Neural Induction of the Blood–Brain Barrier: Still an Enigma

Cellular and Molecular Neurobiology, Vol. 20, No. 1, 2000

Neural Induction of the Blood–Brain Barrier:Still an Enigma

Hans-Christian Bauer1,3 and Hannelore Bauer1,2

Received August 6, 1997; revised March 5 and August 8, 1998; accepted August 28, 1998

SUMMARY

1. The study of the blood–brain barrier and its various realms offers a myriad ofopportunities for scientific exploration. This review focuses on two of these areas inparticular: the induction of the blood–brain barrier and the molecular mechanisms underly-ing this developmental process.

2. The creation of the blood–brain barrier is considered a specific step in the differenti-ation of cerebral capillary endothelial cells, resulting in a number of biochemical andfunctional alterations. Although the specific endothelial properties which maintain thehomeostasis in the central nervous system necessary for neuronal function have been welldescribed, the inductive mechanisms which trigger blood–brain barrier establishment incapillary endothelial cells are unknown.

3. The timetable of blood–brain barrier formation is still a matter of debate, causedlargely by the use of varying experimental systems and by the general difficulty of quantita-tively measuring the degree of blood–brain barrier ‘‘tightness.’’ However, there is a generalconsensus that a gradual formation of the blood–brain barrier starts shortly after intraneu-ral neovascularization and that the neural microenvironment (neurons and/or astrocytes)plays a key role in inducing blood–brain barrier function in capillary endothelial cells.This view stems from numerous in vitro experiments using mostly cocultures of capillaryendothelial cells and astrocytes and assays for easily measurable blood–brain barriermarkers. In vivo, there are great difficulties in proving the inductive influence of theneuronal environment. Also dealt with in this article are brain tumors, the least understoodin vivo systems, and the induction or noninduction of barrier function in the newly estab-lished tumor vascularization.

4. Finally, this review tries to elucidate the question concerning the nature of theinductive signal eliciting blood–brain barrier formation in the cerebral microvasculature.

KEY WORDS: blood–brain barrier; induction; cerebral endothelial cells; brain devel-opment.

INTRODUCTION

Ever since the discovery that cerebral capillary endothelial cells create a differentenvironment within the brain, there has been speculation concerning an active1 Institute fur Molekularbiologie, Osterr. Akad. d. Wissenschaften, Billrothstrasse 11, A-5020 Salz-

burg, Austria.2 Institute fur Zoologie, Universitat Salzburg, A-5020 Salzburg, Austria.3 To whom correspondence should be addressed. Fax: 43 662 6396129. e-mail: [email protected]

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0272-4340/00/0200-0013$18.00/0 2000 Plenum Publishing Corporation

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role of the cellular environment regulating the induction and maintenance of theblood–brain barrier. The developmental switch of cerebral capillary endothelialcells from a nonbarrier to a barrier state requires the acquisition of specific structuraland biochemical properties of these cells such as the loss of fenestrations, theformation of zonulae occludens (tight junctions), a decrease in pinocytotic vesicles,and the establishment of numerous specific transport mechanisms across the endo-thelial plasma membrane (Bradbury, 1979).

In past years, the hypothesis of a neural induction of blood–brain barrierfunction in cerebral capillary endothelial cells has been supported by numerousexperimental findings in vivo and in vitro interpreting and indicating a specificrole of astrocytes and/or neurons in the induction of blood–brain barrier-relatedproperties in cerebral capillary endothelial cells. Although blood–brain barrier-related characteristics in cerebral capillary endothelial cells have been studied exten-sively, only vague ideas exist about the molecular mechanisms which trigger theinitial expression and maintenance of blood–brain barrier function in these cells.Also, the question concerning the exact developmental timing of blood–brain bar-rier functions has not yet been settled and the experimental evidence available sofar has become a matter of ongoing controversy.

In the following article we review various aspects of blood–brain barrier forma-tion, particularly the role neural cells play in this process. Moreover, we commenton the nature of possible effector molecules which might be involved in this develop-mental step.

