Astrocyte Involvement in Blood–Brain Barrier Function: A Critical Update Highlighting Novel, Complex, Neurovascular Interactions

Neurological disorders have been linked to a defective blood–brain barrier (BBB), with dysfunctions triggered by stage-specific disease mechanisms, some of these being generated through interactions in the neurovascular unit (NVU). Advanced knowledge of molecular and signaling mechanisms in the NVU and the emergence of improved experimental models allow BBB permeability prediction and the development of new brain-targeted therapies. As NVU constituents, astrocytes are the most numerous glial cells, characterized by a heterogeneity that occurs as a result of developmental and context-based gene expression profiles and the differential expression of non-coding ribonucleic acids (RNAs). Due to their heterogeneity and dynamic responses to different signals, astrocytes may have a beneficial or detrimental role in the BBB’s barrier function, with deep effects on the pathophysiology of (and on the progression of) central nervous system diseases. The implication of astrocytic-derived extracellular vesicles in pathological mechanisms, due to their ability to pass the BBB, must also be considered. The molecular mechanisms of astrocytes’ interaction with endothelial cells at the BBB level are considered promising therapeutic targets in different neurological conditions. Nevertheless, a personalized and well-founded approach must be addressed, due to the temporal and spatial heterogeneity of reactive astrogliosis states during disease.


Introduction
The blood-brain barrier BBB is a complex, dynamic, selectively permeable structure that lines blood vessels in the brain.It represents the physical and metabolic barrier between the bloodstream and neuroglia from the central nervous system (CNS) parenchyma.The BBB blocks foreign substances in the bloodstream from entering brain tissue and also prevents therapeutic molecules from reaching the brain.Aspects related to BBB disruption should be known in predictions of which drug is best suited to an individual patient [1].
The endothelial cells (ECs) anchored to the basal lamina are critical regulators of the vascular basement membrane's (BM) permeability through tight junctions (TJs) and adherens junctions (AJs) between adjacent ECs, which limit paracellular transport of water-soluble or polar compounds, restrict the transport of small ions, and maintain ECs' polarity [2], whilst a collagen-rich parenchymal extracellular matrix (ECM) provides the scaffold.Pericytes modulate the contractility of the microvasculature and ultimately the BBB, and astrocytes are the 'sensing' cells that react to the metabolic requirements of neurons.The BM proteins, primarily secreted by local astrocytes, together with the astrocytic end-feet, almost completely surround the brain vasculature, forming glia limitans vascularis and, in this way, being responsible for maintaining the BBB's integrity and CNS's homeostasis [3,4].
In this review, we describe the complex interplay associated with maintenance of BBB integrity, which is driven by multiple factors released from heterogeneous astrocyte subtypes that consequently trigger EC activation and participation.

BBB Permeability-Regulating Proteins
Neurological disorders such as ischemia, brain trauma, and neurodegenerative diseases lead to BBB disruption, resulting in brain edema and parenchymal inflammation.In the case of injury, astrocyte phenotype shifts to a complex and heterogeneous reactive form, which can exert a protective role promoting BBB barrier recovery or, conversely, can induce EC apoptosis and decreased expression of proteins involved in BBB barrier integrity and selective permeability.
AJs are composed of cadherin-catenin complexes, together with TJ assembly proteins, that interact by associated adhesive cell-cell interactions.Vascular endothelial (VE)cadherin, expressed by ECs and situated toward the basolateral side of ECs, has an essential role in this BBB permeability regulation [12].
Annexins are Ca2+ and phospholipid-binding proteins that regulate intracellular Ca2+ concentrations and traffic, as well as cellular membrane organization.Annexin A 1, expressed in pericytes and ECs, facilitates TJ function through its connection between the cytoskeleton and the plasma membrane [13].Annexin A2, in contrast, regulates EC phenotype and AJ integrity through interaction with VE-cadherin [14].
Another class of BBB permeability-regulating proteins are the integrins, which are cell-cell or cell-ECM adhesion proteins, existing as heterodimers and formed by combination of 18 different α and β subunits.There are 24 integrin proteins with complex cellular functions, which can activate many intracellular signaling pathways [15].They interact importantly with astrocytic end-feet.For example, the α6vβ8 integrin, expressed by astrocytes induces Transforming Growth Factor-β (TGF-β) production, which stabilizes the endothelium and astrocytic end-feet attachment within the basal lamina, in co-ordination with ECs [16].
Both endothelial and parenchymal BMs contain collagen IV, fibronectin, and laminins.Laminins 1 and 2 are mainly secreted by astrocytes in the parenchymal basement, and laminins 8 and 10 are secreted in the vascular BM.Astrocytic laminins regulate pericyte differentiation.Loss of astrocytic laminin decreases aquaporin-4 expression in astrocytic end-feet and down-regulates TJ protein expression [14].Collagen IV and fibronectin are secreted by ECs, astrocytes, and pericytes, and contribute to BM stability [17].Agrin maintains BBB integrity and function through TJ and AJ protein expression [18].
expression in astrocytic end-feet and down-regulates TJ protein expression [14].Collagen IV and fibronectin are secreted by ECs, astrocytes, and pericytes, and contribute to BM stability [17].Agrin maintains BBB integrity and function through TJ and AJ protein expression [18].
The BBB's selective permeability relies on the fine-tuning and networking of BMECs, pericytes, and astrocytes, and dysregulation in each individual cell type may affect the function of the whole multi-cellular structure, leading to pathological events.

