Effect of astroglial cells on hypoxia-induced permeability in PBMEC cells

Silvia Fischer1, Maria Wobben2, Jan Kleinstück2, Dieter Renz1, and Wolfgang Schaper2

1 Department of Anesthesiology and Intensive Care and 2 Department of Experimental Cardiology, Max-Planck Institute for Physiological and Clinical Research, D-61231 Bad Nauheim, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An in vitro model of the blood-brain barrier (BBB), consisting of porcine brain-derived microvascular endothelial cells (PBMEC), was used to evaluate the effect of astrocytes in the BBB disruption during hypoxia. Hypoxia-induced hyperpermeability was decreased significantly in a coculture model of astroglia cells, either astrocytes or C6 glioma cells, with PBMEC and, to the same extent, when glia cell-conditioned medium was used. Corresponding to effects on hypoxia-induced hyperpermeability, astrocyte- and C6 cell-conditioned medium diminished hypoxia-induced vascular endothelial growth factor (VEGF) mRNA and protein expression, which recently was shown to be responsible for hypoxia-induced permeability changes in vitro. The effect on hypoxia-induced hyperpermeability and VEGF expression was specific for astroglia cells because conditioned medium from bovine smooth muscle cells (BSMC) did not show any effect. Immunocytochemistry revealed that 24 h of hypoxia disrupted the continuity of the tight junction protein, zonula occludens-1 (ZO-1), which lines the cytoplasmic face of intact tight junctions. These changes were prevented when hypoxia was performed in glia cell-conditioned medium. Results suggest that astrocytes protect the BBB from hypoxia-induced paracellular permeability changes by decreasing hypoxia-induced VEGF expression in microvascular endothelial cells.

porcine brain microvascular endothelial cells; vascular endothelial growth factor; astrocytes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE BLOOD-BRAIN BARRIER (BBB) forming a barrier between the circulatory system and the central nervous system is relatively impermeable to ions, many amino acids, small peptides, and proteins. In vertebrates, the BBB exists at the level of the endothelial cells that make up brain capillaries (9). The barrier properties of the BBB result from the absence of fenestrations and the presence of tight intercellular junctions between endothelial cells with extremely high electrical resistance values, which limits the amount of paracellular flux (18). Furthermore, brain endothelial cells undergo a relatively slow rate of fluid-phase endocytosis, as assayed by the uptake of tracer molecules, such as horseradish peroxidase (47). To examine cellular events at the molecular level, cultured brain microvessel endothelial cells were used as model of the BBB. Numerous studies show that porcine brain-derived microvascular endothelial cells (PBMEC) exhibit many in vivo-like qualities in culture, although cultured PBMEC monolayers are considerably more "leaky" than the BBB capillary endothelium in vivo (22, 52, 53).

It is now well established that the signals that induce endothelial cells to express the BBB phenotype result from specific interactions between capillary endothelial cells and brain parenchyma (48, 57) or, more specifically, between capillary endothelial cells and the surrounding perivascular astrocytes (5, 21, 35, 61). Also, the formation of tight junctions between PBMECs is supposed to be maintained by astroglial cells (2, 52, 60). However, the detailed role of astroglial cells in the formation or maintenance of tight junctions between endothelial cells remains to be elucidated, especially in pathological conditions leading to BBB disruption, such as hypoxia/ischemia.

Continuous microvessel endothelial cells are central to the integrity of the BBB, and many pathological conditions like local ischemia or brain tumors lead to an opening of the BBB through transcellular or paracellular pathways (1), which may lead to the development of vasogenic brain edema (3). A likely candidate for the development of ischemia-induced vasogenic brain edema is vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF). VEGF stimulates cell growth and migration in vitro (25, 54), angiogenesis in vivo (16, 39), and increases the vascular permeability (23, 50, 55, 65, 67). VEGF-induced endothelial hyperpermeability is caused by a direct action on endothelial cells (33) and is suggested to mediate enhanced transcytosis and gap formation between endothelial cells (15, 17) and to induce fenestrations in nonfenestrated endothelium (50, 51). VEGF-increased permeability is known to occur in chronic inflammation (20), during wound healing (44), in diabetic rat retinal vessels (43), in tumors, where VEGF production is localized to ischemic areas (45, 56), and during high-altitude edema (66).

