervous tissue, regulated by the end feet of astrocytes that cover the basement membrane of the capillaries [5].

The development of the BBB begins with angiogenesis, started from invasion of the neuroectoderm by endothelial progenitor cells from pre-existing vessels. The endothelia of these vascular sprouts show many characteristics of mature BBB such as TJs, transcytotic vesicles, nutrient transporters and leukocyte adhesion molecules. Afterwards, contact with CNS cells and pericytes (PCs) allows the full functional maturation of the membrane separating nervous Blood-Brain Barrier Breakdown by Combined Detection of Circulating Tumor and Endothelial… http://dx.doi.org/10.5772/intechopen.80594 129

monitor the infiltration of tumor cells in the parenchyma and/or perivascular spaces, even at a single cell level [1–3]. However, the relationship between glioma-induced BBB dysregulation and glioma invasion remains poorly understood. In this chapter we review the natural history of glioblastoma and, on the basis of the scientific evidence published to date, we try to give an explanation and meaning to biomarkers found in the peripheral blood. In particular, we focus our attention on cellular biomarkers, the circulating tumor cells and cellular endothelial progenitors. Our interest is aimed at giving an order in the context of human cell biology of human glioblastoma. This intracranial tumor remains today one of the big killers and represents a major challenge in the field of oncology because it is unresponsive to treatment and

Blood-brain barrier (BBB) separates in a selective manner the nerve tissue of the central nervous system (CNS) from the blood. It is present throughout the CNS, except in the circumventriculars organs, where the capillaries are fenestrated as occurs in choroid plexuses. Indeed, such an anatomical membrane can selectively transport, through the capillary wall, large (>500 Da) or water soluble (hydrophilic) substances, whereas, small, lipid-soluble (hydropho-

Classically, BBB was recognized as the structural base of the so-called *immune privilege* of the brain. Thereby, antigens would be sequestered within the brain and would be invisible to the

This view has been challenged in recent years, due to the discovery of the so-called "glymphatic system" (GS) and of meningeal lymphatics (MLs) of dura mater. GS allow that cerebrospinal fluid flows into brain within periarterial spaces and interstitial fluid and solute clear via perivenous spaces. Furtherly, MLs follow dural blood vessels and cranial nerves and exit the cranium via the foramina together with the venous sinuses, arteries, and cranial nerves in order to join cervical lymph nodes. The relationship of this system with the equilibrium of the bloodbrain barrier is unknown and its role in human pathology has yet to be clarified (**Figure 1**) [6]. Tight junctions (TJs) between endothelial cells (ECs) are the main component of BBB. TJs are occluding cell junctions, which act to seal off the intercellular space and consist of transmembrane proteins such as occludins (Oclns) and claudins (Cldns), connected intracellularly to the

TJs blocks the intercellular pathway and forms a barrier between the arterial blood and the nervous tissue, regulated by the end feet of astrocytes that cover the basement membrane of

The development of the BBB begins with angiogenesis, started from invasion of the neuroectoderm by endothelial progenitor cells from pre-existing vessels. The endothelia of these vascular sprouts show many characteristics of mature BBB such as TJs, transcytotic vesicles, nutrient transporters and leukocyte adhesion molecules. Afterwards, contact with CNS cells and pericytes (PCs) allows the full functional maturation of the membrane separating nervous

able to progress in a way difficult to monitor.

immune system [6].

128 Liquid Biopsy

the capillaries [5].

**2. Development and structure of the blood-brain barrier**

bic) substances can freely pass the endothelium by passive diffusion [4, 5].

actin filaments, forming strands in the plasma membrane.

**Figure 1.** Blood-brain barrier components and their interactions (figure partially modified from Hawkins BT and Davis TP [105]).

tissue from blood. TJs are elaborated and sealed, transcytosis decreases, leukocyte adhesion molecules are downregulated and efflux transporter expression increases [7].

Vascular endothelial growth factor (VEGF), VEGFR-2 and its ligand, play a pivotal role in embryonic angiogenesis and vasculogenesis [8]. In the developing CNS, embryonic brain cells of the subventricular neuroectoderm synthetize VEGF that directs angiogenesis via a concentration gradient through the angiogenic sprouting from vessel networks outside the CNS, in particular, the perineural vascular plexus (PNVP). Within the brain, blood vessels furtherly, then, generate huge networks as the neural tissue grows and concomitantly remodel into a vascular tree with arterial and venous hierarchy [9, 10]. Such a process, when VEGF is reduced or absent, develops in an incorrect way generating decreased blood vessel branching and density in the cortex [11].