Endothelial Cells Interplay with Astrocytes within the Neurovascular Unit (NVU)
EC morphology, gene expression, proliferation, and transport mechanisms responsible for BBB integrity can be modified by biomechanical and biochemical signaling from the blood compartment, as well as by paracrine signaling pathways between microvascular ECs and other cells from the NVU.
Neurovascular coupling modulates the rate of circulating blood flow to adequately match the metabolic needs of active neurons.It is regulated by a signaling network from the NVU that comprises neurons, astrocytes, and vascular cells (ECs, smooth muscle cells, and/or pericytes).
Astrocytes are considered intracranial baroreceptors, which detect brain hypoperfusion, triggering astrocytic Ca2+ signaling and vasoactive molecule release.Adenosine triphosphate (ATP), or neurotransmitters released from active neurons, activates astrocytic P2X receptors which trigger Ca 2+ signals, consequently inducing phospholipase D2-and diacylglycerol lipase-mediated production of arachidonic acid The BBB's selective permeability relies on the fine-tuning and networking of BMECs, pericytes, and astrocytes, and dysregulation in each individual cell type may affect the function of the whole multi-cellular structure, leading to pathological events.

Endothelial Cells Interplay with Astrocytes within the Neurovascular Unit (NVU)
EC morphology, gene expression, proliferation, and transport mechanisms responsible for BBB integrity can be modified by biomechanical and biochemical signaling from the blood compartment, as well as by paracrine signaling pathways between microvascular ECs and other cells from the NVU.
Neurovascular coupling modulates the rate of circulating blood flow to adequately match the metabolic needs of active neurons.It is regulated by a signaling network from the NVU that comprises neurons, astrocytes, and vascular cells (ECs, smooth muscle cells, and/or pericytes).
As NVU constituents, astrocytes secrete numerous paracrine factors, acting mainly on ECs, by changing the expression and distribution of TJ and AJ proteins, and ultimately affecting the BBB's barrier properties.
Astrocytes secrete SHh [23], and BBB ECs express high levels of the Hh receptor Patched-1, the signal transducer Smoothened (Smo), as well as transcription factors of the Gli family.SHh binds to the transmembrane receptor PTCH-1, suppressing the inhibition of Smo, Gli transcription factors are transported to the nucleus, and this sequence of events promotes transcription of SHh target genes.The activation of the Hh pathway induces expression of junctional proteins and promotes BBB phenotype integrity.SHh signaling triggers changes in transendothelial electrical resistance and paracellular permeability due to increased expression of PTCH-1, claudin-3, -5, occludin, junction adhesion molecule-A, VE-cadherin, p120, and laminin [24].
The SHh signaling pathway is active in different brain regions, with different intensities across regions.This heterogeneity correlates with the molecular heterogeneity and differential transcriptional regulation of distinct astrocyte subsets from different brain regions.Reactive astrocytes show stimuli-dependent decreases of SHh activity, which is well defined, both spatially and temporally [25].The astrocytes expressing Gli1 are a particular class of astrocytes, with a dynamic response to environmental stimuli.Different classes of astrocytes can be selectively activated by SHh signals originating from astrocytic or neuronal sources [26].
IL(interleukin)-1β produced by activated microglia suppresses SHh release from astrocytes, leading to an increased BBB permeability [23].IL-6 and Tumor Necrosis Factor (TNF)-α trigger a decrease of TJ protein expression and an increase in reactive oxygen species (ROS).
High mobility group box 1 protein (HMGB1) promotes SHh release in astrocytes through the receptor for advanced glycation end products (RAGE), via downstream phosphorylation of p38, Janus kinase (JAK), and Signal transducer and the activator of transcription 3 (STAT3) [31].
Another member of the Hedgehog family of signaling molecules, Desert Hedgehog (DHh), regulates astrocyte association with blood vessels through PTCH binding on ECs and SMO-dependent Hedgehog target gene transcription.SHh signaling stimulates proliferation of local oligodendrocyte progenitors, adult neural stem cells, and astrocytes after injury, producing a glial scar [32].
ANG-1 is a glycoprotein produced in both ECs and astrocytes, promoting a decrease in endothelial permeability through junctional protein expression [33].ANG-1 binds to Tie-2, a tyrosine kinase receptor expressed in ECs, leading to activation of Phosphoinositide3(PI3)kinase/protein kinase B (AKT), Ras, and mitogen-activated protein (MAP) kinase, involved in ECs' survival and BBB protection against damage [34,35].ANG-1 exerts endothelial anti-apoptotic effects through activated PI3-kinase, which results in phosphorylation and inhibition of the forkhead transcription factor, or in activation of GTP-ases RhoA and Rac1.
When BBB-disrupting factors, such as VEGF, are co-expressed with ANG-1, the barrier integrity is enhanced [40].
Conversely, astrocyte-derived ANG-2 participates in the early phases of disease-BBB disruption, inducing vessels tightening through TJ formation.Astrocytes also produce the angiotensin-converting enzyme-1 (ACE-1) that converts ANG-1 into ANG-2.ACE-1 acts on type 1 angiotensin receptors expressed by BMECs.In the CNS, activation of type 1 angiotensin receptor restricts BBB permeability and stabilizes junctional protein function by promoting their recruitment into lipid rafts [41,42].
RA, a metabolite of vitamin A, is synthesized from retinol by retinaldehyde dehydrogenase, and is highly expressed in reactive astrocytes, triggering enhanced astrocytic RA synthesis [43].RA increases expression of ZO-1 and VE-cadherin [44], and decreases VCAM-1 expression in brain microvascular ECs during inflammation [45].
RA reduces β-catenin expression through transcriptional suppression.Moreover, RA promotes an increase in the phosphorylation of β-catenin (Ser33/Ser37/Thr41) through RA receptor and protein kinase C (PKC) activity, followed by proteasome-mediated degradation of phosphorylated β-catenin.However, Bonney et al. showed that RA inhibition of endothelial Wnt signaling has any effect on claudin-5 expression, due to ectopic vascular Wnt-β-catenin signaling [48].
The Wnt/β-catenin pathway and Wnt growth factors released by astrocytes influence BBB integrity, as shown by Guérit et al. hGFAP-Cre-mediated deletion of the evenness interrupted (Evi) gene, a mediator of Wnt growth factor secretion, leads to brain edema and tracer extravasation.However, effects on EC junction protein expression or transendothelial electrical resistance measurements were not observed.It was presumed that β-cateninmediated Wnt signaling in ECs exceeded Wnt secretion from astrocytes [49].
Canonical and non-canonical Wnts can exert opposing effect in cells from the NVU.The non-canonical Wnt pathways or non-β-catenin-mediated Wnt pathways are involved in cell polarization, cell survival, inflammatory response, and cell migration.The canonical or β-catenin-mediated Wnt signaling pathway is activated after Wnt growth factor binds to Frizzled (Fzd) and the low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6).Wnt binding to its receptor results in the recruitment of the disheveled (Dsh) protein and β-catenin phosphorylation complex, formed by glycogen synthase kinase 3β (GSK-3β), Axin, adenomatous polyposis coli, and casein kinase-1.After the inhibition of β-catenin phosphorylation complex, cytoplasmic β-catenin is translocated to the nucleus, resulting in the transcription of target genes involved in angiogenesis, vascular integrity, and BBB TJ expression.Increased β-catenin activation is followed by endothelial expression of claudin-3 and claudin-5 and reduced BBB permeability.In the absence of Wnt growth factors, β-catenin is phosphorylated by the protein complex, resulting in β-catenin proteasomedependent degradation [50].
