Whispers in the nervous system: Do glia and brain endothelial cells talk to each other, and if so what do they say?

Recent studies show that astrocytic glia communicate with brain endothelial cells, both to induce properties of the blood-brain barrier, and to regulate barrier physiology. In this article Joan Abbott discusses the mechanisms and implications.

N Joan Abbott
Centre for Neuroscience Research, King’s College London

---

https://doi.org/10.36866/pn.49.8

Anatomy and physiology of the blood-brain barrier

The blood-brain barrier (BBB) is formed by brain endothelial cells lining the cerebral vasculature. It is crucial in protecting the brain from fluctuations in plasma composition, e.g. during exercise and after meals, and from circulating agents such as neurotransmitters, metabolites and toxins (Abbott & Romero, 1996; Abbott, 2002). The barrier also contributes to the homeostasis of the brain microenvironment necessary for the healthy function of the CNS. Brain capillaries are up to 100 times tighter than peripheral microvessels as a result of tight junctions (zonulae occludentes) that severely limit paracellular (tight junctional) diffusion, so that molecular movement is predominantly transcellular. Small lipophilic molecules such as oxygen, CO2 and ethanol can freely diffuse across the membranes of the endothelium. Small polar solutes needed for brain function are transported by specific carrier proteins (e.g. GLUT-1 for glucose) and specific carriers meditate the efflux of potentially toxic metabolites (e.g. glutamate). P-glycoprotein is a broad-specificity efflux transporter that limits entry of many hydrophobic molecules including toxins. The brain endothelium has lower levels of endocytosis/transcytosis than peripheral capillaries, but has specific systems for transfer of certain peptides and lipoproteins to the brain. The BBB also contains enzymes (e.g. monoamine oxidase) that reduce the central effects of circulating neuroactive agents. Thus the term ‘BBB’ covers a number of static and dynamic properties that enable the endothelium to protect and regulate the brain microenvironment (Abbott & Romero, 1996).

Induction of the BBB phenotype: role of astrocytes

Certain features of the BBB are also expressed to some degree in peripheral capillary endothelium, but they are upregulated in brain endothelium and hence used as ‘markers’ of BBB phenotype. There is great interest in the mechanism(s) for this upregulation (Bauer & Bauer, 2000; Abbott, 2002), both for physiological understanding, and to gain insight into pathological conditions, as an important step in designing effective therapies.

The endfeet of astrocytic glia form a lacework of fine lamellae closely apposed to the surface of the endothelium (Kacem et al 1998) (figure 1), suggesting that influences from astrocytes could contribute to induction of the specialised BBB phenotype. Early grafting experiments showed that brain vessels growing into grafts of peripheral tissue became less tight to intravascular tracers, while the relatively leaky vessels of peripheral tissues became tighter on growing into grafts of brain tissue (reviewed in Bauer & Bauer, 2000). Later studies showed that cultured astrocytes could mimic many of the inductive influences of neural tissue. Successful barrier induction and maintenance appear to depend critically on the local conditions and maturational state, with important implications for human therapeutic grafting and for treating brain tumours.

During development the barrier becomes relatively impermeable to large proteins such as albumin and horseradish peroxidase before it can effectively exclude smaller solutes such as mannitol and ions; there is also evidence for gradual maturation of BBB transporters.

In vitro studies have confirmed the key inductive role of astrocytes (Bauer & Bauer, 2000; Abbott, 2002). Freshly- isolated brain endothelial cells and some immortalised brain endothelial cell lines will grow as a monolayer on plastic or on porous filters, and will retain aspects of a BBB phenotype, but generally with some loss of barrier properties (Krämer, Abbott & Begley, 2001). Many barrier features can be upregulated by co-culture with glial cells, including tight junctions, gamma-glutamyl transpeptidase (γ-GTP), GLUT-1, the L- and A-system amino acid carriers, P-glycoprotein, and some endocytotic systems. Inter- estingly, certain transporters and clotting factors associated with peripheral endothelium are down-regulated in brain endothelium, which could indicate suppression by neighbouring glial cells. Some cultured non-brain endothelial cells also show upregulation of BBB markers under glial influence, indicating that the underlying processes involve more general cellular mechanisms.

What are the signals from glia to endothelium?

A key question is the nature of the glial influence responsible for induction of BBB features. Some of the inductive effects can be produced by conditioned medium taken from growing glial cells, evidence for action of a soluble factor(s). However, induction is generally more effective where glial cells contact the endothelial basal lamina. Induction appears to depend on the correct apical/basal polarity between endothelium and glia, and may involve the extracellular matrix.

