Regulation of a Blood-Brain Barrier—Specific Enzyme Expressed by Cerebral Pericytes (Pericytic Aminopeptidase N/pAPN) under Cell Culture Conditions

Abstract

In this study we show that the aminopeptidase N of cerebral pericytes (pAPN) associated with the blood—brain barrier (BBB) is downregulated in pericytic cell cultures. This observation is in accordance with previous data describing comparable in vitro effects for BBB-specific enzymes of endothelial or pericytic origin, such as γ-glutamyl transpeptidase or alkaline phosphatase. By polymerase chain reaction and in situ hybridization we were able to determine that the downregulation of pAPN occurs at the posttranscriptional level. The mRNA of pAPN was found to be constitutively expressed even when the protein is no longer detectable. Culturing the pericytes in an endothelial cell—conditioned medium allowed pAPN to be reexpressed. However, the reexpression effect depended largely on the culturing conditions of the pericytes. Although purified pericytes deprived of endothelial cells did not reveal a reexpression effect, pericytes that were kept in contact with endothelial cells were able to acquire a pAPN-positive phenotype, indicating that endothelial cells constitute an essential requirement for the in vitro reexpression of pAPN. Astrocytes, however, were insufficient in exerting any reexpression effect.

Keywords

Blood—brain barrier Pericytes Aminopeptidase N Coculture conditions

The blood—brain barrier (BBB) regulates the exchange of blood solutes with the interstitial cerebral fluid and is composed of a physical and metabolic barrier. The BBB consists of a microvascular endothelium with its cellular adjuncts (Reese and Karnovsky, 1967; Dermietzel, 1975; Crone and Levitt, 1984; Dermietzel and Krause, 1991). In recent years considerable efforts have been focused on models that mimic BBB conditions in vitro (Audus and Borchardt, 1987; Greenwood, 1991; Rubin et al., 1991; Joo, 1992; Abbott et al., 1992). The results of these experiments have strengthened the essential role of endothelial cells for BBB mechanisms. They have also provided evidence that endothelium-associated cells, like astrocytes and pericytes, are required for the establishment and maintenance of intrinsic BBB functions (Farrell et al., 1987; Maxwell et al., 1987, 1989; Meyer et al., 1990, 1991; Frey et al., 1991; Risau et al., 1992).

Astrocytes, which tightly clasp cerebral microvessels by their endfeet, have been demonstrated to exhibit inductive capabilities on the formation and functional efficiency with respect to the tightness of the cerebral endothelium (Janzer and Raff, 1987), the most prominent physiologic feature of the intact BBB (Reese and Karnovsky, 1967; Stewart and Wiley, 1981; Janzer and Raff, 1987; Rutten et al., 1987). Furthermore, the expression of metabolic components of the BBB is suggested to be governed by regulatory influences provided by astrocytes. Prominent metabolic components of the BBB are the glucose transporter(s) (Glut-1) (Maxwell et al., 1989), alkaline phosphatase (Meyer et al., 1991; Roux et al., 1994), γ-glutamyl transpeptidase (Maxwell et al., 1987; Meyer et al., 1991; Mizuguchi et al., 1994; Roux et al., 1994), the low-density lipoprotein-receptor (Dehouck et al., 1994), the transferrin transporter system (Roberts et al., 1993), acetylcholinesterase (Pakaski and Kasa, 1992), and the Na-K-Cl-cotransporter (Sun et al., 1995). Recent findings, however, have led to a reconsideration of the role that astrocytes play in the process of BBB induction. For instance, the studies of Holash et al. (1993) substantiate the general concept that astrocytes exhibit significant effects that maintain or influence functional properties of the BBB, but they lack the ability to initially induce BBB formation. The simplistic view of a bilateral axis in BBB induction and maintenance involving exclusively astrocytes and endothelial cells has become debatable (see Dermietzel and Krause, 1991).

Another cell type coming into focus in BBB research is the cerebral pericyte, a morphologic, biochemical, and physiologic heterogeneous cell population. Pericytes, which share a common basement membrane with the cerebral endothelium, are suggested to have phagocytotic potency (Cancilla et al., 1972; Jordan and Thomas, 1988), which operates as a “second line of defense” at the boundary between blood and brain (Farrell et al., 1987). Furthermore, pericytes are considered to be influential in the regulation of capillary blood flow (Le Beux and Willemot, 1980; Joyce et al., 1985a, 1985b; Herman and D' Amore, 1985). In peripheral tissues pericytes have been reported to provide (1) potential modulators of endothelial permeability (Imayama and Urabe, 1984), (2) stabilizing effects on microvessel walls (Nehls and Drenckhahn, 1993), and (3) promoting activity on angiogenic processes (Orlidge and D' Amore, 1987; Klagsbrun and D' Amore, 1991) and capillary sprouting (Verhoeven and Buyssens, 1988; Nehls et al., 1992, 1994). Evidence is also accruing that cerebral pericytes constitute a major component of the metabolic BBB by expressing specific enzymes, such as γ-glutamyl transpeptidase (Frey et al., 1991; Risau et al., 1992), or enzymes involved in neurotransmitter metabolism, like glutamyl aminopeptidase (Bausback et al., 1988; Healy and Wilk, 1993; Song et al., 1993).

Consistent with these observations, we recently reported on the expression of a specific isoform of aminopeptidase N (CD 13) in cerebral pericytes (pAPN) (Krause et al., 1988; Kunz et al., 1994). The lack of pAPN expression in brain regions devoid of a tight endothelium, like the circumventricular organs, suggested that pAPN is a member of the metabolic complement of the BBB that is involved in neuropeptide degradation (Mizutani et al., 1993), i.e., APN has been suggested to be involved in the degradation of endogenous endorphins, like enkephalins (De la Baume et al., 1983). We also acquired evidence that pAPN responds to pathologic disturbances of the BBB (Kunz et al., 1995).

In essence, cerebral pericytes are an important member of the cellular complex that constitutes the BBB. However, detailed data on the cell biology of this cell class of the BBB are still lacking.

To gain a better insight into the cellular biologic properties of cerebral pericytes, we designed a method to purify pericytes of brain microvessels. By exploiting pAPN as a specific marker for cerebral pericytes, we found that (1) purified cerebral pericytes are unable to preserve their brain-specific phenotype in culture, as shown by loss of pAPN expression, (2) downregulation of pAPN occurs at the translational level, (3) (re)induction of pAPN can only be achieved in coculture with endothelial cells, (4) cultured primary astrocytes are insufficient to (re)induce the pericytic phenotype, and (5) (re)induction requires a soluble factor (or factors) provided by endothelial cells.