Ligands inducing Wnt/β-catenin signaling suppression increased both caveolin-1mediated transcellular transport in ECs and BBB transcellular permeability.Wnt factors secreted by astrocytes were associated with low caveolin-1 expression and decreased transcellular vesicular traffic in brain ECs.A reduction of Wnt factor release from astrocytes decreased Wnt pathway target gene expression in ECs and astrocytes.When astrocytic Wnt factor was suppressed, end-feet coverage of brain microvessels was impaired [49].
IGF-1 is a neurotrophic factor produced in astrocytes, neurons, ECs, and other glia.Bake et al. reported that astrocyte-derived IGF-1 exerted protective effects against EC death, thus attenuating BBB disruption [51].
Astrocytes, microglia, ECs, leukocytes, neurons, and other cells interact at different stages of the response to injury.In vivo administration of FGFs after injury can affect astrocyte phenotype directly or through changes in other cell types.FGFs act in injury in a stage-dependent manner to either activate or suppress astrocyte activation.Deletion of FGFs, β-integrin, and sHh lead to reactive astrogliosis [55].FGF receptors (FGFRs) are expressed in neurons, astrocytes, and oligodendrocytes.FGF ligand binding on FGFRs activates downstream PLC (Phospholipase) γ, MAP kinase/extracellular signal-regulated kinase (ERK), and STAT3 pathways.FGF-2 and FGFRs are involved in communication between degenerating neurons, microglia, and astrocytes.FGF signaling provides a supportive ECM and increased astrocytic proliferation [56].Cyclin D1 and E are required for cell cycle progression and proliferation.c-Myc regulates gene expression through binding on the enhancer sequence and recruiting histone acetyltransferases.FGF-2 increases Cyclin D1 and c-Myc expression and mediates astrocyte proliferation [57].Kang et al. showed that FGF8 biding on FGFR3 was responsible for an astrocyte morphology with more branches than that produced via FGF2 signaling.FGFR3 reduces astrocyte branch formation and minimizes hypertrophic responses in the site of reactive gliosis [58].A study performed by Reuss et al. with mice lacking FGF-2 or FGF-5 showed that FGF signaling regulates astrocytes differentiation in a region-specific manner, this being demonstrated by reduced levels of glial fibrillar acidic protein (GFAP) in FGF-2-and FGF-5-deficient mice.Reduced levels of GFAP were also observed in perivascular astroglial end-feet, accompanied by albumin extravasation from brain capillaries and reduced levels of the TJ proteins ZO-1 and occludin in ECs from brain capillaries.These results showed that FGF signaling may have a role in the regulation of BBB permeability in vivo [59][60][61].
However, Baumann et al. found that astrocyte-derived HIF-1α-dependent paracrine signaling did not contribute to the modulation of EC barrier function under normoxic or hypoxic conditions.Astrocytes prevented hypoxia-induced increased permeability, but through an HIF-1 independent pathway, which triggers increased astrocytic VEGF levels [67].The effects of astrocytic VEGF on ECs has been described previously as being partially attributed to NO generation in these cells, through a NO synthase/cGMP-dependent pathway [68][69][70].
Glutamate is a major excitatory transmitter which exerts its excitatory effects via glutamatergic receptors, including the N-methyl-D-aspartate (NMDA) receptor and the αamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor.Astrocyte-derived glutamate binds on NMDA receptors from ECs and induces vasodilatation that is dependent on NOS-3.NMDA receptors are ionotropic receptors and act as ion channels after glutamate binding, increasing the intracellular calcium concentration.Elevated cytoplasmic Ca 2+ triggers mitochondrial production of ROS, disrupting endothelial junctions and the cerebral endothelial barrier.ROS can mobilize intracellular Ca 2+ stores and lead to the opening of calcium channels, both of which contribute to endothelial barrier leakage [71].
MMPs are zinc-endopeptidases released from reactive astrocytes, and are involved in degradation of endothelial TJ and ECM remodeling.The pathological role of MMPs in acute and chronic neurodegenerative disorders is reflected by BBB disruption [72,73].Claudin-5 and occludin degradation occurs through MMP-2 action in the early phase of BBB disruption, and through MMP-9 in the delayed phase [74].Increased MMP-9 expression is responsible for EC apoptosis through laminin degradation following subarachnoid hemorrhage in rats [75].MMP-9 and MMP-2 contribute to caspase3-mediated brain EC death after hypoxia-reoxygenation [76].
Glutamate released during epileptic seizures triggers increased MMP-2 and MMP-9 expression and activity, leading to decreased TJ protein levels and thereby resulting in BBB leakage [77,78].
ET-1, -2 and -3 are endogenous vasoconstrictors that regulate EC function through Endothelin receptor (ETR) type A and type B. ETRB is expressed in astrocytes and upregulated in brain disorders.ET-1 is synthesized by ECs, macrophages, cardiomyocytes, astrocytes, microglia, and neuronal cells [79].
ET-1 activates the ETRB receptor, followed by PLC and then PKC activation.Activation of the PKC pathway leads to ROS release, which increases the production of MMP-9.Exposure of astrocytes to ET-1 also triggers the up-regulation of NO synthase and NO production [80].Increased production of MMP-9 and VEGF stimulated by ETs induces brain edema [81][82][83][84].
In summary, astrocytes, as the main secretory cells in CNS, produce paracrine factors that can be expressed at low levels or even not expressed in a healthy brain.Under pathological conditions, the expression of BBB permeability-inducing factors is increased in the reactive astrocytes, as summarized by Figure 2.After CNS injury, the astrocytes' functional polarization may also be set to up-regulate BBB integrity-restoring factors.Local environmental stimulation may lead to changes in the secretion of paracrine factors by astrocytes, triggering a dynamic regulation of the BBB.Astrocytic soluble factors with physiological roles in BBB maintenance, as well as soluble factors secreted in reactive astrocytosis triggering BBB disruption and vascular inflammation, are described in Table 1.Several potential molecular mechanisms underlying BBB structural and functional modulation in health and disease under astrocyte control are also summarized.
in the reactive astrocytes, as summarized by Figure 2.After CNS injury, the astrocytes' functional polarization may also be set to up-regulate BBB integrity-restoring factors.
Local environmental stimulation may lead to changes in the secretion of paracrine factors by astrocytes, triggering a dynamic regulation of the BBB.Astrocytic soluble factors with physiological roles in BBB maintenance, as well as soluble factors secreted in reactive astrocytosis triggering BBB disruption and vascular inflammation, are described in Table 1.Several potential molecular mechanisms underlying BBB structural and functional modulation in health and disease under astrocyte control are also summarized.