THE NATURE OF BLOOD–BARRIER INDUCTION AND THETEMPORAL OCCURRENCE OF THE BLOOD–BRAIN BARRIER

Developmental biologists speak generally of induction when a specific develop-ment of one tissue occurs only in the presence of another tissue, the inductor, butnot in its absence. Inductive events were categorized by some authors as directiveand permissive events, the latter meaning that the inductor acts upon a tissue thatis already determined toward its final fate but still needs an exogenous stimulusfor the expression of its phenotype (Saxen, 1977; Kratochwil, 1983). These defini-tions seem to apply only to the embryonic development of tissues, i.e., the cells ofwhich are not determined, but apparently not to the induction of blood–brainbarrier characterization in the cerebral microvasculature. Cerebral capillary endo-thelial cells are already terminally differentiated when the blood–brain barrier isformed, and blood–brain barrier induction does not seem to result in a final andirreversible status, i.e., when the neural environmental is lacking, blood–brain bar-rier characteristics disappear. Thus, the stimulus must be continuous in order tomaintain the blood–brain barrier functional.

Engelhardt and Risau (1995) proposed a ‘‘two-phase model’’ for that inductiveprocess: in the first phase sprouting capillaries move into the neuroectoderm follow-ing a concentration gradient provided by vascular endothelial growth factor, whichis produced by neuroepithelial cells and is bound to flk-1 expressed by cerebralcapillary endothelial cells. This early contact with neuroepithelial cells makes cere-

Blood–Brain Barrier Induction 15

bral capillary endothelial cells ‘‘committed.’’ Further contact with neuronal cellsinduces th e development of the blood–brain barrier as characterized by formationof tight junctions and expression or upregulation of specific molecules.

The developing cerebral corx is vascularized by angiogenesis, and not by vascu-logenesis, which starts out from pluripotent endothelial cells (Risau and Wolburg,1990). From a preexisting perineural vascular plexus, cerebral capillary endothelialcells start invading the neuroectoderm. At that early stage (around day 10 in themouse and day 11 in the rat) perineural vessels still exhibit a more embryonicappearance, that is, their lumena are not yet round, they lack the extracellularmatrix, and they have thick walls and a high number of pinocytotic vesicles (Baueret al., 1993).

It is still unclear when the blood–brain barrier is established in the invadingcerebral capillary endothelial cells, but certainly the timetable of blood–brain barrierformation varies from species to species (for review see Saunders, 1992; Saunderset al., 1999). In the literature, controversial views exist concerning the exact time-point of initiation and development of the blood–brain barrier.

From histochemical and physiological studies it has been concluded that theonset of blood–brain barrier function coincides with the neovascularization of thedeveloping cerebral cortex (Saunders, 1977; 1992; Bauer et al., 1995). On the otherhand, there are several lines of evidence indicating that a fully developed blood–brain barrier does not exist before birth in rodents (Vorbrodt and Dobrogowska,1994; Fabian and Hulsebosh, 1989). In spite of these timing differences, there is ageneral agreement that blood–brain barrier formation is a gradual process (Delormeet al., 1970; Wakai and Hirokawa, 1979; Roncali et al., 1986; Risau et al., 1986; Risauand Wolburg, 1990; Kniesel et al., 1996).

So, why are the indications for blood–brain barrier development in mammals socontroversial? There appear to be several reasons: (i) the diversity of experimentalanimal species, (ii) varying assay systems for testing blood–brain barrier permeabil-ity (e.g., perfusion versus injection), and (iii) the use of different tracer substances(ions, dyes, lipids, sugars, proteins), which determines the nature of the barrier.

Most of the studies on blood–brain barrier formation were done with birdsand mammals, the latter including some species of marsupials (metatheria) (TableI). Marsupials have the advantage of being born prematurely and then developingex utero. Thus, their developing cerebral cortex is easily accessible and relativelyrobust. In these animals, neuronal differentiation starts around birth. For example,the developmental status of a newborn South American opossum (Monodelphisdomesticus) is comparable to that of a 13-day-old embryonic rat. Or, the forebrainof a newborn tammar wallaby corresponds to the forebrain of a 6-week-old humanembryo (Reynolds et al., 1985). Dziegielewska et al. (1988) have demonstrated thatplasma proteins or horseradish peroxidase injected intravenously into newborntammar wallabies did not penetrate into the brain. In addition, tight junctions werefound between cerebral capillary endothelial cells of the cerebral microvasculatureand between epithelial cells of the choroid plexus, suggesting an early start ofblood–brain barrier function.