MATERIALS AND METHODS

Antibodies and immunologic techniques

A number of monoclonal and polyclonal antibodies were applied for the characterization of cell specificity under immunofluorescence conditions. The following antibodies were applied:

Monoclonal antibodies. The antibody directed against pAPN was generated in our laboratory and has been extensively characterized (Krause et al., 1988; Kunz et al., 1994, 1995). Undiluted hybridoma supernatants were used for immunocytochemical detection as well as for solid-phase isolation (see below). Anti-EDI (MCA 341), an antibody recognizing reactive cells of monocytic origin (macrophages) was purchased from Serotec (Wiesbaden, Germany) and applied in a dilution of 1:250. The anti-MUC 102 antibody, which is regarded to be specific for microglial cells (Gehrmann and Kreutzberg, 1991), was kindly provided by G. Kreutzberg (Max-Planck Institute of Psychiatry, Munich, Germany). The working solution of this monoclonal antibodies (mAb) was 1:700. Monoclonal antibodies against smooth muscle (sm)-actin and vimentin were purchased from Sigma (Munich, Germany) and Boehringer (Mannheim, Germany), respectively. The applied dilutions were 1:400 for anti-sm actin and 1:150 for anti-vimentin.

Polyclonal antibodies. For recognition of cerebral endothelial cells an antibody directed against the BBB-specific Glut-1 (dilution 1:50) was used. The anti-Glut-1 antibody was prepared and affinity-purified as described earlier (Wang, 1987; Dermietzel et al., 1992). An antibody against the factor VIII-related antigen (Dakopats, Hamburg, Germany) was applied in a dilution of 1:1,000 and served as a general marker for endothelial cells. Primary astrocytes were identified by the expression of glial fibrillary acidic protein (GFAP) by means of an anti-GFAP antibody (dilution 1:200; Sigma).

Fluorescein isothiocyanate- (FITC) and Texas red—coupled secondary antibodies (Sigma) were used in a dilution of 1:400.

Immunocytochemistry. For immunologic detection of cell-specific proteins, indirect immunocytochemistry was performed as described earlier (Kunz et al., 1994). In brief, cells were washed twice with 0.1 mol/L phosphate-buffered saline (PBS) to remove culture medium, followed by a brief fixation in absolute ethanol for 10 minutes at room temperature. After several brief washes with 0.1 mol/L PBS, nonspecific binding sites were saturated by incubation of the cells in 0.1 mol/L PBS containing 0.1% bovine serum albumin (BSA) and 10% horse serum as additives. Blocking time was 30 minutes. For double immunostaining, cells were sequentially incubated with a monoclonal and a polyclonal primary antibody for 60 minutes each at room temperature, followed by thorough rinsing with 0.1 mol/L PBS with 0.1% BSA, and subsequent incubation with the FITC- or Texas red-coupled secondary antibodies, or both, in the dark for 20 minutes each at room temperature. After three additional washes with 0.1 mol/L PBS with 0.1% BSA, specimens were embedded in FITC-Guard (Testog, Chicago, IL, U.S.A.) and subjected to immunofluorescence microscopy on an Axiovert 35 microscope (Zeiss, Oberkochen, Germany).

Isolation of cells and cell culture procedures

Cerebral microvascular cells were isolated as described earlier (Kunz et al., 1994). In brief, cells were obtained from the cerebral cortex of 8 to 10 rat brains (Wistar strain, 8 to 12 weeks old) by collagenase digestion (collagenase type CLS II; Seromed, Berlin, Germany) followed by a subsequent 50% Percollgradient (Pharmacia, Freiburg, Germany), and centrifuged at 1,250g (4°C) for 10 minutes (Risau et al., 1992). The collected microvascular cells were washed twice with 0.1 mol/L PBS. The isolated cells were then used for cell culture as crude microvascular cell fraction (CMF) or further subjected to an additional purification step to isolate purified cerebral pericytes (see below). Samples of the CMF were seeded on poly-l-lysine-precoated coverslips or cultured in plastic culture flasks (25 cm²) and maintained at 37°C (5% CO₂) in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 1% glucose, 10% fetal calf serum (Boehringer), 1% bovine retina extract (Risau et al., 1992), 100 U/mL penicillin (Gibco, Eggenstein, Germany), 100 U/mL streptomycin (Gibco), 1 mmol/L sodium pyruvate (Seromed), and 2 mmol/L glutamine (Gibco). Culture medium was changed three to four times weekly. Under these culture conditions microvascular cells reached confluency within 10 to 20 days. All cultures used for experimentation were from the first to fourth passages.

Purification of cerebral pericytes was achieved by a solid-phase technique making use of immunomagnetic beads. For purification microvascular cells were prepared as described above. The collected fraction was then resuspended in 50 µL DMEM supplemented with 4.5% glucose. Dissociation was achieved with a dissociation solution (Sigma), used in a concentration of 4 mL per 50 µL of cell suspension, by repeated tituration with a glas pipette at room temperature. After centrifugation (160g, 5 minutes), samples were incubated with 500 µL of the undiluted supernatant of anti-pAPN antibody for 30 minutes at room temperature, washed twice with DMEM with 4.5% glucose, and finally resuspended in another 500 µL DMEM with 4.5% glucose. A Dynabead M-450 solution (Dynal, Oslo, Norway) coated with goat anti-mouse IgG was added, achieving a total concentration of 10⁷ beads/mL cell suspension. After incubation for 45 minutes at 4°C under constant shaking, samples were placed in a Dynal magnet (MPC; Dynal) to separate immunoadsorbed cells. Separation time was 2 minutes. Supernatants containing free microvascular cells not immunoadsorbed to the Dynabeads were soaked off and further used as mixed culture samples (MCS). The purified pericytes were collected, washed twice with DMEM with 4.5% glucose, and seeded into plastic culture dishes (25 cm²). Both the purified pericyte cultures (PPC) and MCS, which consist chiefly of microvascular fragments and free unbound pericytes, were grown under the same culture conditions as described above for CMF.

Cerebral astrocytes were isolated from cerebral cortices of 1-to 2-day-old rats (Wistar strain) for coculture with PPC and MCS as described previously (Dermietzel et al., 1991). To achieve highest purity of astrocytic cell cultures (>99%), contaminating macrophages and oligodendrocytes were separated by a series of continuous shaking for 6, 18, and 24 hours. Contaminating cells were removed by changing the culture medium after each agitation step. For coculture experiments first passage astrocytes were used.

A cerebral endothelial cell line bEnd.3 (Montesano et al., 1990; kindly provided by D. Männel, University of Regensburg) was further used for coculture experiments. The bEnd.3 cells were cultured at 37°C (5% CO₂) in DMEM containing 4.5% glucose and 5% fetal calf serum in plastic culture flasks. After acquiring confluency (1 to 2 days after seeding), bEnd.3 cells were passaged for coculturing with microvascular cell fragments or purified pericytes.

Coculture conditions. Primary astrocytes and bEnd.3 were subjected to coculture with PPC and MCS.