Reactive Astrogliosis in BBB Dysfunction
Oxidative or chemical stress and a pro-inflammatory milieu elicit astrocyte reactivity, reflected in transcriptional changes.Astrocyte exposure to a multitude of extracellular signals induces the hypertrophy, proliferation, and secretion of pro-inflammatory molecules.The intensity and duration of transcriptional changes depend on the type and power of different stimuli eliciting an array of changes, from some reversible ones to glial scar formation.Astrocytes exhibit dynamic differences in morphology, physiological properties, function, and response to injury and disease [91].
Escartin et al. have defined the term "reactive astrogliosis" which describes changes in transcriptional regulation, as well as biochemical, morphological, metabolic, and physiological states in response to pathological stimuli.In contrast, astrocyte plasticity has been defined as changes in astrocyte gene expression and phenotype in response to physiological signals [92].
Reactive astrocytes have pro-inflammatory and oxidative profiles, and increased expression of vasoactive mediators, triggering endothelial junction disruption, immune cell infiltration, and Endothelial β-catenin down-regulation of WNT/β-catenin signaling, leads to decreased levels of TJs proteins and disruption of intercellular junctions, enhancing BBB destabilization and breakdown [62,93].
Activation of NF-κB in astrocytes by deletion of IκBα triggers brain inflammation and BBB disruption.NF-κB inhibition in astrocytes, conferred by transgenic expression of a degradation-resistant form of IκBα (IκBα-dn), elicits down-regulation of chemokine C-C motiv ligand (CCL)2, CCL5, ICAM-1, VCAM-1, and Itg that are essential for leukocyte adhesion to brain microvessels and migration into the CNS through BBB [85,90].
NF-κB activation in astrogliosis decreases expression of TJs, AJs, and endothelial efflux transporters, but meanwhile increases endothelial inflammatory response and oxidative stress [86].In acute inflammatory reactions, NF-κB is also activated in ECs through IL-1β and TNF-α, leading to decreased expression and an altered cellular localization of TJ proteins ZO-1 and claudin-5.Inhibition of IKK or protein kinase C zeta (PKCζ) blocks this effect on TJ proteins [97].
Conversely, as Ridder et al. described, NF-κB basal activity is needed for brain EC survival and function.Inflammatory mediators activate the protein kinase TAK1 and subsequently TAK1 stimulates IKK.TAK1 and the NEMO component of IKK can prevent EC death independent of the NF-Kb pathway components p65 and IKK2.This NF-κBmediated endothelial pathway seems to revoke the increased permeability of the BBB in the presence of inflammation [98].
STAT3 may also switch gene transcription in reactive astrocyte subsets in a contextdependent manner [99].STAT3 activation in astrocytes via phosphorylation by JAK is associated with reactive astrogliosis.STAT3 regulates the production of cytokines and chemokines in reactive astrocyte.Inhibition of the JAK-STAT3 pathway reduces mRNA levels of IL 6, IL-1b, IL-4, and VEGF in cell cultures [100].Lipopolysaccharide (LPS)mediated induction of the chemokines CCL20, CX3CL1, CXCL5, and CXCL10 has been described in astrocytes [101].TNF induces a reactive astrocyte phenotype that result in BBB dysfunction through activation of STAT3 and increased expression of serpinA3, which encodes alpha 1-antichymotrypsin [102].
Calcineurin (also known as protein phosphatase 3), a serine/threonine-protein activated by increased intracellular Ca 2+ levels, binds and dephosphorylates nuclear factor of activated T-cells (NFAT), resulting in NFAT nuclear translocation in the nucleus followed by conditioned activation of gene transcription.NFAT signaling relies on the activation of PLC through different receptors, like T-cell receptor (TCR) or B-cell receptor (BCR).This activation leads to the release of PI3 and diacylglycerol.The PI3 is especially important for calcium influx because it binds to a PI3 receptor located in the membrane of ER.Calcineurinexpressing reactive astrocytes are present in neurodegenerative diseases [107,108].
Reactive astrocytes can affect BBB endothelial integrity through secreted proteins [30,109,110].Astrocytes have also been shown to secrete factors that interfere with leukocyte recruitment and migration into the CNS through BBB [111,112].The presence of an increased number of reactive astrocytes, which is disease-associated, is involved in BBB disruption [113,114].Contrarily, some studies have shown that reactive astrocytes are sustaining BBB function after neural injury [115,116].