Vasculogenesis and angiogenesis have been extensively studied in experimental setting in mouse retina and hindbrain and in zebrafish with the development of the "tip-stalk" model of angiogenic sprouting, which describes the different precursor of endothelial cells within newly formed vascular sprouts:


Once mature connections and blood flow have been established, the proliferation and the migration of "activate endothelium" ceases. Tip and stalk cells display differential gene expression profiles [12]. The Notch signaling pathway is a very important tool in the regulation of the tip and stalk cells specification. The activation of Notch signaling inhibits tip cells differentiation and promotes the stalk cell phenotype [13].

for TJs formation. Cldn5 is regulated by the Wnt/β-catenin pathway, however activation of VEGF, or other signaling pathways, can oppose the action of Wnt/β-catenin pathway. The Embryonic ablation of Cldn5 in mice induces early postnatal brain edema and death [21]. Claudin-5 deficient mice exhibit an increased leakiness for small-molecular compounds (<800 Da) [22]. BBB leakiness can be tolerated during embryogenesis as long as the placental barrier is functional. A post-natal maturation of brain circulation is a more than likely fact. Indeed, in the mammalian

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Interestingly, in the regions where the corners of three epithelial cells meet, TJs have a specialized structure, the so-called tricellular junctions (tTJs) which contain the tetraspan Marvel-domain protein tricellulin. Such a protein has been detected in the rat and human brain [24, 25]. Tricellular junctions may be critical for the BBB formation. Moreover, lipolysisstimulated lipoprotein receptor (LSR), a component of paracellular junctions in their three cell membranes meeting points, expression follows CNS angiogenesis and correlates with

PCs are contractile cells surrounding the endothelium of capillaries and postcapillary venules, which are enclosed within the basal lamina of the endothelium along the vessels. They behave as mesenchymal multipotent stem cells giving rise to ECs or smooth muscle cells. PCs play an important role in the angiogenesis and in the BBB integrity. Pericytes show specialized characteristics and roles in different organs such as kidney, liver and brain. Moreover, density

A direct contact between pericytes and ECs is established where the basement membrane is absent via the "peg-and-socket" junctions which are formed by n-cadherin and connexin-43 hemichannels. Adherent junctions between PCs and ECs are also present. Interactions of ECs with PCs and SMCs are pivotal processes in the regularization, remodeling, stabilization and function of vascular wall and BBB, i.e., by the regulation of the transcellular barrier [27–29]. Moreover, the single adhesion receptor CD146 functions on PCs as a co-receptor for Plateletderived growth factor receptors beta (PDGFR-β) to regulate interactions between ECs and PCs. CD146, shows an initial expression on ECs, during BBB maturation, that slopes down upon PCs recruitment and BBB maturation [30]. Interestingly, astrocytic laminin induces pericyte differentiation from the resting stage to the contractile stage, switching pericyte function

Astrocytes (ACs) surround microvessels and capillaries and interact with endothelial cells through the end-feet of their processes. ACs play critical roles in regulating cerebral blood flow in response to neuronal activity by relaying signals and maintain BBB function by inducing barrier properties and the polarization of transporters [28]. Under steady-state conditions ACs promote BBB homeostasis through soluble factors such as Sonic hedgehog (Shh), retinoic acid (RA), glial-derived neurotrophic factor (GDNF) and angiopoietin1 (Ang-1), which interact with receptors on ECs to increase junctional protein expression, raise transendothelial electrical resistance (TEER), and reduce permeability. Furthermore, knocking out a-dystrobrevin (a-DB), a scaffolding protein of the astrocytic endfeet, or astrocyte-secreted laminin a2, leads to down-regulation of junctional proteins and a leaky BBB [32]. Interactions between ACs and ECs are very important not only for formation and maintenance of BBB, but also for

brain, angiogenesis in the cortex well proceeds until 2–3 weeks after birth [23].

BBB formation during embryogenesis [26].

of pericytes and vessel coverage vary among tissues [27].

from stabilizing the BBB to compromising it [31].

astrocytic differentiation [21].

Wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt) pathway plays a pivotal role in angiogenesis both physiological and pathological and in vessels remodeling [14, 15]. Three Wnt signaling pathways are known: the so-called "canonical" Wnt/βcatenin and the two "noncanonical" pathways: the Wnt/calcium (Wnt/Ca2+) and the Wnt/ planar cell polarity (Wnt/PCP) [16].

The canonical Wnt/β-catenin signaling pathway targets the regulatory molecule β-catenin. The so-called cytoplasmic destruction complex, consisting of glycogen synthase kinase-3β (GSK3β), Axin, Casein kinase1/2 (CK1/2), Protein phosphatase 2A (PP2A), and adenomatous polyposis coli (APC) leads to a post-translational modification status of β-catenin. In the absence of Wnt signaling, cytosolic β-catenin is phosphorylated by CK1 at Thr41, and GSK3β at Ser33 and Ser37. Phosphorylated β-catenin is then ubiquitinated via the E3 ligase, β-transducing-repeatcontaining protein (β-TrCP), and thereby prepared for proteasomal degradation. Conversely, Wnt binding to Frizzled proteins, a Wnt receptors family recruits the co-receptor LRP5/6, that causes an activation of the cytoplasmic phosphoprotein Disheveled. These events lead to the inhibition of GSK3β, thereby promoting the accumulation of unphosphorylated β-catenin and its subsequent translocation to the nucleus, where it binds to a variety of transcription factors, including T-cell factor/lymphocyte enhancing factor (TCF/LEF) and forkhead box (in particular, the FOXO subtypes) family proteins. TCF/LEF represses targets in the absence of signaling, but beta-catenin, when its pathway is activated, enters into the nucleus, binds to TCF on the chromatin and allows the transcription of a number of Wnt target genes involved in cell proliferation, Wnt signal transduction and vascular growth [17, 18].

Tight junctions are organized to form a paracellular seal in transporting epithelia in order to allow the directional transfer of ions and solutes across cell layers.

Tight junctions comprise several trans-membrane proteins (TMPs) which interact with adaptor proteins of the cytoplasmic plaque via their C-terminal domains. TMPs are classified on the basis of their number of transmembrane domains (tetraspan, trispan, and single-span domains) and include the tetraspan Marvel-domain proteins (occludin, tricellulin, and Mar velD3), the claudin family of proteins, the trispan BVES (blood vessel epicardial substance) protein, the single-span JAMs (junctional adhesion molecule-A, -B, and -C), and the polarity determinant Crumbs3. The cytoplasmic plaque is composed of Zona occludens proteins (ZO-1, ZO-2, and ZO-3), multi-PDZ domain protein 1 (MUPP1), cingulin, protein associated with Lin-7 (PALS1), Pals1 associated tight junction (PATJ), protease activated receptor 3 (PAR3) and protease activated receptor 6 (PAR6), which interact in the intracellular space with the cytoskeleton. Moreover, several signaling molecules are associated to the proteins of the cytoplasmic plaque [19].

Oclns and Cldns mainly characterize TJs. The exact role of Ocln is not well known, but it may be a structural component important in in the formation of TJs. Its function is regulated via cytokines, proteases and GTPases [20].

The Wnt/β-catenin pathway, play role in BBB differentiation and may be fundamental in BBB maintenance. Such a pathway is also active in endothelial cells of the adult CNS, providing an essential tool for BBB maintenance [21]. Claudin family member claudin-5 (Cldn5) is important for TJs formation. Cldn5 is regulated by the Wnt/β-catenin pathway, however activation of VEGF, or other signaling pathways, can oppose the action of Wnt/β-catenin pathway. The Embryonic ablation of Cldn5 in mice induces early postnatal brain edema and death [21]. Claudin-5 deficient mice exhibit an increased leakiness for small-molecular compounds (<800 Da) [22]. BBB leakiness can be tolerated during embryogenesis as long as the placental barrier is functional. A post-natal maturation of brain circulation is a more than likely fact. Indeed, in the mammalian brain, angiogenesis in the cortex well proceeds until 2–3 weeks after birth [23].

of the tip and stalk cells specification. The activation of Notch signaling inhibits tip cells dif-

Wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt) pathway plays a pivotal role in angiogenesis both physiological and pathological and in vessels remodeling [14, 15]. Three Wnt signaling pathways are known: the so-called "canonical" Wnt/βcatenin and the two "noncanonical" pathways: the Wnt/calcium (Wnt/Ca2+) and the Wnt/

The canonical Wnt/β-catenin signaling pathway targets the regulatory molecule β-catenin. The so-called cytoplasmic destruction complex, consisting of glycogen synthase kinase-3β (GSK3β), Axin, Casein kinase1/2 (CK1/2), Protein phosphatase 2A (PP2A), and adenomatous polyposis coli (APC) leads to a post-translational modification status of β-catenin. In the absence of Wnt signaling, cytosolic β-catenin is phosphorylated by CK1 at Thr41, and GSK3β at Ser33 and Ser37. Phosphorylated β-catenin is then ubiquitinated via the E3 ligase, β-transducing-repeatcontaining protein (β-TrCP), and thereby prepared for proteasomal degradation. Conversely, Wnt binding to Frizzled proteins, a Wnt receptors family recruits the co-receptor LRP5/6, that causes an activation of the cytoplasmic phosphoprotein Disheveled. These events lead to the inhibition of GSK3β, thereby promoting the accumulation of unphosphorylated β-catenin and its subsequent translocation to the nucleus, where it binds to a variety of transcription factors, including T-cell factor/lymphocyte enhancing factor (TCF/LEF) and forkhead box (in particular, the FOXO subtypes) family proteins. TCF/LEF represses targets in the absence of signaling, but beta-catenin, when its pathway is activated, enters into the nucleus, binds to TCF on the chromatin and allows the transcription of a number of Wnt target genes involved

Tight junctions are organized to form a paracellular seal in transporting epithelia in order to

Tight junctions comprise several trans-membrane proteins (TMPs) which interact with adaptor proteins of the cytoplasmic plaque via their C-terminal domains. TMPs are classified on the basis of their number of transmembrane domains (tetraspan, trispan, and single-span domains) and include the tetraspan Marvel-domain proteins (occludin, tricellulin, and Mar velD3), the claudin family of proteins, the trispan BVES (blood vessel epicardial substance) protein, the single-span JAMs (junctional adhesion molecule-A, -B, and -C), and the polarity determinant Crumbs3. The cytoplasmic plaque is composed of Zona occludens proteins (ZO-1, ZO-2, and ZO-3), multi-PDZ domain protein 1 (MUPP1), cingulin, protein associated with Lin-7 (PALS1), Pals1 associated tight junction (PATJ), protease activated receptor 3 (PAR3) and protease activated receptor 6 (PAR6), which interact in the intracellular space with the cytoskeleton. Moreover, several

Oclns and Cldns mainly characterize TJs. The exact role of Ocln is not well known, but it may be a structural component important in in the formation of TJs. Its function is regulated via

The Wnt/β-catenin pathway, play role in BBB differentiation and may be fundamental in BBB maintenance. Such a pathway is also active in endothelial cells of the adult CNS, providing an essential tool for BBB maintenance [21]. Claudin family member claudin-5 (Cldn5) is important

in cell proliferation, Wnt signal transduction and vascular growth [17, 18].

signaling molecules are associated to the proteins of the cytoplasmic plaque [19].

cytokines, proteases and GTPases [20].

allow the directional transfer of ions and solutes across cell layers.

ferentiation and promotes the stalk cell phenotype [13].

planar cell polarity (Wnt/PCP) [16].

130 Liquid Biopsy

Interestingly, in the regions where the corners of three epithelial cells meet, TJs have a specialized structure, the so-called tricellular junctions (tTJs) which contain the tetraspan Marvel-domain protein tricellulin. Such a protein has been detected in the rat and human brain [24, 25]. Tricellular junctions may be critical for the BBB formation. Moreover, lipolysisstimulated lipoprotein receptor (LSR), a component of paracellular junctions in their three cell membranes meeting points, expression follows CNS angiogenesis and correlates with BBB formation during embryogenesis [26].