Coculturing was performed under the following conditions. First, conditions with unrestricted physical contact were achieved by seeding PPC or MCS on a subconfluent layer of astrocytes (alternatively, bEnd.3 cells). Second, culture conditions with limited cell-cell contact were realized using perforated polycarbonate membranes (pore size approximately 1 µm; Becton-Dickinson, Lincoln Park, NJ, U.S.A.). Membranes were loaded on one side with the feeder layer (astrocytes or bEnd.3 cells, respectively), which was then cultured to confluency, subsequently turned around and further loaded with purified pericytes or MCS. Membranes were fixed in a special sheath-holder device (Minucells; Minuth et al., 1992) throughout the experiments. Third, cocultures with exclusion of physical contact were obtained by cultivation of cells on both sides of glass coverslips in the same sheath-holder device.

Preparation of cerebral microvessels for Western blotting. To achieve sufficient amounts of pAPN from cerebral microvessels for Western blotting vessels were isolated as described by Mrsulja et al. (1976). This protocol achieves a higher concentration of pAPN than the Percoll method described above. All steps of isolation were performed at 4°C. In brief, cerebrum of one adult rat (Wistar strain) was homogenized in 20 volumes of Ringer's solution with 1% BSA and 10 mmol/L HEPES, pH 7.4, in a glass homogenizer with a tight-fitting pestle (10 strokes for each sample). After homogenization, the samples were centrifuged at 1,500g for 15 minutes. The pellets were resuspended in Ringer's solution with 1% BSA and centrifuged for another 10 minutes at 1,500g. The collected pellets were suspended in 10 mL of 0.25 mol/L sucrose solution, layered over a two-step discontinuous sucrose gradient of 1.0 mol/L and 1.75 mol/L sucrose (wt/vol; 12 mL each), and centrifuged at 58,000g for 30 minutes in a Beckman ultracentrifuge (rotor SW-28). The interphase between the 1.0 mol/L and 1.75 mol/L sucrose was collected and checked for microvessel purity by phase-contrast microscopy.

Biochemical and molecular biologic techniques

Preparation of kidney brush-border membranes. Preparation of the brush-border membranes from rat kidney proximal tubules was done as described by Biber et al. (1981). All steps were performed at 4°C. Cortices of two kidneys from one adult rat (Wistar strain, 3 to 6 months old) was homogenized and centrifuged for 15 minutes at 1,800g. The supernatant was centrifuged for a further 30 minutes at 22,300g. Collection of pellets and homogenization and centrifugation steps were repeated twice, as described above. The pellets of the final centrifugation step were highly enriched in brush-border membranes.

Polyacrylamide gel electrophoresis and Western blotting. Samples of kidney brush-border membranes, homogenates of freshly isolated cerebral microvessels, and cultivated PPC were separated on a one-dimensional continuous 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane (0.45 µm; Millipore, Eschborn, Germany) by the semidry blotting method. For immunoincubation, the membrane was blocked in PBS with 0.05% Tween 20 at 4°C overnight. The incubation with the monoclonal anti-pAPN antibody was performed at room temperature for 2.5 hours, followed by the secondary antibody (anti-mouse IgG antibody coupled to 10-nm gold beads; Aurion, Wageningen, the Netherlands) overnight. Bands labeled with immunogold were visualized by silver enhancement according to the recommendation of the manufacturer (Aurion).

Glycosidase digestion. Kidney brush-border membranes were digested with endoglycosidase F (Boehringer) to evaluate binding efficiency of the mAb anti-pAPN to the glycosylated and nonglycosylated forms of APN. For this purpose 5 µL of washed brush-border membranes were solubilized in 50 µL 1% SDS sample buffer. After heating the brush-border membranes at 100°C for 1 minute in 1% SDS sample buffer and removing the insoluble residue by centrifugation, the extract was adjusted to 25 µL endo F buffer (50 mmol/L potassium phosphate, 1% N-octylglycoside, 25 mmol/L EDTA, pH 6.5). After a further incubation at 100°C for 1 minute and cooling at 37°C, 250 mU endoglycosidase F were added. The reaction mixture was then incubated for 60 minutes at 37°C before gel electrophoresis. Gels of treated and untreated fractions were subjected to Western blotting (see above).

RNA isolation. Total RNA was isolated from purified pericytes and from microvascular cells (42 days in vitro, third passage, one culture flask of 25 cm², each) according to Chomczynski and Sacci (1987) with slight modification. In brief, cell lysis was performed using TRIzol reagent (total RNA isolation reagent; GibcoBRL, Berlin, Germany). After addition of chloroform and centrifugation (15 minutes, 12,000g, 4°C), the aqueous phase containing the total RNA was aspirated and precipitated in isopropyl alcohol.

Reverse transcriptase polymerase chain reaction. Total RNA from PPC and MCS was used as template for amplifying a fragment of pAPN. Primer sets for pAPN cDNA amplification and Reverse transcriptase polymerase chain reaction (RT-PCR) conditions were identical to those described recently (Kunz et al., 1994).

Southern blotting. For Southern blotting, an agarose gel (2%) was loaded with 20 ng of each RT-PCR cDNA sample obtained from purified pericytes or MCS. After electrophoresis, the cDNA was blotted onto nitrocellulose membranes according to standard techniques (Sambrook et al., 1989). The membranes were then hybridized overnight at 42°C to a digoxygenin (DIG; Boehringer)-labeled pAPN cDNA probe (Kunz et al., 1994). Final washes were performed under high stringency conditions in 0.1× saline sodium citrate buffer with 0.1% SDS at 65°C. The hybridized DIG-labeled complex was detected by means of an anti-DIG antibody coupled to alkaline phosphatase (Boehringer).

Nonradioactive in situ hybridization. For in situ hybridization PPC grown on poly-l-lysine-coated coverslips (50 days in vitro, second passage) were fixed in 0.1 mol/L PBS with 4% formaldehyde for 60 minutes at 4°C and subsequently postfixed for 10 minutes at −20°C in 95% ethanol–5% glacial acetic acid. After rinsing with 0.1 mol/L PBS with 0.1 mol/L glycine, cells were equilibrated in proteinase-K buffer (PKB; 50 mmol/L EDTA, 100 mmol/L Tris-HCl, pH 8.0) for 10 minutes, followed by protein digestion with proteinase K (3 µg/mL PKB; Sigma) for another 20 minutes at 37°C under gentle agitation. Proteinase K activity was stopped by postfixation in 0.1 mol/L PBS with 4% formaldehyde (10 minutes at room temperature), and cells were washed at room temperature in 0.1 mol/L PBS with 0.1 mol/L glycine and diethyl pyrocarbonate-treated water (DEPC-H₂O; 0.02% DEPC; Sigma). Acetylation of the samples was achieved by incubation in 0.1 mol/L triethanol-amine, pH 8.0, supplemented with 0.25% acidic acid anhydride for 10 minutes, followed by two further washes with DEPC-H₂O. Before prehybridization, cells were dehydrated in an ascending series of ethanol baths (70% to 100%), followed by subsequent air drying. For prehybridization, specimens were incubated in a humid chamber containing 50% formamide, 0.3 mol/L sodium chloride, 20 mmol/L Tris, pH 8.0, 1 mmol/L EDTA, 10% dextran sulfate, 1× Denhardt's solution, and 500 µg/mL yeast tRNA for 1 to 2 hours at 42°C. Thereafter the probes were washed with DEPC-H₂O, dehydrated in an ascending series of ethanol baths (50% to 100%), and air dried. Hybridization was performed overnight in a humid chamber at 42°C, using 200 pg/mL of DIG-labeled anti-sense cRNA probes (≈,372 bp) (in prehybridization mixture) matching to the mRNA of APN. Control experiments were performed by application of corresponding DIG-labeled sense cRNA probes.