Astrocyte Functional Identity Is Driven by Transcriptional and Epigenetic Changes
The astrocyte end-feet are an important component of BBB, together with brain endothelial cells, vascular smooth muscle cells, pericytes, and the vascular basement membrane.The dynamic changes of BBB functions are fundamentally influenced by the direct influence of the heterogeneous astrocyte subsets.A mechanistic binary division of astrocytes into neurotoxic (A1) and neuroprotective (A2) phenotypes is considered outdated.The research on reactive astrocytes' influence on BBB permeability is actually based on multiple functional parameters used to obtain relevant data concerning the detrimental versus protective roles of the astrocytes.
Astrocyte heterogeneity and plasticity in different brain regions, as well as within a brain region, are owed to developmental and context-based gene expression profiles.The large-area spatial transcriptomic (LaST) map developed by Bayraktar et al. quantifies different gene expressions at the single-cell level.With this approach, the authors demonstrated gene expression gradients in astrocytes across large tissue areas in mouse brains.Whole tissue sections were examined with a high 3D resolution in spinning-disk confocal microscopy, then single-cell (sc)RNA-seq data, followed by spatial reconstruction analysis, confirmed the presence of superficial, mid, and deep astrocyte layers in the adult mouse cortex.Screening 46 candidate astrocytic genes with potential differential expression, the authors found layer-independent heterogeneity, detected by clustering of scRNAseq data, and layer-dependent heterogeneity, demonstrated by single-molecule fluorescent in situ hybridization (smFISH) and spatial reconstruction of scRNA-seq data [117].
Transcription factor motif analysis showed the presence of multiple transcriptional layers in astrocytes across different brain regions.Lozzi et al. identified generalized astrocytic transcriptional regulators, as well as region-specific transcription factors in adult astrocytes.The region-specific transcription factors control regulatory networks and contribute to the distinct molecular signatures and functional profiles of astrocyte heterogeneity [118].
In neurodegenerative diseases, a spectrum of reactive-astrocyte phenotypes from different brain regions respond to pathological stimuli at a given time point, and thus astrocytic involvement might also be disease stage-dependent [119].
sc-RNA-seq and single-nucleus (sn)-RNA-seq combined with multidimensional data and clustering analysis described distinct and complex stage-dependent transcriptomic profiles in chronic neurodegenerative disease.Further, sc-RNA-seq and sn-RNA-seq highlighted astrocytes exhibiting alternative phenotypes, regenerative or degenerative, in a context-dependent manner, across different CNS regions [120].
Extrinsic signals and/or interaction with CNS-resident or non-resident cells can trigger alteration of epigenetic states that, in turn, can modify intrinsic transcriptional programs, with long-term influence on astrocyte identity [121,122].
Gene transcription is a complex process which is dependent not only on chromatin structure but also on its accessibility for transcription regulators.The assay for transposaseaccessible chromatin (ATAC-seq) is used for identifying genome-wide accessible regions of chromatin [123].Genome-wide chromatin analysis highlights the importance of spatial chromatin architecture.Structures like chromatin loops can bring distant promoters and regulatory elements involved in transcriptional regulation into close proximity [124].Chromatin structure can be modified through histone rearrangements as follows: it can be (1) catalyzed by histone acetyltransferases (HATs) and methyltransferases (HMTs) and (2) reduced by histone demethylases (HDMs) and deacetylases (HDACs).Increased levels of acetylated histone 3 at lysins 9, 14, and 27 (H3K9K14ac and H3K27ac, respectively) were detected in reactive astrocytes after LPS exposure in the presence of microglia.Additionally, NF-κB-dependent histone acetylation in active transcriptional sites is a marker for pro-inflammatory astrocyte phenotypes.H3K9me3 is a marker of transcription silencing and decreased astrocyte reactivity initially derived from LPS exposure.Moreover, some astrocyte subsets are characterized by active transcription, whereas others are repressed due to H3K27ac and H3K9me3 changes [125] (Figure 3).
Histone phosphorylation on serine, threonine, and tyrosine is involved in chromatin condensation and transcription silencing.Histone ubiquitination is associated with genome stability, cell cycle, and transcription.Other histone modifications such as crotonylation, lactylation, and serotonylation, identified by mass spectrometry, are involved in neurodevelopment and neurological disease [126].
Another epigenetic mechanism involved in gene transcription regulation is DNA methylation.Cytosine methylation represses gene expression through the addition of methyl groups within CpG dinucleotides, and 5-methylcytosine is an epigenetic marker found in CpG islands from about 60% of promoter sequences.Methylated CpG islands from gene promoters prevent interactions with transcriptional regulators or, conversely, recruit proteins involved in gene repression, such as co-repressors [127].
DNA methylation and open chromatin maps identified with genome-wide next generation sequencing (NGS) techniques showed functional changes of the promoters and distal or proximal cis regulatory regions in astroglia from different brain regions in young adult mice, leading to functional, epigenomic, and regional diversity.Hierarchical clustering of the total mRNA-Seq, ATAC-Seq, and RRBS datasets highlighted transcriptomic-and epigenomic-specific changes from cortical to cerebellar astrocytes [128].
There is no clear demarcation between the pro-and anti-inflammatory status of astrocyte subsets in the brain, and the differential expression of non-coding RNAs occurring in astrocytes contributes to their heterogeneous phenotypes and context-dependent response.DNA methylation and open chromatin maps identified with genome-wide next generation sequencing (NGS) techniques showed functional changes of the promoters and distal or proximal cis regulatory regions in astroglia from different brain regions in young adult mice, leading to functional, epigenomic, and regional diversity.Hierarchical clustering of the total mRNA-Seq, ATAC-Seq, and RRBS datasets highlighted transcriptomic-and epigenomic-specific changes from cortical to cerebellar astrocytes [128].