A considerable body of literature has been published emerging from studieson blood–brain barrier formation in the mouse (Bauer et al., 1993, 1995), rat (Grazer

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Table I. Detection of Blood–Brain Barrier Function in Vivoa

Developmental RouteSpecies stage Material (injected/perfused) Volume Reference

Chick E 4.5–10 HRP i.v. n.r. Delorme et al. (1970)Chick E 9 HRP i.v. 5–10% Wakai & Hirokawa

(1979)Chick E 6–12 HRP Intracardiac 40% Roncali et al. (1986)Tammar NB HRP/HSA i.v. 10–23% Dziegielewska et al.

wallaby (1988)Sheep E 60 Human i.v. 7.5% Dziegielewska et al.

protein (1979)Rabbit NB Sugars, Carotid artery 100 ml Braun et al. 1980

aminesRat E 10.51 Trypan i.p./i.v. n.r. Grazer & Clemente

blue (1957)Rat E 151 Albumin Umbilical vein 5% Olsson et al. (1968)Rat NB IgG i.p. Up to 40% Fabian & Hulsebosch

(1989)Mouse NB HRP i.v. Total blood Vorbrodt et al.

volume (1986)Mouse E 13 HRP Intracardiac 1–3 3 total Risau et al. (1986)

bloodvolume

Mouse E 15 HRP i.p. n.r. Stewart & Hayakawa(1987)

Mouse E 12–17 Evans blue, Perfused Bauer et al. (1995)trypanblue, HRP

a The injection volume (when given as a %) has been compared to estimates of circulating blood volume(10% of body weight). See also Saunders et al. (1992) E, day of embryonic development; n.r., notreported; HSA, human serum albumin.

and Clemente, 1957; Olsson et al., 1968; Fabian and Hulsebosch, 1989; Yoshida etal., 1988), and sheep (Dziegielewska et al., 1979). By injecting human plasma proteinsinto the vasculature of a 60-day-old sheep fetus, for example, it was demonstratedthat none of the injected proteins penetrated into the intraneural domains; theblood–brain barrier seemed to be tight and functional at that stage (Dziegielewskaet al., 1979).

Experimental evidence from studies with embryonic and newborn rats hassuggested that blood–brain barrier-related structures were first detectable at E 10.5(Grazer and Clementi, 1957). This is in contrast to other reports demonstrating theappearance of blood–brain barrier-related structural characteristics only duringadvanced embryonic development (E 17) and attributing blood–brain barrier func-tion to stages from E 15 to neonatal. There is only one report on blood–brainbarrier development in rabbits (Braun et al., 1980). The findings suggest a ratherslow development, a maturation which takes place from the neonatal stage ofdevelopment of adulthood.

Similar discrepancies concerning the onset of blood–brain barrier functionexist when dealing with mouse brain development. Using intracardiac horseradishperoxidase (HRP) injection, Risau et al. (1986) found a functional blood–brainbarrier in distinct areas of the embryonic mouse central nervous system from E 13


to E 16, illustrating a rather slow maturation of the blood–brain barrier, which lastswell into the neonatal stages. This is consistent with data reported by Stewart andHayakawa (1987). In contrast, Bauer et al. (1993, 1995) have shown that morphologi-cal blood–brain barrier-related characteristics in cerebral capillary endothelial cellsof the developing mouse appear simultaneously with intraneural capillary formation(E 10.5), basically allowing a very early beginning of blood–brain barrier function.Perfusion of mouse embryos using dyes and horseradish peroxidase revealed thatlarge areas of central nervous system were already ‘‘tight’’ as early as day E 12 ofgestation. However, a weak blue staining (stemming from Evans blue) was observedin the cerebellum and frontal cortex at that stage, which might be due to free dyewhich was in excess of the binding capacity of plasma proteins.

Varying results from tracer experiments may be explained by the differentexperimental methods used. In order to avoid high pressure on the fragile embryonicvessel walls, Bauer et al. (1995) applied a perfusion technique instead of injectionof dyes and HRP. Unlike HRP injection generally used (see Table I), perfusion ata very low rate minimizes the risk of too high a pressure leading to an unintentionallyincreased leakiness of the cerebral capillaries (Bauer et al., 1995).