After hybridization, cells were washed thoroughly with 2× SSC (0.3 mol/L NaCl, 30 mmol/L citrate buffer, pH 7.0) and 0.1 × SSC at room temperature. To remove nonhybridized cRNA, specimens were incubated with 10 mmol/L Tris and 0.5 mol/L NaCl containing ribonuclease A (6 µg/mL; Boehringer) for 10 minutes at 37°C. Cells were finally rinsed three times in 0.1× SSC at 55°C and one time in 0.1× SSC at room temperature.

For immunodetection of the DIG-labeled hybridization products, specimens were washed twice in 0.1 mol/L PBS. Nonspecific binding sites were saturated with 0.1 mol/L PBS with 1% BSA, and cells were incubated for 2 hours with a polyclonal anti-DIG antibody (1:50 in 0.1 mol/L PBS with 0.2% BSA and 0.1% Triton × 100) (Boehringer). Immunostaining was performed by means of a FITC-coupled secondary antibody as described above.

RESULTS

Isolation and characterization of cerebral microvascular cells

The history of a particular cell type is considered to be of primary importance in the process of dedifferentiation or redifferentiation events under in vitro conditions (Koechlin et al., 1991). To achieve a reproducible collection of pericytes, we first characterized fractions of these cells that differed significantly in their cell composition.

Crude microvascular cell fraction. Crude microvascular cell fraction was obtained by isolating microvessels from cortical rodent brains after subjecting them to collagenase treatment (Risau et al., 1992). By applying a number of different cell-specific antibodies, the CMF was determined to consist of a collection of endothelial cells (factor VIII-related antigen-positive; Fig. 1A), pericytes (pAPN- and sm-actin-positive; Fig. 1B,C), and to a minor degree, contaminating astrocytes (GFAP-positive; Fig. 1D). Microglial cells (MUC 102-positive; Fig. 1E) and macrophages (EDI-positive; Fig. 1F) were also found in varying amounts. In addition, the CMF contained small fragments of intact microvessels that resisted collagenase digestion (not shown). When grown under supplemented culture conditions (10% BSA and 1% bovine retina extract), pericyte proliferation was favored over the other cell species. A uniform pericyte-like cell type was achieved after 14 days in culture (Swinscoe and Carlson, 1992). This cell type was characterized by intracellular fibers of sm-actin (stress fibers; see Fig. 1C) and the formation of multicellular nodules (Schor and Schor, 1986; Schor et al., 1990). Because endothelial cells are inhibited in growth by proliferating pericytes (Orlidge and D' Amore, 1987; Swinscoe and Carlson, 1992), the formation of endothelial “cobblestone-like” monolayers was rare and transient in CMF.

FIG. 1.

Immunofluorescent characterization of the crude microvascular cell fraction (CMF) after 20 days in vitro. A cluster of factor VIII-related antigen (F-VIII ra)-positive endothelial cells in primary culture are shown forming a typical contact-inhibited monolayer (a). Single pericyte aminopeptidase N (pAPN)-positive cells (b) and some smooth muscle (sm)-actin-expressing cells (c) are shown. Astrocytes staining positively for glial fibrillary acidic protein (GFAP) are shown (d). Contaminating microglial cells and macrophages are detected with antibodies directed against MUC 102 (e) and ED1 (f). Scale bar = 50 µm, Original magnification ×350.

Purified pericyte cultures. To obtain purified cultures of cerebral pericytes, we designed a solid-phase isolation technique using our mAb directed against the pericyte-specific enzyme pAPN. The isolation protocol consisted of immunoadsorption of antibody-labeled pericytes to immunomagnetic beads (for detail see Materials and Methods). The samples of immunoadsorbed cells contained a small number of individual pAPN-positive cells (Figs. 2a and 3a) and few microvascular segments (Figs. 2b and 3b). Contaminations with macrophages, free microglial cells, or astrocytes, as described above for the CMF, were not detected in PPC. Microvascular segments did not attach to the tissue culture supports and were lost after the first passage. After 15 to 40 days in culture, purified pericytes had grown in small islets (Fig. 2C) reaching area-restricted confluency (Fig. 2D) with the formation of nodules (Fig. 2E). The pericytic plaques resembled in both cellular morphology and their growth pattern the pericytes obtained from the CMF. All purified pericytes expressed sm-actin (Fig. 2F) and vimentin (Fig. 2G).

FIG. 2.

Isolation and culture of purified cerebral pericytes (PPC) achieved after separation by immunomagnetic beads. Acute isolated single cells (a) and small capillary fragments (b) are immunobound to Dynabeads (arrowheads). (c) Phase-contrast microscopy indicates that the majority of isolated cells (3 days after plating) in PPC have adsorbed immunobeads to their surface. Arrowheads point to immunocoupled beads. In confluent culture (15 days after plating) PPC reveal a characteristic irregular morphology and overlapping processes (d). Purified pericyte cultures (25 days after plating) exhibit noticeable retraction and form numerous multicellular nodules (e). Immunofluorescent localization of cytoskeleton proteins sm-actin (f) and vimentin (g) in PPC (7 days after plating). See Fig. 1 for other abbreviations used. Scale bar = 50 µm. Original magnification a and b, ×550; c-e, ×175; f and g, ×350.

FIG. 3.

Immunofluorescent staining of PPC. Pericyte aminopeptidase N was expressed by acutely isolated pericytes (a) and microvascular fragments (b). Loss of pAPN immunoreactivity was observed when cells became adherent within 3 days of culture (c); (d) phase-contrast of (c). Arrowheads (a-c) depict fluorescent Dynabeads. (e) Cells (asterisks) from mixed culture samples (MCS) in long-term culture (>40 days) were positive for pAPN. Arrows point to nonspecific staining in (e). See Figs. 1 and 2 for other abbreviations used. Scale bar = 50 µm. Original magnification a and b, ×550; c-e, ×350.