There is no clear demarcation between the pro-and anti-inflammatory status of astrocyte subsets in the brain, and the differential expression of non-coding RNAs occurring in astrocytes contributes to their heterogeneous phenotypes and contextdependent response.
Non-coding RNAs represent an epigenetic mechanism that leads to gene silencing, affecting protein translation without altering the DNA sequence.MicroRNAs (miRNAs) are a large group of small endogenous molecules of single strand non-coding RNA, approximately 21-25 nucleotides in length, and their fundamental role is to regulate gene expression at the post-transcriptional level.They act as negative regulators by binding through the 3′ untranslated region (UTR) of mRNA targets and determining its degradation and/or the inhibition of its translation.MiRNAs can either directly preserve BBB integrity by targeting endothelial junction protein translation, or indirectly by down- Non-coding RNAs represent an epigenetic mechanism that leads to gene silencing, affecting protein translation without altering the DNA sequence.MicroRNAs (miRNAs) are a large group of small endogenous molecules of single strand non-coding RNA, approximately 21-25 nucleotides in length, and their fundamental role is to regulate gene expression at the post-transcriptional level.They act as negative regulators by binding through the 3 untranslated region (UTR) of mRNA targets and determining its degradation and/or the inhibition of its translation.MiRNAs can either directly preserve BBB integrity by targeting endothelial junction protein translation, or indirectly by down-regulating NF-Kb, mitogen-activated protein kinases, and PI3/Akt signaling pathways to reduce inflammation and apoptosis, or by promoting the expression of ANG-1/Tie-2 axis in ECs.Additionally, miRNAs may also have an impact on the crosstalk between brain ECs and supporting cells, which is critical for the maintenance of BBB function [129].
Astrocyte activation induces up-regulation of miR-155, which, by inhibiting the mRNA of SOCS-1, determines pro-inflammatory cytokine expression.SOCS-1 regulates the immune response through direct inhibition of JAK and consequent inhibition of STAT [131,132].Korotkov et al. showed that miR-155 could be induced in astrocytes by pro-inflammatory TNF-α through NF-κB and activator protein 1 (AP-1) pathways, and that this effect is potentiated in the presence of miR-142.Increased expression of IL-1β and PTGS2 was seen in astrocytes following stimulation via the medium from the miR-142-overexpressing cells [133].
miR-181 expressed in astrocytes has an important role in the post-transcriptional regulation of reactive astrogliosis phenotypes.miR-181 directly targets the mRNA of Bcl-2, a mitochondrial membrane-associated protein that inhibits apoptosis, and heat-shock protein 70, expressed in response to cellular stress.A remarkable change in astrocyte phenotype has been observed after LPS exposure through miR-181 down-regulation.A reduced level of miR-181 triggers pro-inflammatory cytokine release, including leukemia inhibitory factor or LIF, IL-6, TNF-α, IL-1β, IL-8, and HMGB1.In contrast, levels of the anti-inflammatory cytokine IL-10 increase in astrocytes when miR-181 is over-expressed.Knockdown of miR-181 resulted in elevated FGF2 levels in LPS-treated astrocytes [134].miRNAs involved in astrocytic pro and anti-inflammatory function are depicted in Figure 4.
Astrocyte activation induces up-regulation of miR-155, which, by inhibiting the mRNA of SOCS-1, determines pro-inflammatory cytokine expression.SOCS-1 regulates the immune response through direct inhibition of JAK and consequent inhibition of STAT [131,132].Korotkov et al. showed that miR-155 could be induced in astrocytes by proinflammatory TNF-α through NF-κB and activator protein 1 (AP-1) pathways, and that this effect is potentiated in the presence of miR-142.Increased expression of IL-1β and PTGS2 was seen in astrocytes following stimulation via the medium from the miR-142overexpressing cells [133].
miR-181 expressed in astrocytes has an important role in the post-transcriptional regulation of reactive astrogliosis phenotypes.miR-181 directly targets the mRNA of Bcl-2, a mitochondrial membrane-associated protein that inhibits apoptosis, and heat-shock protein 70, expressed in response to cellular stress.A remarkable change in astrocyte phenotype has been observed after LPS exposure through miR-181 down-regulation.A reduced level of miR-181 triggers pro-inflammatory cytokine release, including leukemia inhibitory factor or LIF, IL-6, TNF-α, IL-1β, IL-8, and HMGB1.In contrast, levels of the anti-inflammatory cytokine IL-10 increase in astrocytes when miR-181 is over-expressed.Knockdown of miR-181 resulted in elevated FGF2 levels in LPS-treated astrocytes [134].miRNAs involved in astrocytic pro and anti-inflammatory function are depicted in Figure 4. Mouse brain microvascular ECs in co-culture with astrocytes were subjected to oxygen-glucose deprivation followed by reperfusion in the absence or presence of VEGF.VEGF increased the level of LOC102640519, which in turn positively regulated the expres-sion of HOXC13 and negatively regulated TJ-associated proteins (ZO-1, Occludin, and Claudin-5), leading to BBB leakage [135].
In physiological conditions, the molecules from the cargo of astrocyte-derived exosomes contribute to the maintenance of CNS homeostasis.Astrocytic exosomes are enriched with miR-195, which improves BBB integrity [136].
Reactive astrocytes accelerate secretome release, including extracellular vesicles, which transfer neurotoxic molecules that trigger neurodegeneration, or pro-inflammatory molecules causing neuroinflammation.Astrocyte-derived extracellular vesicles pass throughout the BBB, reaching the periphery, where pro-inflammatory factors promote leukocyte migration from the periphery to brain parenchyma [137].
Jovičić et al. showed that astrocytes exclusively secrete 12 miRNAs via exosomes; meanwhile, 217 different miRNAs have been exclusively detected in astrocytes.Of these, 61 miRNAs were more than two-fold enriched in astrocytes compared to exosomes, and 42 miRNAs had higher than two-fold enrichment in exosomes compared to astrocytes.The authors also suggested the existence of a yet-undecrypted mechanism through which miRNAs are selected for inclusion or exclusion from exosomes [138].