Chick embryos were used by several authors, again with somewhat divergingresults. Whereas Delorme et al. (1970) observed an ‘‘immature’’ blood–brain barrierin the chicken around E 4–5, other authors found the onset of blood–brain barrierfunction (exclusion of HRP or dye) between E 6 and E 12 of chick development(Wakai and Hirokawa, 1979, 1981; Roncali et al., 1986) (see Table I). The use of vari-ous tracers which determine the barrier to macromolecules or smaller hydrophylicmoleculessuch asglucoseor aminoacidsandions is important for drawingconclusionsabout the onset of a functional blood–brain barrier. Early studies from Stern and co-workers (1927, 1929) already tried to address this question and have shown in variousanimal models that there is a difference in the penetration of crystalloids (smallermolecules like sodium ferrocyanide) and colloids (larger macromolecules, i.e., dyesbound to proteins) into developing brain versus the adult brain, the first showing ahigher permeability in young animals and the latter showing none. Later on, thesefindings were confirmed by many authors showing age related differences betweenthe uptake of smaller molecules and macromolecules (Tuor et al., 1992; reviewed bySaunders, 1977, 1992; Saunders et al., 1999; Dziegielewska et al., 1997, 1999). However,a closer look at the uptake of the particular small molecules may be needed in orderto get to a proper assessment of blood–brain barrier development. For example,Braun et al. (1980) found no differences in glucose uptake between newborn and adultrabbits but significant higher uptake rates for choline and adenine, the latter being dueto specific uptake mechanisms. The authors conclude that already in young animals aselective blood–brain barrier is working which is adjusted to the needs of the growingbrain. Thus, the concept of an immature blood–brain barrier in developing animalsmay be misleading (see also Saunders 1977).

Further questions about blood–brain barrier development exist concerning thefindings of endothelial cell differentiation in the perineural domain. Capillaries inthat cell layer, where no glial cells or neurons are present, also exhibit blood–brainbarrier characteristics (e.g., loss of fenestrations or new junctional complexes) inthe course of later embryonic development (Bauer et al., 1993; Balslev et al., 1997).

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INDUCTION OF THE BLOOD–BRAIN BARRIER IN VIVOAND IN VITRO

More than 20 years ago the hypothesis was postulated that the morphologicalproperties of microcapillary endothelial cells are governed by the type of tissuefrom which they originate (Svendgaard et al., 1975). When transplanted into thecoelomic cavity of a different species, embryonic brain tissue induced the expressionof blood–brain barrier characteristics in the newly formed blood vessels originatingfrom the host tissue (Stewart and Wiley, 1981). On the other hand, when nonbarriertissue such as skin had been transplanted into the brain, the graft endothelium wasnot replaced by neural blood–brain barrier endothelium. Instead, graft vesselssurvived and anastomosed with host vessels without acquiring blood–brain barrierfeatures (Stewart et al., 1984). Similar experiments have been repeatedly performedusing various experimental systems, and some of the data reported have confirmedthis hypothesis. So have Janzer and Raff (1987), who injected astrocytes into theanterior eye chamber of syngenic rats and found that at least macromolecules didnot penetrate the newly formed astrocytic aggregates. They interpreted this asindicating that a functional blood–brain barrier had been induced in nonneuralendothelial cells which had vascularized the astrocytic aggregates. This interpreta-tion has been challenged by Small et al. (1993) and Holash et al. (1993); see below.Wakai et al. (1986) have grafted nonneural tissue like muscle and skin into thefourth ventricle of rat brain, and despite innervation from the host brain, the graftsnever acquired tightness for HRP. Strong evidence for the blood–brain barrier-inductive power of cerebral tissue comes also from experiments using central ner-vous system grafts transplanted into the fourth ventricle which kept their blood–brain barrier features like tight junctions (Broadwell et al., 1989).

However, in spite of this intriguing experimental evidence, there are alsofindings supporting arguments against the neural-induction theory: Rosenstein(1987a, b) observed that neocortical rat tissue transplanted into a host rat brainlost its blood–brain barrier to HRP or IgG-peroxidase and never regained it.

These contradictory findings may be due to the different grafting methods andin the analysis of the grafts after vascularization (Broadwell, 1988; Rosenstein,1988). Only additional experiments using host animals of various ages and maybeother physiological methods will shed light on this question, which is of great interestfor scientists and of importance from a therapeutic point of view.