The chemical nature of the glial- produced inductive signal(s) is unclear; it is much harder to detect and identify these signals than for classical neuro- transmitters (they ‘whisper’ rather than ‘shout’), since changes are often slow and subtle. Several candidate molecules have been identified (apparently acting on different aspects of the BBB phenotype) including TGF-β, GDNF, βFGF, IL-6 and hydrocortisone. Attempts to isolate the inductive influence(s) from glial conditioned medium have been only partially successful; earlier studies attributed induction to peptides/ proteins, while recent work implicates non-proteinaceous agents of <1kDa molecular weight. Many of the inducing factors have potential as differentiating agents, so the BBB phenotype may represent an enhanced state of differentiation, which can be triggered and maintained by a number of influences, some derived from glia. Indeed, the differentiating effects of intraluminal flow, raised intracellular cAMP or application of retinoic acid are more effective in inducing the BBB phenotype in endothelial cells co- cultured with glia.

In some parts of the nervous system, a blood-tissue barrier is present without astrocytic contact. Thus the microvessels on the pial surface show some BBB features, likely to be due to soluble factors acting via the subarachnoid CSF. Peripheral nerves have effective barriers in the endoneurial capillary endothelium and in the outer perineurium. Since peripheral nerves lack astrocytes, Schwann cells or axons may have equivalent inductive potential (Allt & Lawrenson, 2000). In the CNS, the predominant influence maintaining the mature BBB appears to be astrocytic.

Inductive influence of brain endothelium on astrocytes

Given the complexity of BBB induction by glial cells, it is clear that close communication between endothelium and glial cells must occur. It is therefore not surprising to find that endothelial cells have a reciprocal inductive influence on astrocytes – i.e. endothelium talks to glia. Thus arrays of particles on astrocytic end feet associated with aquaporin-4 are upregulated in co- culture with endothelium. The observed upregulation of γ-GTP in endothelial cells by glia involves a two-way exchange of signals. When endothelial and glial cells are grown together there is a mutual upregulation of antioxidant enzymes so the endothelial-glial partnership copes better with oxidative stress, e.g. in hypoxia/reperfusion injury (Schroeter et al 1999). Recently, leukaemia inhibitory factor (LIF) released by endothelial cells of the optic nerve has been shown to induce astrocytic differentiation. Thus maintenance of the adult BBB appears to depend on continuing exchange of inductive signals between glia and endothelium, and disturbance of this induction may be crucial in several neuropathologies involving BBB dysfunction, such as tumours and multiple sclerosis.

Short-term communication between glia and endothelium

In addition to the long-term processes involved in induction via altered gene expression/protein synthesis, glial- endothelial interactions also occur over a shorter time-scale (seconds-minutes). This has been most clearly documented by monitoring intracellular calcium waves, with evidence for ATP acting as a glial-endothelial signalling molecule (Paemeleire & Leybaert, 2000). Such signalling may enable astrocytes to modulate the energy supply to neurons, and to regulate endothelial transport in a way that supports neuronal function.

Humoral modulation of brain endothelial permeability

A number of chemical agents have been shown to modulate the permeability of the blood-brain barrier, including some released by astrocytic glial cells (table 1). The list includes several inflammatory mediators, consistent with evidence for increased BBB permeability in CNS inflammation (Abbott, 2000). Where the increase in permeability is transient, opening of the paracellular (tight junctional) pathway appears to be responsible. Attention has thus focused on the cellular mechanisms controlling the tight junction and associated cytoskeleton.

Several of the molecules causing barrier opening act via receptor- mediated signal transduction pathways within the endothelium, some involving elevation of intracellular calcium concentration ([Ca2+]i). Although relevant calcium-dependent changes in endothelial proteins have been reported, the details of the molecular cascade are not clear. Examination both in situ and in vitro is beginning to reveal the details of the signal transduction pathways involving particular receptors and their interactions, for example for histamine, bradykinin, and nucleotides. In pial microvessels studied in situ, B2 bradykinin receptor activation resulted in increased perme- ability involving free radicals. Activation of cultured brain endothelium by ATP and related nucleotides caused elevation of [Ca2+]i predominantly via P2U (=P2Y2) receptors. Interestingly, additional P2Y1 receptor activation could be detected in cells grown on a biological matrix, mimicking the in vivo condition (figure 2), evidence that the endothelial receptor phenotype is influenced by its local environment.

Both in situ and in vitro, the receptor- mediated barrier opening is generally reversible (Abbott, 2000). This raises the interesting possibility that barrier opening can occur as a well-regulated process under physiological conditions, in response to agents released locally. As the barrier appears designed to protect the brain and maintain CNS homeostasis, would not barrier opening be harmful? There may be physiological advantages in local and reversible barrier opening. Thus the plasma is a rich source of factors required for normal brain repair, including growth factors supporting neurite sprouting and outgrowth in regions of neuronal damage and death. In addition, transient barrier opening could be a good way to maintain immunologic surveillance of the CNS, and for neurons to ‘sample’ plasma composition as part of the brain’s key function in regulatory control of the body.