Mixed culture samples. The MCS consisted of the supernatants obtained after solid-phase adsorption of pericytes. In contrast to the PPC, the MCS revealed endothelial cells and contaminating macrophages, as well as microglial cells and very few astrocytes, but had in general a lower percentage of pericytes than the crude CMF (not shown).

pAPN protein expression is downregulated in vitro

The two cell fractions (PPC and MCS), which differed significantly in their cellular composition, were then exploited for studies of pAPN expression.

Expression of pAPN was high in acute isolated pericytes (Fig. 3A,B), as revealed by immunocytochemistry. In PPC, 2 to 5 days after plating, pAPN protein expression was completely downregulated as indicated by loss of pAPN immunoreactivity (Fig. 3C,D). In MCS, however, pAPN expression was still preserved at a lower level. Singular pAPN -positive cells were found in MCS even after long-term culture (>40 days; Fig. 3E). Expression and downregulation of pAPN appeared to be independent of the substrate used for both cell culture fractions (not shown). The downregulation effect was also confirmed by Western blots of freshly isolated microvessels and PPC (11 days in vitro). Purified pericyte cultures revealed no immunodetectable staining of pAPN as compared with the microvessel fraction (Fig. 4A).

FIG. 4.

(a) Immunoblots of kidney brush-border membranes (lane 1), cerebral microvessels (lane 2), and PPC after 11 days in vitro (lane 3). Immunoincubation with anti-pAPN antibody yielded a single band of 140 kDa in brush-border membranes and cerebral microvessels, but not in PPC (lane 3). (b) Pericyte aminopeptidase N was sensitive to endoglycosidase F digestion. Immunoblot of kidney brush-border membrane with anti-pAPN (lane 1) displays a single band of 140 kDa. Lane 2 exhibits a shift of the band of kidney brush-border membranes after endoglycosidase F digestion. Numbers at left refer to the molecular mass standards (in kDa). Lanes were loaded with 20 µg of protein each.

To address the question of which level the downregulation of pAPN occurred on, we performed RT-PCR with subsequent Southern blotting of the pAPN amplicon (Fig. 5A) and in situ hybridization (Fig. 5B,C) to detect pAPN mRNA. Surprisingly both approaches revealed that mRNA of pAPN is still detectable in PPC and MCS even in long-term cultures (40 to 50 days in vitro, two passages).

FIG. 5.

Detection of pAPN mRNA in long-term cultures of cerebral pericytes (>40 days). Southern blot analysis was performed using a digoxygenin-labeled reverse transcriptase polymerase chain reaction (RT-PCR) fragment amplified from RNA of acute isolated cerebrovascular cells (a). Lane 1 shows the hybridization product with the pAPN amplicon isolated from PPC. The band at about 370 bp documents the existence of pAPN cDNA in PPC in spite of the fact that the protein is no longer detectable (see Fig. 3c). Lane 2 exhibits the hybridization product from MCS. Lane 3 displays the positive control after hybridization of the purified pAPN amplicon with itself. Positions of the cDNA fragment are indicated on the left side in kilobases. In situ hybridization of PPC was performed using an anti-sense cRNA probe for pAPN (b). Hybridized probes were visualized with a fluorescein isothiocyanate (FITC)-coupled secondary antibody. Strong reaction product of the cRNA-antidigoxygenin hybrid is prevalent in the perinuclear region. (c) Negative control after application of corresponding digoxygenin-labeled sense cRNA probe is shown. See Figs. 1 –3 for other abbreviations. Scale bar = 50 µm. Original magnification b and c: ×350.

These findings strongly indicate that the regulation of pAPN expression is most likely to occur at the posttranscriptional level.

One reasonable possibility of posttranscriptional modification during tissue culturing is a change of the glycosylation pattern that is no longer detectable by the mAb. To exclude this possibility we performed endoglycosidase F digestion on kidney brush-border fractions and subjected the digested and undigested samples to Western blotting with the mAb. Kidney brush-border preparations were used to achieve a sufficient amount of digestible APN. As can be seen in Fig. 4B, glycosidase F treatment leads to an expected shift in APN migration but does not exert a loss of binding of the mAb.

Cocultures

Astrocytes have been documented to possess an inductive potency on the expression of BBB-specific features in cultured endothelial and pericytic cells (Maxwell et al., 1987, 1989; Cecchelli et al., 1992). We therefore investigated the effect of coculturing PPC and MCS with astrocytes on pAPN expression. When both preparations were kept in coculture with first-passaged astrocytes, no effect on pAPN expression in pericytes was achieved. In particular, PPC remained negative after 6 days of coculture (Fig. 6A,B), and MCS did not show any change in their low-level expression of pAPN (Fig. 6C—E). The lack of reinduction of pAPN expression in astrocytic cocultures was independent of the culture condition. Neither intimate contact in mixed cultures when both cell types were plated on one side of a glass support nor cultivation with limited cell-cell contact when cells were grown on separate sides of a polycarbonate filter led to a reappearance of pAPN immunodetectability (not shown). Apparently, pAPN expression in cerebral pericytes is not under the control of astrocytic factors.

FIG. 6.

Cocultures of PPC (a and b) and MCS (c-e) with first-passaged astrocytes. (a) A pericyte from PPC (asterisk) grown on the astrocytic monolayer for 3 days reveals no pAPN expression. (b) shows the corresponding GFAP-stained field. Arrowheads point to scattered Dynabeads. (c) Coculture of pericytes from MCS with astrocytes express low levels of pAPN (arrowhead) that do not differ from noncoculture conditions (see Fig. 3e). (d) Corresponding GFAP immunostaining of (c). (e) Phase-contrast view of the same field depicts the morphology of the marked pericyte. Nonspecific autofluorescence is indicated by arrows in (c). See Figs. 1 –3 for other abbreviations. Scale bar = 50 µm. Original magnification ×350.

Alternatively, we used the brain endothelial cell line bEnd.3 for cocultures. In contrast to the astrocytic cocultures, bEnd.3 cells exhibited a significant effect on pAPN reexpression. After 40 days of culture, when MCS were transferred to socio-culture with bEnd.3 cells, a significant increase in the intensity of pAPN immunostaining was achieved (Fig. 7A,B). Frequency of pAPN-positive cells also increased by a factor of 10 as compared with the void MCS. To assess whether this effect was attributable to a humoral factor produced by the bEnd.3 cells, we cultured MCS in bEnd.3-conditioned medium. Conditioning of the medium was performed for 3 days. When MCS were cultured in the conditioned medium, the same effect was achieved as under coculture conditions (Fig. 7C,D).

FIG. 7.