BARRIER PROPERTIES IN BLOOD VESSELS OFINTRACEREBRAL TUMORS

Another puzzling facet of blood–brain barrier induction comes from observa-tions of intracerebral tumors. Vascular endothelial cells invading cerebral tumorsloose blood–brain barrier features and become more permeable to proteins. Shiverset al. (1984) introduced C6 glioma cells into host rat brains, where they formedastrocytomas whose vessels proved to be highly permeable to tracer molecules.Moreover, the degree of barrier loss appears to correlate with the malignancy of


tumor cells, so that heterogeneous zones of different blood–brain barrier intactnessare observable in a single tumor (see review by Dermietzel and Krause, 1991).These findings are reminiscent of those of Rosenstein (1987b) indicating that, incertain circumstances, astrocytes may fail to induce or maintain a blood–brainbarrier. On the other hand, there are reports of an intact blood–brain barrier inprimary glial tumors (Sage; 1982). In this context, it has been suggested that it isthe presence of distinct glia-derived factors which maintain the blood–brain barrierin tumors. When the metastatic cells, rather than astrocytes, are the immediateneighbors of the tumor’s capillaries, they do not express barrier properties. Endothe-lial cells from tumors of nonbrain origin, such as meningiomas and metastatictumors, lack blood–brain barrier structures as evidenced by computer tomographicscans (Sage, 1982, Pardridge, 1988). However, there may be doubts concerning thepower of resolution of these scans, which might not be sufficient to detect blood–brain barrier structures.

FORMATION OF THE BLOOD–BRAIN BARRIER IN VITRO

A more fruitful approach for a detailed study of blood–brain barrier inductionhas been the use of in vitro systems. Starting from the first cerebral capillaryendothelial cells in culture (Joo and Karnushina, 1973), numerous studies havebeen published describing culture systems for studying blood–brain barrier functionand/or induction (see reviews by Joo, 1992, 1996). The aim of all these studies wasto mimic the in vivo situation by establishing a monolayer of pure cerebral capillaryendothelial cells, which could then be cocultured with astrocytes and tested formorphological (tight junction formation), biochemical (expression of transportproperties), and functional (electrical resistance) alterations. Though perhaps themost convincing evidence for blood–brain barrier induction is the formation oftight junctions, this parameter was used only sparsely simply because of the tediouselectronoptical analysis. Nevertheless, it was shown repeatedly (Arthur et al., 1987;Tao-Cheng et al., 1987; Shivers et al., 1988) that cocultivation of cerebral capillaryendothelial cells with astrocytes or conditioned media from astrocytes led to asignificant increase in interendothelial tight junction formation.

Another experimental approach to study blood–brain barrier induction in vitrowas the use of assay systems monitoring the activation of blood–brain barrier-related enzymes and other specific transport mechanisms or the increase in electricalresistance of endothelial plasma membranes. Some of the earliest in vitro studieswere done by DeBault and Cancilla (1979) and Cancilla and DeBault (1983),demonstrating an increase in the activity of c-glutamyl transpeptidase (a transportsystem for neutral amino acids) in cultured cerebral capillary endothelial cells aftercocultivation with glial cells. This early finding has been confirmed in several otherstudies (Maxwell et al., 1987; Vorbrodt et al., 1986; Dehouck et al., 1990; Tontschand Bauer, 1989; Roux et al., 1994). Additionally, other blood–brain barrier-relatedmarkers have been studied including activity and polar expression of the Na1K1

ATPase and alkaline phosphatase (Beck et al., 1986; Bauer et al., 1990; Meyer etal., 1991; Roux et al., 1994); glucose uptake (Maxwell et al., 1989); expression of

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HT7, a blood–brain barrier-associated plasma membrane glycoprotein (Lobrinuset al., 1992; Seulberger et al., 1990); expression of the multidrug resistanceP-glycoprotein; and expression of glucose transporter 1 (GLUT1) and transferrinreceptor in cerebral microvascular endothelial cells (Boado et al., 1994; Lechardeuret al., 1995; Hayashi et al., 1997).

Electrical resistance was measured on confluent monolayers cultured ontranswell filters. Again, following cocultivation with astrocytes or treatment withconditioned medium from astrocytes and cyclic adenosine monophosphate (Rubinet al., 1991) the resistance was found to be increased dramatically, up to 1000 V/cm2, normally obtained only in vivo (Butt et al., 1990).