Quite small stresses to the CNS such as minor injury or surgery cause transient increases in the permeability of the BBB, typically appearing microscopically as focal leaks. We lack non- invasive ways of identifying such BBB leaks, but it is possible that local cyclic opening and closing of the barrier is a normal physiological process. Cyclic barrier activity would have a negligible effect on the general homeostasis of the brain microenvironment, but could satisfy the local requirements that triggered the barrier opening. This idea prompts a closer examination of the cellular mechanisms that can modulate BBB permeability.

Source of BBB permeability- modulating agents

Since the mechanisms controlling the tight junctions of the brain endothelium are the ‘effectors’ of BBB permeability modulation, and the chemical agents listed in Table 1 are capable of exerting the modulation, it is useful to identify possible sources of these molecules. In some cases, the endothelium is both able to release and respond to the agent, e.g. endothelin (ET-1), acting on ETA receptors, and ATP acting on nucleotide receptors. Under pathological conditions, mast cells and perivascular microglia (resident macrophages of the CNS) may release inflammatory agents close to the endothelium. Fine nerve terminals of a number of neuronal populations run close to microvessels and release agents such as histamine, 5-HT, substance P and glutamate. Astrocytes are able to release several humoral agents (table 1) although the regulation of this release is not well understood. Furthermore, in response to some of the agents able to open the BBB, astrocytes can upregulate and release modulating factors, e.g. bradykinin causes upregulation of astrocytic expression and release of interleukin-6 (IL-6), with a potentiating action on bradykinin- mediated BBB opening. Thus in addition to a role in barrier induction and maintenance, astrocytes may play active roles in modulating BBB permeability over shorter time scales. Such potentiating mechanisms also mean that agents present at concentrations too low to open the barrier are able to exert an effect in the presence of low concentrations of potentiating agents.

Conclusions

The development and maintenance of the BBB formed by brain endothelium, and the specialisations of perivascular astrocytes that enable them to act in partnership with the endothelium, involve complex cell:cell exchange of chemical signals, inducing BBB features in the longer term, and modulating cellular physiology in the shorter term. Investigation of this mutual interaction is important for understanding the biological basis of neuropathies in which BBB dysfunction occurs, and in development of effective therapeutic strategies. Physiologists need to listen not only to neuronal ‘shouting’ but also to non-neuronal ‘whispering’.

Acknowledgements

Supported by The Wellcome Trust, MRC, and the KCL Blood-Brain Barrier Consortium with Industry (Lilly, Astra Zeneca, Aventis, Mindset and GlaxoSmithKline). A more detailed version of this article has been published (Abbott, 2002).

References

Abbott, NJ (2000). Inflammatory mediators and modulation of blood-brain barrier permeability. Cellular and Molecular Neurobiology 20, 131-147.

Abbott, NJ (2002). Astrocyte-endothelial interactions and blood-brain barrier permeability. Journal of Anatomy 200, 629-638.

Abbott, NJ & Romero, I.A. (1996). Transporting therapeutics across the blood-brain barrier. Molecular Medicine Today 2, 106-113.

Allt, G & Lawrenson, JG (2000). The blood-nerve barrier: enzymes, transporters and receptors – a comparison with the blood-brain barrier. Brain Research Bulletin 52, 1-12.

Bauer, HC & Bauer, H (2000). Neural induction of the blood-brain barrier: still an enigma. Cellular & Molecular Neurobiology 20, 13-28.

Kacem, K, Lacombe, P, Seylaz, J & Bonvento, G (1998). Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: a confocal microscopy study. Glia 23, 1-10.

Krämer, SD, Abbott, NJ & Begley, DJ (2001). Biological models to study blood-brain barrier permeation. In Pharmacokinetic Optimization in Drug Research: Biological, Physicochemical and Computational Strategies. (eds. B Testa, H van de Waterbeemd, G Folkers and R Guy), pp. 127-153. Weinheim, Wiley-VCH.

Paemeleire, K & Leybaert, L (2000). ATP-dependent astrocyte-endothelial calcium signalling following mechanical damage to a single astrocyte in astrocyte- endothelial co-cultures. Journal of Neurotrauma 17, 345-358.

Schroeter, ML, Mertsch, K, Giese, H, Muller, S, Sporbert, A, Hickel, B & Blasig, IE (1999). Astrocytes enhance radical defence in capillary endothelial cells constituting the blood-brain barrier. FEBS Letters 449, 241-244.

Abbreviations

BBB, blood-brain barrier; bFGF, basic fibroblast growth factor; [Ca2+]i , intracellular calcium concentration; CSF, cerebrospinal fluid; ET-1, endothelin-1; γ-GTP, γ -glutamyl transpeptidase; GDNF, glial-derived neurotrophic factor; IL-1β, IL-6, interleukins; LIF, leukaemia inhibitory factor; MIP2, macrophage inflammatory factor-2; 2-MeSATP, 2-methylthio ATP; NO, nitric oxide; TGF-β, transforming growth factor β; TNFα, tumour necrosis factor-α.