Effect of cocultures of MCS (a-c) with the cerebral endothelial cell line bEnd.3 and after treatment with a bEnd.3-conditioned medium (d). (a) Pericytes originating from MCS display increased pAPN immunoreactivity. (b) Same field as (a) double-immunolabeled with anti-glucose transporter (Glut-1) antibody to visualize bEnd.3 cells. (c) Effect of coculturing MCS and bEnd.3 cells for 2 days without physical cell contact. Cells were seeded on both sides of a coverslip. (d) Pericytes of the MCS treated with bEnd.3-conditioned medium. In all cases significantly higher levels of pAPN expression were achieved as compared with noncoculture conditions of MCS (see Fig. 3e). (e) Pericytes from PPC (asterisks) cocultured with bEnd.3 do not reveal reexpression of pAPN. (f) Corresponding Glut-1 staining of bEnd.3 cells. Arrows point to nonspecific staining. See Figs. 1 –3 for other abbreviations. Scale bar = 50 µm. Original magnification ×350.

Finally, we evaluated whether the in vitro history of the pericytes is essential for the humoral reinducibility of pAPN. For this purpose PPC were cocultured with bEnd.-3 cells. In cocultured pericytes deprived of endothelial cells no expression of pAPN was obtained (Fig. 7E,F). We therefore consider the permanent presence of coisolated cells, presumably endothelial cells, in the MCS responsible for the latent inducibility of pAPN expression in pericytes.

DISCUSSIONS

Evidence is accruing that the BBB is functionally and developmentally constituted by a multicellular complex involving endothelial cells as well as astrocytes, pericytes, and neurons, and that cooperative interaction among these cells is required to guarantee the functional integrity of the BBB (Dermietzel and Krause, 1991). This cooperation becomes most apparent when the maturation of the BBB during brain development is considered.

Morphogenetic factors required for the establishment of blood-brain barrier features

In the embryonal and postnatal brain, maturation of the BBB is considered to be guided by inductive and permissive interactions between pluripotent invading endothelial cells (Bär, 1980) and its neuroectodermal environment (Risau, 1995; Stewart and Wiley, 1981). According to this paradigm, several studies revealed a differentiation of BBB features and the onset of brain-specific endothelial protein expression concurrent with the differentiation of the brain (Qin and Sato, 1995; Dermietzel et al., 1992; Risau et al., 1986a, 1986b). Apparently, the final acquisition of the BBB depends on a morphogenetic program that involves the cooperation of several cellular components. Information on the molecular factors participating in these processes are rare but a recent example dealing with the expression of the vascular endothelial growth factor-2 (VEGFR-2) receptor provides support for the idea that a temporal morphogenetic regimen governs BBB maturation. Although the expression of VEGFR-2 is high during brain angiogenesis, which necessitates the potent mitogenic activity of VEGF, the receptor density is downregulated when BBB maturation is initialized (Plate et al., 1994), a necessary prerequisite for the acquisition of BBB tightness because VEGF is known to be a vascular permeability factor (Millauer et al., 1993; Ferrara et al., 1992).

With respect to the cellular entities primarily involved in BBB induction, astrocytes have been considered to represent the key cellular component, both in situ (Janzer and Raff, 1987) and in vitro (see introduction). However, recent investigations have challenged the simplistic view of astrocytic induction of the BBB. Immature neuroepithelial cells or glial progenitor cells are now considered to be responsible for the initiation of BBB development (Holash et al., 1993).

In this context, the role of pericytes in BBB differentiation and establishment should be reconsidered. Recent in vitro studies have shown that pericytes and endothelial cells of CNS origin have mutual influence on each other's replicative and biosynthetic behavior. Retinal capillary endothelial cells, for example, secrete a heparin-binding factor that promotes pericyte proliferation (Swinscoe and Carlson, 1992). On the other hand, coculturing endothelial cells with pericytes results in the activation of a latent form of transforming growth factor-β1 (TGF-β1) and in the subsequent suppression of endothelial cell growth (Orlidge and D' Amore, 1987). However, TGF-β1 also increases endothelin-1 expression in vascular endothelium, which in turn operates as a pericytic mitogen and causes contraction of pericytes (Chakravarthy et al., 1992). These results suggest complex biologic feedback mechanisms between endothelial cells and pericytes.

Cerebral pericytes, like endothelial cells, are substituted with a specific set of proteins that are functionally involved in the enzymatic component of the BBB. Aminopeptidase A (Healy and Wilk, 1993; Song et al., 1993), γ-glutamyl transpeptidase (Risau et al., 1992), and pAPN (Kunz et al., 1994) are well-studied representatives of the pericytic enzymatic barrier, which is primarily involved in proteolytic activities.

Pericyte aminopeptidase N has recently been revealed to be a late BBB marker in rodent brain development occurring around embryo day 18 of brain angiogenesis (Dermietzel and Krause, 1991). Thus, the final establishment of the pericytic BBB feature can be considered to succeed in most of the endothelial BBB components, in particular, in the achievement of endothelial tightness (Dermietzel and Krause, 1991; Bauer et al., 1992).

In vitro expression of pAPN requires a soluble endothelial factor and depends on coculturing conditions

To gain more information on the inductive mechanisms involved in the expression of this prominent BBB enzyme, we studied the expression of pAPN under various culture conditions.

Downregulation of pAPN occurred within 2 to 3 days when pericytes became adherent. However, as shown by RT-PCR and subsequent Southern blotting, mRNA for pAPN remained detectable even in long-term cultures. This finding strongly suggests a posttranscriptional regulation for pAPN expression in vitro. One reasonable explanation for the loss of detectability of pAPN is a stress-induced switch in the glycosylation pattern of pAPN (O'Connell et al., 1990), resulting in a modified isoform that is no longer accessible to our mAb.

However, this possibility could be ruled out by glycosidase treatment of an APN-enriched membrane fraction. Kidney cells of the proximal tubules that have been shown to express the same isoform of APN (Kunz et al., 1994; Watt and Yip, 1989) showed no loss of binding of the mAb after glycosidase digestion when subjected to Western blotting. These findings are indicative for a persistent recognition of the pAPN epitope by the mAb. It seems therefore more likely that APN gene expression is controlled by posttranscriptional regulation mechanisms, as has been shown recently for the same enzyme in T lymphocytes, natural killer cells, and permanently growing tumor cells (Wex et al., 1997).

By using coculture systems of purified pericytes with both primary astrocytes and immortalized endothelial cells, we further examined the possible inductive and modulating effects of cellular components on pAPN expression. The most important finding of these experiments revealed that pAPN expression is enhanced by one or more soluble endothelial factors. Interestingly, this inductive effect depended on the socio-culture conditions of the pericytes. Only when mixed pericytic cultures were grown in coculture with endothelial cells could pAPN expression be significantly enhanced by endothelial cell-conditioned medium. Purified pericytes grown in complete isolation from endothelial cells revealed no pAPN reexpression potency at all. Although primary astrocytes from neonatal rat brains have been described to induce various endothelial BBB features in vitro (see above), we found no effect on pAPN expression in our cell culture model. A reasonable explanation for this effect is that the differentiation program of pericytes is under control of endothelial cells and, moreover, that a deprivation of pericytes from this supportive “string” leads to an irreversible loss of pericytic differentiation potency. Thus, our experimental design provides stringent in vitro evidence that BBB differentiation requires the cooperation of different vascular cellular components and that the achievement of functionally differentiated cerebral pericytes is closely related to endothelial cells.