Generally, in vitro experiments concerning blood–brain barrier induction haveto be judged critically and the results may not reflect the situation in vivo sincecerebral capillary endothelial cells used in vitro are mostly derived from adultbrain capillaries which have already been blood–brain barrier functional. Thus, anyinduction of blood–brain barrier-associated features in cultured cerebral capillaryendothelial cells may just be an upregulation of blood–brain barrier marker expres-sion which was downregulated during culturing. The upregulation of blood–brainbarrier features may be interpreted as maintenance rather than induction of thebarrier. This caution in interpreting in vitro experiments has been mentioned byHolash et al. (1993) in a review reevaluating the effect of astrocytes on blood–brainbarrier formation.

THE ROLE OF NEURAL CELLS ON BLOOD–BRAINBARRIER INDUCTION

As depicted in the previous section, the neural environment appears to becrucial for the induction of blood–brain barrier function in cerebral capillary endo-thelial cells. Besides the seminal experiment of Stewart and Wiley (1981), numerousstudies have been published dealing with the role of astrocytes or neurons ininducing blood–brain barrier features in cerebral capillary endothelial cells in vivoand in vitro (for review see Wolburg, 1995). Interestingly, not all of these studiesconfirmed the importance of astrocytes for barrier formation in cerebral capillaryendothelial cells.

Janzer and Raff (1987) claim to have shown that astrocytes, when transplantedinto the anterior eye chamber of the rat, exert a barrier-inducing action on invadingblood vessels originating from the host tissue. In contrast, when fibroblasts weretransplanted, the invading vessels were found to be equally permeable to vasculartracers as in the control animals. Strong arguments against the importance ofastrocytes in that particular experimental system were raised by Small et al. (1993)and Holash et al. (1993). Small et al. (1993) found that iris vessels are impermeableto dyes even in the absence of astrocytes and, therefore, considered this experimentalmodel to be inappropriate for studies of blood–brain barrier induction. Anotherreport, raising reservations about assuming a vital role of astrocytes for in vivoinduction or at least maintenance of blood–brain barrier function, came from Krumand Rosenstein (1989), who showed that in transplanted superior cervical ganglia,


astrocytes did not invade grafts, nor did they influence barrier properties of thehost endothelium. In another set of experiments, the authors used a gliotoxin topartly destroy astrocytic endfeet and subsequently could not find any change incerebral capillary endothelial cells permeability for HRP (Krum and Rosenstein,1993).

INTERACTION OF NEURAL CELLS AND ASTROCYTES IN VITRO

It is definitely easier, albeit less conclusive, to study the role of astrocytes and/or neurons on cultured cerebral capillary endothelial cells. Cancilla and DeBault(1983) were the first to coculture glial cells with primary cerebral endothelial cells.C6 glioma cells were commonly used as a substitute for astrocytes since C6 cellsproliferate quickly and are similar to astrocytes in their marker profiles and pheno-typic expression. DeBault and Cancilla (1979) and DeBault (1981) have demon-strated that after 3 days of cocultivation with C6 glioma cells, c-glutamyl transpepti-dase in cerebral capillary endothelial cells was increased, suggesting an inductionof blood–brain barrier function in the cells. This experiment was repeated by otherresearchers with more or less comparable results (Bauer et al., 1990; Tontsch andBauer, 1991).

Besides the activity of c-glutamyl transpeptidase, other markers for blood–brain barrier function were tested in cerebral capillary endothelial cells after cocul-ture with glial cells, including Na1K1 ATPase and alkaline phosphatase (Beck etal., 1986; Bauer et al., 1990; Meyer et al., 1991; Roux et al., 1984), an increasedappearance of tight junctions (Tao-Cheng et al., 1987; Meresse et al., 1989; Neuhauset al., 1991), and transmembrane resistance (Dehouck et al., 1990; Kasa et al., 1990,1991). In addition, increased transport of neutral amino acids, formation of capillary-like structures, and upregulation of the low-density lipoprotein (LDL) receptorswere observed (Cancilla and DeBault, 1983; Laterra et al., 1990; Dehouck et al.,1994).