Conclusions
BMECs are endowed with a complex network of intercellular tight and adherens junctions which, together with a low transcytosis rate and a specific transport system, confer to the BBB the role of a selectively permeable barrier.The structural and functional integrity of this barrier ensures the protection of the CNS from endogenous and exogenous threats, maintaining the brain parenchyma's homeostasis and immune privilege, effective for neuron functioning.
BBB permeability is affected by the surrounding microenvironment, which is dynamically changed by the interactions and signaling between neurons and non-neuronal cells such as ECs, pericytes, and astrocytes in NVU.
Astrocytes form the perivascular end-feet at the BBB and secrete many factors that modulate the integrity of this barrier in the healthy adult brain, as well as factors that change permeability in pathological conditions.
Under neurological disorders, a loss in the permeability of the barrier is associated with phenotypical changes in both the ECs and astrocytes.Astrocytic structural and functional changes occur via signaling events modulated in a disease-dependent manner, leading to reactive astrocytosis.Reactive astrocytes exhibit temporal and spatial heterogeneity and a spectrum of activation states via differentially expressed genes and via changes in gene expression at the post-transcriptional level under non-coding RNA regulation.
In this review, we showed detailed aspects of ECs, their interaction with astrocytes in NVU, and the subsequent effects on BBB permeability.We also highlighted mechanisms leading to astrocyte heterogeneity, these being more complex than the simple phenotype dichotomy of astrocytes with neurotoxic and pro-inflammatory, or neuroprotective and anti-inflammatory features.The BBB microenvironment is a complex and versatile one, in which heterogeneous astrocyte subpopulations have a central role, contributing to the known multifaceted neurological pathology and patient-personalized responses to therapy.