PCs are contractile cells surrounding the endothelium of capillaries and postcapillary venules, which are enclosed within the basal lamina of the endothelium along the vessels. They behave as mesenchymal multipotent stem cells giving rise to ECs or smooth muscle cells. PCs play an important role in the angiogenesis and in the BBB integrity. Pericytes show specialized characteristics and roles in different organs such as kidney, liver and brain. Moreover, density of pericytes and vessel coverage vary among tissues [27].

A direct contact between pericytes and ECs is established where the basement membrane is absent via the "peg-and-socket" junctions which are formed by n-cadherin and connexin-43 hemichannels. Adherent junctions between PCs and ECs are also present. Interactions of ECs with PCs and SMCs are pivotal processes in the regularization, remodeling, stabilization and function of vascular wall and BBB, i.e., by the regulation of the transcellular barrier [27–29].

Moreover, the single adhesion receptor CD146 functions on PCs as a co-receptor for Plateletderived growth factor receptors beta (PDGFR-β) to regulate interactions between ECs and PCs. CD146, shows an initial expression on ECs, during BBB maturation, that slopes down upon PCs recruitment and BBB maturation [30]. Interestingly, astrocytic laminin induces pericyte differentiation from the resting stage to the contractile stage, switching pericyte function from stabilizing the BBB to compromising it [31].

Astrocytes (ACs) surround microvessels and capillaries and interact with endothelial cells through the end-feet of their processes. ACs play critical roles in regulating cerebral blood flow in response to neuronal activity by relaying signals and maintain BBB function by inducing barrier properties and the polarization of transporters [28]. Under steady-state conditions ACs promote BBB homeostasis through soluble factors such as Sonic hedgehog (Shh), retinoic acid (RA), glial-derived neurotrophic factor (GDNF) and angiopoietin1 (Ang-1), which interact with receptors on ECs to increase junctional protein expression, raise transendothelial electrical resistance (TEER), and reduce permeability. Furthermore, knocking out a-dystrobrevin (a-DB), a scaffolding protein of the astrocytic endfeet, or astrocyte-secreted laminin a2, leads to down-regulation of junctional proteins and a leaky BBB [32]. Interactions between ACs and ECs are very important not only for formation and maintenance of BBB, but also for astrocytic differentiation [21].

Morphology and function of BBB are linked as shown by microscopic observation. Early ultrastructural studies where performed by administration of silver nitrate in the drinking water of rodents [33]. They showed the presence of very scarce quantity of silver around the capillaries in the cerebral cortex, medulla, and cerebellum. In such brain regions, capillaries are continuous. In other areas, such as neurohypofisis, area postrema, pineal body and intercolumnar tubercle, heavy accumulation of silver was present around fenestrated capillaries [34]. Using lanthanum and horseradish peroxidase as tracers, via intravenous injection, it has been demonstrated that these substances are unable to penetrate between the endothelial cells because of the tight junctions presence (zonulae occludentes). Regarding the contribution of astrocytes to the BBB, it is well known that its end-feet form a relatively complete layer, but the junctions between are gap junctions and not of the occluding kind [35]. Perivascular endfeet of astrocytes do not provide an effective barrier even if substances should pass through the endothelial cells into the brain. Indeed, after intravenous infusion of peroxidase, endothelial cells show micropinocytotic vesicles containing the tracer [36]. Such a transendothelial cell barrier is very selective and based on carrier-mediated transports, but is not furtherly mechanically regulated.

nidogen, osteonectin and laminin. BM functions as a charge and molecular weight barrier and is able to interact with integrins in order to regulate permeability and cellular transport also across the BBB. Dystroglycans and integrins are transmembrane receptors that allow BBB cells to interact with BM. During brain angiogenesis in mice, ECs show α4β1 and α5β1 integrin, whereas in adult animals α4 and α5β1integrins promote stabilization of vessels [42], α4β1 and α5β1 integrin induce cell proliferation through MAPK signaling in human ECs. Moreover, β1 integrin interaction with laminin maintains levels of claudin-5 in TJs [43, 44].