The question arises whether or not inductive signals from neural cells alsoelicit blood–brain barrier features in endothelial cells of other tissues. The answeris ambiguous since Maxwell et al. (1987) and Tontsch and Bauer (1991) did notfind blood–brain barrier marker induction in nonbrain endothelial cells. Lately,however, Hayashi et al. (1997) could show an induction of blood–brain barrierfeatures in human umbilical vein endothelial cells. Using a heterologous culturesystem which allowed only astrocytic endfeet to contact the umbilical vein endothe-lial cells, Hayashi et al. have shown that a number of blood–brain barrier-specificmarkers such as glucose transporter, transferrin receptor, c-glutamyl transpeptidase,and P-glycoprotein were upregulated in their transcription. Even astrocyte-derivedextracellular matrix had similar effects on these cells. This inductive effect onnonbrain endothelial cells may be due to the specific culture method, which is closerto the in vivo situation compared to those applied by the other researchers, whofound no effects. This might indicate that indeed endothelial cells do have a highplasticity and may not be so different from tissue to tissue after all.

Astrocytic endfeet membranes differ from those of the astrocytic cell body by

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an increased number of structures (termed orthogonal array of particles) identifiedby means of freeze–fracture technique. When the blood–brain barrier breaks down,this high number of orthogonal array of particles in endfeet membranes was foundto be greatly reduced or even absent (Dermietzel and Leibstein, 1978; Wolburg etal., 1986). Thus, it appears plausible to suggest a specific role of the orthogonalarray of particles in astrocyte–endothelial interactions. Unfortunately, astrocytesin culture lose this particle-related polarity, so their function in blood–brain barrierinduction in vitro is difficult to determine (Landis and Weinstein, 1983; Neuhauset al., 1991).

Besides using glial/endothelial cocultures, numerous blood–brain barrier in-duction experiments in vitro were done using conditioned media from astrocytes.Results from these studies suggested a specific role of glia-derived soluble factorsin blood–brain barrier induction, a debated topic which is elaborated on in thefollowing section.

When the blood–brain barrier is formed, the cerebral microvasculature issurrounded by neurons (or neuroblasts) and radial glial cells. Experiments em-ploying cocultivation of cerebral capillary endothelial cells and neurons or cerebralcapillary endothelial cells and neuronal plasma membranes have indeed shown adose-dependent increase in c-glutamyl transpeptidase enzyme activity, higher thanwith cocultured glial cells (Tontsch and Bauer, 1991), indicating an inductive effectof neurons.

THE INDUCTIVE SIGNAL: CELL–CELL CONTACT ORSOLUBLE FACTOR?

The question as to the nature of the glia/neuron-mediated blood–brain barrierinduction in cerebral capillary endothelial cells remains open. When the cerebralcapillaries migrate from the perineural domain into the intraneural tissue, theyseem to be in close contact with the surrounding neuroepithelial cells. Later on, inthe more mature animal the near-proximity of astroglial endfeet is obvious (Bar,1980; Bauer et al., 1993). While in the adult animal the extracellular matrix ensheath-ing the cerebral capillaries is a continuous layer and separates the cerebral capillaryendothelial cells from the astrocytic endfeet (Bar, 1980), cerebral capillary endothe-lial cells in the early embryo possess hardly any extracellular matrix (Caley andMaxwell, 1970; Bar and Wolff, 1972). Thus, direct cell–cell contact between migrat-ing cerebral capillary endothelial cells and neuroblasts or radial glial cells in theembryonic central nervous system seems possible, whereas any interaction betweencerebral capillary endothelial cells and astrocytes in the adult brain tissue is influ-enced by the extracellular matrix, which either transmits messages or plays aninductive role itself.

Several studies have demonstrated that conditioned media from astrocytes doelicit blood–brain barrier characteristics in cultured cerebral capillary endothelialcells. These blood–brain barrier characteristics comprise enhanced activity of theATPase, an increase in tight junction formation (Arthur et al., 1987; Rubin et al.,1991), enhanced expression of HT7 and of neurothelin (Lobrinus et al., 1992), and


an increased electrical resistance, obviously due to tight junction formation (Raubet al., 1992).

Rubin et al. (1991) have used conditioned media derived from primarycultures of rat astrocytes and cyclic AMP (cAMP) and have shown convincinglythat after treatment with these two factors, bovine cerebral capillary endothelialcells form tight junctions and exhibit an increased electrical resistance. Interest-ingly, conditioned media from astrocytes itself did not exert a significant effecton junction formation but enhanced the effects of cAMP. The role of cAMPin blood–brain barrier induction has also been confirmed by Beuckmann et al.(1995), demonstrating that the downregulated activity of alkaline phosphatasein cultured cerebral capillary endothelial cells is restored in in vivo levelsfollowing treatment with cAMP.