Figure 1 .
Figure 1.Graphical representation of the BBB structure.BBB function is supported by the presence of TJs and AJs, together with cytoplasmatic proteins, ZO.The ECs are anchored to the basal lamina.The pericytes act as contractile components in the brain's microvascular BBB, sensible to astrocyte inputs.The parenchymal BM made from ECM secreted by astrocytes and the astrocytic end-feet form the glia limitans vascularis.(TJ: Tight Junctions, AJ: Adherent Junctions, ZO: Zonula Occludens, EC: Endothelial Cells, BBB: blood-brain barrier, BM: basement membrane, ECM: extracellular matrix).

Figure 1 .
Figure 1.Graphical representation of the BBB structure.BBB function is supported by the presence of TJs and AJs, together with cytoplasmatic proteins, ZO.The ECs are anchored to the basal lamina.The pericytes act as contractile components in the brain's microvascular BBB, sensible to astrocyte inputs.The parenchymal BM made from ECM secreted by astrocytes and the astrocytic end-feet form the glia limitans vascularis.(TJ: Tight Junctions, AJ: Adherent Junctions, ZO: Zonula Occludens, EC: Endothelial Cells, BBB: blood-brain barrier, BM: basement membrane, ECM: extracellular matrix).

Figure 2 .
Figure 2. Graphical representation of the most important astrocytic roles in BBB and CNS function.(1) Neurovascular unit.ATP from active neurons activates astrocytic P2X receptors that induce PL-D2 and diacylglycerol lipase-mediated production of arahidonic acid and generation of prostaglandin E2.Prostaglandin E2 is secreted by the astrocytic end-feed and induces pericyte relaxation by binding to EP4-R.(2) Apolipoprotein E (ApoE).The astrocytes are the main source of ApoE in the CNS.ApoE-4 activates cyclophilin A (MMP-9) in the pericytes which disrupts the BBB.ApoE3, produced by astrocytes, blocks the APOE-4-associated BBB disruption.(3) Hedgehog Family.Sonic Hedgehog (SHh).SHh signaling through endothelial receptors decreases TNF-alfa, IL-6, and IL-1b transcription factors, increases the expression of TJs (occludin, ZO-1, etc.), and reduces ICAM-1 expression on ECs.Desert Hedgehog (DHh) regulates the binding between astrocytic end-feet and protein-patched receptors (PTCH-R).(4) Astrogliosis.STAT3 activation in reactive astrocytes (via phosphorylation by Janus kinase) leads to reactive astrogliosis.STAT3 regulates the survival of proliferating reactive astrocytes.Additionally, MMP-2 and -9 are released from reactive astrocytes, favoring BBB disruption.Janus kinase-STAT3 inhibition has been shown to reduce levels of IL-6, IL-1b, and IL-4.(5) NF-Kb activation.Astrogliosis is accompanied by NF-kB activation in reactive astrocytes which triggers BBB disruption.The canonical NK-κB pathway is induced by TLRs, pro-inflammatory cytokines, ROS, and NO.The alternative NK-κB pathway is activated by the B cell activating factor of the TNF family (BAFF), lymphotoxin β, and CD40L.NF-kB pathway activation leads to glutamine, ATP, IFNγ, TGF-β, IL-1α, TNFα, and iNOS production, promoting a pro-inflammatory environment and leading to BBB disruption.

Figure 2 .
Figure 2. Graphical representation of the most important astrocytic roles in BBB and CNS function.(1) Neurovascular unit.ATP from active neurons activates astrocytic P2X receptors that induce PL-D2 and diacylglycerol lipase-mediated production of arahidonic acid and generation of prostaglandin E2.Prostaglandin E2 is secreted by the astrocytic end-feed and induces pericyte relaxation by binding to EP4-R.(2) Apolipoprotein E (ApoE).The astrocytes are the main source of ApoE in the CNS.ApoE-4 activates cyclophilin A (MMP-9) in the pericytes which disrupts the BBB.ApoE3, produced by astrocytes, blocks the APOE-4-associated BBB disruption.(3) Hedgehog Family.Sonic Hedgehog (SHh).SHh signaling through endothelial receptors decreases TNF-alfa, IL-6, and IL-1b transcription factors, increases the expression of TJs (occludin, ZO-1, etc.), and reduces ICAM-1 expression on ECs.Desert Hedgehog (DHh) regulates the binding between astrocytic end-feet and protein-patched receptors (PTCH-R).(4) Astrogliosis.STAT3 activation in reactive astrocytes (via phosphorylation by Janus kinase) leads to reactive astrogliosis.STAT3 regulates the survival of proliferating reactive astrocytes.Additionally, MMP-2 and -9 are released from reactive astrocytes, favoring BBB disruption.Janus kinase-STAT3 inhibition has been shown to reduce levels of IL-6, IL-1b, and IL-4.(5) NF-Kb activation.Astrogliosis is accompanied by NF-kB activation in reactive astrocytes which triggers BBB disruption.The canonical NK-κB pathway is induced by TLRs, pro-inflammatory cytokines, ROS, and NO.The alternative NK-κB pathway is activated by the B cell activating factor of the TNF family (BAFF), lymphotoxin β, and CD40L.NF-kB pathway activation leads to glutamine, ATP, IFNγ, TGF-β, IL-1α, TNFα, and iNOS production, promoting a pro-inflammatory environment and leading to BBB disruption.

Figure 3 .
Figure 3. Graphical representation of the epigenetic landscape of astrocyte reactivity.Inside the CNS, the astrocytes present two phenotypes: A1, neurotoxic and A2, neuroprotective.The chromatin structure can be modified by alterations in the histone structures, catalyzed by histone acetyltransferases (HATs) and histone methyltransferases (HMTs), and diminished by histone demethylases (HDMs) and histone deacetylases (HDACs).When exposed to LPS in the presence of microglia, reactive astrocytes exhibit elevated levels of acetylated histone 3. The acetylation of histone 3, driven by NF-κB signaling, particularly at active transcriptional sites, serves as a hallmark indicating a pro-inflammatory astrocyte phenotype.

Figure 3 .
Figure 3. Graphical representation of the epigenetic landscape of astrocyte reactivity.Inside the CNS, the astrocytes present two phenotypes: A1, neurotoxic and A2, neuroprotective.The chromatin structure can be modified by alterations in the histone structures, catalyzed by histone acetyltransferases (HATs) and histone methyltransferases (HMTs), and diminished by histone demethylases (HDMs) and histone deacetylases (HDACs).When exposed to LPS in the presence of microglia, reactive astrocytes exhibit elevated levels of acetylated histone 3. The acetylation of histone 3, driven by NF-κB signaling, particularly at active transcriptional sites, serves as a hallmark indicating a pro-inflammatory astrocyte phenotype.

Table 1 .
Astrocytic factors involved in BBB structural and functional modulation.