Footnotes

Abbreviations used

References

Abbott

Hughes

CCW

Revest

Greenwood

(1992) Development and characterization of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier. J Cell Sci 103:23–37

Audus

Borchardt

(1987) Bovine brain microvessel endothelial cell monolayers as a model system for the blood-brain barrier. Ann NY Acad Sci 507:9–18

Bär

(1980) The vascular system of the cerebral cortex. Adv Anat Embryol Cell Biol 59:1–62

Bauer

Steiner

Bauer

(1992) Embryonic development of the CNS microvasculature in the mouse: new insights into the structural mechanisms of early angiogenesis. EXS 61:64–68

Bausback

Churchill

Ward

(1988) Angiotensin metabolism by cerebral microvascular aminopeptidase A. Biochem Pharmacol 37:155–160

Biber

Stieger

Haase

Murer

(1981) A high yield preparation for rat kidney brush border membranes. Biochim Biophys Acta 647:169–176

Cancilla

Baker

Pollock

Frommes

(1972) The reaction of pericytes of the central nervous system to exogenous protein. Lab Invest 26:376–383

Cecchelli

Dehouck

(1992) Inter-communication between capillary endothelial cells and astrocytes: up-regulation of the LDL receptor at the blood-brain barrier. In: Frontiers in cerebral vascular biology. Transport and its regulation, ( Drewes

Betz

, eds) New York, Plenum Press, p 26

Chakravarthy

Gardiner

Anderson

Archer

Trimble

(1992) The effect of endothelin I on the retinal microvascular pericyte. Microvasc Res 43:241–254

10.

Chomczynski

Sacci

(1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-cholorophorm extraction. Anal Biochem 162:156–159

11.

Crone

Levitt

(1984) Capillary permeability to small solutes. In: Handbook of Physiology, Vol. IV: The Cardiovascular System ( Renkin

Michel

, eds), Bethesda, MD, American Physiological Society, pp 411–466

12.

De la Baume

Schwartz

Chaillet

Marcais-Collado

Costentin

(1983) Participation of both “enkephalinase” and aminopeptidase activities in the metabolism of endogenous enkephalins. Neuroscience 8:143–151

13.

Dehouck

M-P

Fruchart

J-C

Cecchelli

(1994) Upregulation of the low density lipoprotein receptor at the blood-brain barrier: intercommunications between brain capillary endothelial cells and astrocytes. J Cell Biol 126:465–473

14.

Dermietzel

(1975) Junctions in the central nervous system of the cat. IV. Intraendothelial junctions of cerebral blood vessels from selected areas of the brain. Cell Tiss Res 164:45–62

15.

Dermietzel

Krause

(1991) Molecular anatomy of the blood-brain barrier as defined by immunocytochemistry. Int Rev Cytol 127:57–109

16.

Dermietzel

Hertberg

Kessler

Spray

(1991) Gap junctions between cultured astrocytes: immunocytochemical, molecular and electrophysiological analysis. J Neurosci 11:1421–1432

17.

Dermietzel

Krause

Kremer

Wang

Stevenson

(1992) Pattern of glucose transporter (Glut 1) expression in embryonic brains is related to maturation of blood-brain barrier tightness. Dev Dyn 193:152–163

18.

Farrell

Steward

Farrell

Del Maestro

(1987) Pericytes in human cerebral microvasculature. Anat Rec 218:466–469

19.

Ferrara

Houck

Jakeman

Leung

(1992) Molecular and biological properties of the vascular endothelial growth-factor family of proteins. Endocr Rev 13:18–32

20.

Frey

Meckelein

Weiler-Güttier

Mockel

Flach

Gassen

(1991) Pericytes of the brain microvasculature express γ-glutamyl transpeptidase. Eur J Biochem 202:421–429

21.

Gehrmann

Kreutzberg

(1991) Characterization of two new monoclonal antibodies directed against rat microglia. J Comp Neurol 313:409–430

22.

Greenwood

(1991) Astrocytes, cerebral endothelium and cell culture: the pursuit of an in vitro blood-brain barrier. Ann NY Acad Sci 633:426–431

23.

Healy

Wilk

(1993) Localization of immunoreactive glutamyl aminopeptidase in rat brain. II. Distribution and correlation with angiotensin II. Brain Res 606:295–303

24.

Herman

D'Amore

(1985) Microvascular pericytes contain muscle and nonmuscle actins. J Cell Biol 101:43–52

25.

Holash

Noden

Steward

(1993) Re-evaluating the role of astrocytes in blood-brain barrier induction. Dev Dyn 197:14–25

26.

Imayama

Urabe

(1984) Pericytes on the dermal microvasculature of the rat skin. Anat Embryol 169:271–274

27.

Janzer

Raff

(1987) Astrocytes induce blood—brain barrier properties in endothelial cells. Nature 325:253–257

28.

Joo

(1992) The cerebral microvessels in culture, an update. J Neurochem 58:1–17

29.

Jordan

Thomas

(1988) Brain macrophages: questions of origin and inter relationship. Brain Res 472:165–178

30.

Joyce

Haire

Palade

(1985a) Contractile proteins in pericytes. I. Immunoperoxidase localization of tropomyosin. J Cell Biol 100:1379–1385

31.

Joyce

Haire

Palade

(1985b) Contractile proteins in pericytes. II. Immunocytochemical localization of tropomyosin. J Cell Biol 100:1387–1395

32.

Klagsbrun

D'Amore

(1991) Regulators of angiogenesis. Ann Rev Physiol 53:217–23

33.

Koechlin

Pisam

Poujeol

Tauc

Rambourg

(1991) Conversion of a rabbit proximal convoluted tubule (PCT) into a cell monolayer: ultrastructural study of cell dedifferentiation and redifferentiation. Eur J Cell Biol 54:224–236

34.

Krause

Vatter

Dermietzel

(1988) Immunochemical and immunocytochemical characterization of a novel monoclonal antibody recognizing a 140 kDa protein in cerebral pericytes of the rat. Cell Tiss Res 252:543–555

35.

Kunz

Krause

Kremer

Dermietzel

(1994) The 140 kDa protein of blood-brain barrier-associated pericytes is identical to aminopeptidase N. J Neurochem 62:2375–2386

36.

Kunz

Krause

Gehrmann

Dermietzel

(1995) Changes in the expression pattern of blood—brain barrier-associated pericytic aminopeptidase N (pAPN) in the course of acute experimental autoimmune encephalomyelitis. J Neuroimmunol 59:41–55

37.

Le Beux

YVI

Willemot

(1980) Actin- and myosin-like filaments in retinal pericytes and endothelial cells. Invest Ophthalmol Vis Sci 19:1433–1441

38.