So far, nothing is known about secreted or membrane-bound glia/neuron-derived molecules or factors which could be responsible for the blood–brain barrier-inducing effect on cerebral capillary endothelial cells. Interestingly, Tontsch andBauer (1991) have shown that the isolated plasma membranes derived fromastrocytes and neurons were active in inducing blood–brain barrier features incultured cerebral capillary endothelial cells. Subsequently, a glycoprotein fractionin the range of 150 kDa was isolated from purified plasma membranes, furtherpurified, and found to induce c-glutamyl transpeptidase activity in cultured cerebralcapillary endothelial cells. However, this increase in enzyme activity never exceededthat elicited by crude glial or neuronal plasma membranes. On the other hand, asynergistic effect was observed when, in addition to the active protein fraction,interleukin-1 (IL-1) or laminin was added. IL-1 and laminin alone did not affectc-glutamyl transpeptidase or Na1K1 ATPase activity, nor did other matrix compo-nents added to the culture medium of the cerebral capillary endothelial cells (Baueret al., unpublished). The inability of extracellular matrix components to induce orrestore blood–brain barrier characteristics in cultured endothelial cells was alsodemonstrated by Meyer et al. (1990, 1991), who found that downregulation ofc-glutamyl transpeptidase or of alkaline phosphatase was not halted or reversedby various extracellular matrix components coated on the culture dish. By the sameauthors it has been shown that only cell–cell contact had an inductive effect, andnot glia-conditioned medium. However, the susceptibility of the cultured cerebralcapillary endothelial cells to neural induction depends on their proliferative state,i.e., on the degree of confluency of the culture.

Also, the inductive signal did not work for other blood–brain barrier-associatedcharacteristics such as GLUT1 expression, which remained unchanged (Bauer etal., unpublished). This was also found by Boado et al. (1994), showing that neitherconditioned media nor plasma membrane fractions from C6 glioma cells inducedenhanced expression of the GLUT1 gene in cerebral capillary endothelial cells, butbrain homogenate from bovine brain did. Most probably it is the growth factorspresent in the homogenate which stimulate GLUT1 expression, since addition ofbFGF and TNFa to the culture medium of cerebral capillary endothelial cells alsoexerted an inducing effect. The effect of both soluble factors and astroglia-derivedmembranous components on blood-brain barrier-associated marker enzyme activityhas been shown by Roux et al. (1994) in a rat brain capillary endothelial cell line,

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suggesting that indeed a complex stimulus is needed for blood–brain barrier in-duction.

Taking all these diverging reports into account, inductive signals or factorsmay be both secreted molecules (growth factors or interleukins) diffusing over ashort range of up to several cell layers’ distance and membrane-associated compo-nents which work through or via the basal lamina.

CONCLUSION

Upon close inspection, the actual knowledge about blood–brain barrier induc-tion is rather sparse and many open questions still remain. In spite of the consider-able progress that has been made in the field of blood–brain barrier research sincethe earliest injection studies, there has yet to be a major breakthrough addressingthe crucial questions as to the inductive mechanisms leading to blood–brain barrierformation. The timing of blood–brain barrier formation, the mechanisms of itsinitial induction, and the role of astrocytes and neurons in this process are stillunsolved topics. Most elusive is the molecular mechanism and thus the nature ofthe inductive signal.

Additional experiments will be needed using refined techniques to elucidatethe intra- and extracellular events surrounding the critical developmental tighteningof the embryonic blood–brain barrier. Also, extended comparative studies wouldbe helpful to shed light on the timing blood–brain barrier function. One will haveto await the purification and subsequent characterization of the inducing factor(s).Molecular evidence of the genes encoding blood–brain barrier-inducing moleculesin neural cells should provide the basis for knockout experiments as demonstratedby the work with the multidrug resistance protein, which resulted in mice left onlywith a partially functional blood–brain barrier (Schinkel et al., 1994).

ACKNOWLEDGMENTS

This paper was supported in part by the Austrian Ministry of Science (49869/1), the Austrian FWF (Projects 7654, 8289, and 9195), and the Austrian NationalBank. Hannelore Bauer is currently financed by the Austrian Programme for Ad-vanced Research and Technology (APART). The help of Mrs. Madeleine Bohrerand Mr. Bernhard Hennig in reading the manuscript is gratefully acknowledged.

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Neural Induction of the Blood–Brain Barrier: Still an Enigma