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The endothelial cells (ECs) compose the wall of vessels and capillaries and represent the primary blood-tissue barrier. The ECs acting as a protective filter are able to regulate the passage of molecules and immune cells and the level of specialization of each blood-barrier is determined by the functions of endothelial-wall. In the brain, there is the higher level of specialization of the endothelial wall. The filter function of BBB is carried out by ECs strictly

Pathological conditions within the central nervous system like ischemia, inflammation or tumor growth lead to blood-brain barrier (BBB) dysfunction, emphasizing that the permeability barrier regulation is principally provided by the local microenvironment and its maintenance is a necessary condition in any circumstance. In many brain tumors morphological

Wolburg et al. [48] found that claudin-3, a key component of BBB tight junctions, is lost in glioblastoma. Further evidence on claudin-1 loss in tumor microvessels, as well as downregulation of claudin-5 and occludin in hyperplastic vasculature, result in a phenotypic change in

A recent study by Watkins et al. [49] using a mouse model demonstrates that astrocytic endfeet displace from their position alongside endothelial cells with disruption of the communication between the astrocytes and vasculature and that single glioma cells were sufficient to

The structural perturbations of the vascular barrier during tumor progression, in other organs was demonstrated, are successive to the release in the tumor microenvironment of specific

The crucial point is the vascular damage that the tumor direct and indirect operas through the release on the next microenvironment of chemical factors able to increase the permeability of the tissue vessels reducing their protective barrier function. This could explain the pathogenesis of the BBB damage occurring during the development of glioblastoma. The BBB damage induced by the glioblastoma represents a strategy employed to control the BBB opening to

The detection of endogenous circulating molecules normally restricted by the BBB, namely albumin, immunoglobulin G, or fibrinogen, in the brain parenchyma, using immunohistochemistry

irregularities of the perivascular space correlate with a breakdown of the BBB [45–47].

BBB function due to leaky tight junctions and hyperpermeable endothelial cells.

cytokines that downregulate the transcription of these structural proteins [50].

**3. Blood-brain barrier damage**

interconnect with numerous tight junctions.

produce local BBB opening.

allow the passage of drugs [51].

Caveolae are small, bulb-shaped, plasma membrane invaginations. They have been described to have a function in endocytosis and transcytosis and in in maintaining the lipid composition of the membrane, as well as acting as signaling background.

While endothelial cells in peripheral organs, such as the lung and heart, are enriched in caveolae, in BBB only a small number of caveolae are detectable [24]. Mfsd2a (transporter of the major facilitator superfamily domain-containing (Mfsd) family) contributes to the regulation of vesicular traffic in BBB endothelial cells [37] through the transport of the essential omega-3 fatty acid docosahexaenoic acid (DHA). The expression of Mfsd2a becomes upregulated in ECs with the maturation of BBB. Gene ablation of Mfsd2a in mice results in BBB leakiness and increased vesicular traffic in ECs.

Caveolins (Cavs) are thought to play a role in the regulation of BBB function. Cavs, are a family of integral membrane proteins which represent both positive and negative regulators of intracellular signaling as scaffolding proteins that regulate the intracellular distribution of the signaling molecules. Cav-1 overexpression protects the integrity of the BBB mainly by preventing the degradation of TJ proteins in rats [38].

Cav-1 is a marker of caveolae in endothelial cells and is important in the regulation of various functions like endocytosis, transcytosis, signal transduction, and molecular transport. Recent studies on mice indicate that the suppression of the caveolae pathway requires the transport of lipids, notably DHA-containing phospholipids, by Mfsd2a to regulate CNS endothelial cell plasma membrane composition and to inhibit caveolae vesicle formation [39].

Moreover, Cav-1 regulate the angiogenic response by influencing VEGF receptor 2 (VEGFR2) phosphorylation and internalization [40, 41].

In BBB the basement membrane (BM) represents the noncellular component. Astrocytes, PCs and ECs synthetize and secrete molecules which constitute the BM surrounding the external surface of the endothelial cell, composed by type IV collagen, fibronectin, heparan sulfate, nidogen, osteonectin and laminin. BM functions as a charge and molecular weight barrier and is able to interact with integrins in order to regulate permeability and cellular transport also across the BBB. Dystroglycans and integrins are transmembrane receptors that allow BBB cells to interact with BM. During brain angiogenesis in mice, ECs show α4β1 and α5β1 integrin, whereas in adult animals α4 and α5β1integrins promote stabilization of vessels [42], α4β1 and α5β1 integrin induce cell proliferation through MAPK signaling in human ECs. Moreover, β1 integrin interaction with laminin maintains levels of claudin-5 in TJs [43, 44].