Maxwell

Berliner

Cancilla

(1987) Induction of gamma glutamyltranspeptidase in cultured cerebral endothelial cells by a product released by astrocytes. Brain Res 410:309–314

39.

Maxwell

Berliner

Cancilla

(1989) Stimulation of glucose analogue uptake in cerebral microvessel endothelium by a product released by astrocytes. J Neuropathol Exp Neurol 48:69–80

40.

Meyer

Mischek

Vehyl

Henzel

Galla

H-J

(1990) Blood—brain barrier characteristic enzymatic properties in cultured brain capillary endothelial cells. Brain Res 514:305–309

41.

Meyer

Rauh

Galla

H-J

(1991) The susceptibility of cerebral endothelial cells to astroglial induction of blood—brain barrier enzymes depends on their proliferate state. J Neurochem 57:1971–1977

42.

Millauer

Wizigmann-Voos

Schnurch

Martinez

Moller

NPH

Risau

Ullrich

(1993) High affinity VEGF binding and developmental expression suggests Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 72:835–846

43.

Minuth

Stöckl

Kloth

Dermietzel

(1992) Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions. Eur J Cell Biol 57: 132–137

44.

Mizuguchi

Hashioka

Fujii

Utoguchi

Kubo

Nakagawa

Baba

Mayumi

(1994) Glial extracellular matrix modulates γ-glutamyl transpeptidase activity in cultured bovine brain capillary and bovine aortic endothelial cells. Brain Res 651:155–159

45.

Mizutani

Goto

Nomur

Ino

Kikkawa

Kurauchi

Goldstein

Tomoda

(1993) Possible action of human placental aminopeptidase N in feto-placental unit. Res Commun Chem Pathol Pharmacol 82:65–80

46.

Montesano

Pepper

Möhle-Steinlein

Risau

Wagner

Orci

(1990) Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene. Cell 62:435–445

47.

Mrsulja

Fujimoto

Klatzo

Spatz

(1976) Isolation of brain capillaries: a simplified technique. Brain Res 110: 361–365

48.

Nehls

Drenckhahn

(1993) The versatility of microvascular pericytes: from mesenchyme to smooth muscle? Histochem 99:1–12

49.

Nehls

Denzer

Drenckhahn

(1992) Pericyte involvement in capillary sprouting during angiogenesis in situ. Cell Tiss Res 270: 469–474

50.

Nehls

Schuchardt

Drenckhahn

(1994) The effect of fibroblasts, vascular smooth muscle cells, and pericytes on sprout formation of endothelial cells in a fibrin gel angiogenesis system. Microvasc Res 48:349–363

51.

O'Connell

Gerkis

D'Apice

AJF

(1990) Variable O-glycosylation of CD 13 (aminopeptidase N). J Biol Chem 266:4593–4597

52.

Orlidge

D' Amore

(1987) Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells. J Cell Biol 105: 1455–1462

53.

Pakaski

Kasa

(1992) Glial cells in coculture can increase the acetylcholinesterase activity in human brain endothelial cells. Neurochem Int 21:129–133

54.

Plate

Breier

Weich

Mennel

Risau

(1994) Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int J Cancer 59:520–529

55.

Qin

Sato

(1995) Mouse multidrug resistance '1a/3 gene is the earliest known endothelial cell differentiation marker during blood-brain barrier development. Dev Dyn 202: 172–180

56.

Reese

Karnovsky

(1967) Fine structural localization of a blood—brain barrier to exogenous peroxidase. J Cell Biol 34:207–217

57.

Risau

(1995) Differentiation of endothelium. FASEB J 9:926–933

58.

Risau

Hallmann

Albrecht

(1986a) Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier. Dev Biol 117:537–545

59.

Risau

Hallmann

Albrecht

Henke

(1986b) Brain induces the expression of an early cell surface marker for blood—brain barrier—specific endothelium. EMBO J 5:3179–3183

60.

Risau

Dingler

Albrecht

Dehouck

M-P

Cecchelli

(1992) Blood—brain barrier pericytes are the main source of γ-glutamyltranspeptidase activity in brain capillaries. J Neurochem 58:667–672

61.

Roberts

Fine

Sandra

(1993) Receptor-mediated endocytosis of transferrin at the blood brain barrier. J Cell Sci 104:521–532

62.

Roux

Durieu-Trautmann

Chaverot

Claire

Mailly

Bourre

J-M

Strosberg

Couraud

P-O

(1994) Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain micro vessel endothelial cells. J Cell Physiol 159:101–113

63.

Rubin

Hall

Porter

Barbu

Cannon

Horner

Janatpour

Liaw

Manning

Morales

Tanner

Tomaselli

Bard

(1991) A cell culture model of the blood—brain barrier. J Cell Biol 115:1725–1735

64.

Rutten

Hoover

Karnovsky

(1987) Electrical resistance and macromolecular permeability of brain endothelial monolayer cultures. Brain Res 425:301–310

65.

Sambrook

Fritsch

Maniatis

(1989) Molecular cloning. A Laboratory Manual. New York, Cold Spring Harbor, pp 9.38–9.40

66.

Schor

(1986) The isolation and culture of endothelial cells and pericytes from the bovine retinal microvasculature: a comparative study with large vessel vascular cells. Microvasc Res 32:21–38

67.

Schor

Allen

Canfield

Sloan

Schor

(1990) Pericytes derived from the retinal microvasculature undergo calcification in vitro. J Cell Sci 97:449–461

68.

Song

Wilk

Healy

(1993) Localization of immunoreactive glutamyl aminopeptidase in rat brain. I. Association with cerebral microvessels. Brain Res 606:286–294

69.

Stewart

Wiley

(1981) Developing nervous tissue induces formation of blood-brain barrier characteristics in invading cells: a study using quail-chick transplantation chimeras. Dev Biol 84: 183–192

70.

Sun

Lytle

O'Donnell

(1995) Astroglial cell-induced expression of Na-K-Cl cotransporter in brain microvascular endothelial cells. Am J Physiol 269:C1506–C1512

71.

Swinscoe

Carlson

(1992) Capillary endothelial cells secrete a heparin-binding mitogen for pericytes. J Cell Sci 103:453–461

72.

Verhoeven

Buyssens

(1988) Desmin-positive stellate cells associated with angiogenesis in a tumour and non-tumour system. Virch Arch 54:263–272

73.

Wang

(1987) The d-glucose transporter is tissue specific. J Biol Chem 262: 15689–15695

74.

Watt

Yip

(1989) Amino acid sequence deduced from a rat kidney cDNA suggests it encodes the Zn-peptidase aminopeptidase N. J Biol Chem 264:5480–5487

75.

Wex

Bühling

Arndt

Frank

Ansorge

Lendeckel

(1997) The activation-dependent induction of APN-(CD13) in T-cells is controlled at different levels of gene expression. FEBS Lett 412: 53–56