We have demonstrated in previous studies that hypoxia-induced permeability in vitro is mediated by the VEGF/VEGF receptor system in an autocrine manner via the release of nitric oxide (NO) (26). The release of NO alone is not sufficient to induce hyperpermeability and requires conditions stabilizing the second messenger NO, like hypoxia or the presence of antioxidants. In this study, the role of astroglia cells to influence permeability and VEGF expression during hypoxia of PBMEC will be investigated, because there is still no knowledge about the mechanism of the effect of glia cells on hypoxia-induced permeability changes. Until now, astrocytes have been shown to have a protective effect to the BBB disruption following ischemia/reperfusion probably by a radical scavenging mechanism (36). Because it is possible that cell-cell communication exists in both directions (31), the noncontact coculture method was used where glia cell-conditioned medium (glia cell CM) is conditioned either continually (46) or glia cell CM is added to PBMEC cultures. This report demonstrates that hypoxia-induced hyperpermeability is decreased in response to coculture with rat brain astrocytes or cells of the C6 line, which display astrocytic properties (8, 37). Corresponding to the effect of glia cell-conditioned medium on hypoxia-induced hyperpermeability, the VEGF expression is decreased.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Capillary endothelial cells were isolated from porcine brains as described previously (42). The gray matter was digested with dispase II (0.8 mg/100 ml) for 3 h at 37°C. Capillaries were isolated after centrifugation with 15% dextran. The pellet was resuspended in medium M199 and treated with collagenase/dispase (1 mg/ml) for 5 h at 37°C. Endothelial cells were obtained after Percoll gradient centrifugation. Cells were seeded on Biocoat petri dishes (Becton-Dickinson, Heidelberg, Germany) or on rat tail collagen I-coated (Becton-Dickinson) polycarbonate transwell inserts with a pore size of 0.4 µm and a diameter of 12 mm (Millipore, Eschborn, Germany) and incubated at 37°C in a 5% CO2-humidified incubator. The cells were grown in M199 medium supplemented with 20% (vol/vol) FCS, 200 U/ml penicillin, 200 U/ml streptomycin, and 2.5 µg/ml amphotericin B. All ingredients were purchased from Life Technologies (Eggenstein, Germany). After 7 days, the primary cultures formed confluent monolayers. Cells were characterized by analyzing endothelial marker enzymes like alkaline phosphatase and gamma -glutamyltransferase activity using assay kits from Sigma (Munich, Germany). Furthermore, the uptake of acetylated low-density lipoprotein (Dil-Ac-LDL; Paesel and Lorei, Frankfurt, Germany) (63) and the expression of the glucose transporter I were used as a marker for cerebral endothelial cells. Pericytes and astrocytes were characterized by immunofluorescence staining with anti-alpha -smooth muscle actin (Sigma) and anti-glial fibrillary acidic protein (GFAP; Boehringer, Mannheim, Germany) antibodies. Cultures used for our experiments contained less than 5% pericytes. Cells of astroglial origin could only be detected in some cultures, but their content was always less than 1%.

Neonatal rat forebrain astrocytes were isolated as described (22). Briefly, cerebral hemispheres were removed from 1- to 2-day-old rats, cleaned of meninges and choroid plexus, chopped, and digested with trypsin. After centrifugation, the cell pellet was resuspended in BME medium containing 10% (vol/vol) FCS and antibiotics. At confluence, oligodendroglia cells were removed by shaking, and astrocytes were passaged by trypsinization. For the described experiments, cells of the third to fourth passages were used. These cultures were ~95% GFAP positive by indirect immunofluorescence.

C6 glioma cells (6) obtained from the American Type Culture Collection (ATCC) were cultured in M199 medium containing 10% (vol/vol) FCS and antibiotics.

Bovine smooth muscle cells (BSMC) were isolated and cultured as described (24).

Preparation of CM. The medium of confluent cultures of astrocytes or C6 cells was replaced by fresh one without FCS. Cells were either cultured for 24 h of normoxia (normoxic CM) or hypoxia (hypoxic CM). The CM was harvested, filtrated with a sterile 0.2 µm filter, and stored frozen at -20°C until use.

Cocultures. The influence of astrocytes and C6 cells on PBMEC monolayer integrity was studied using the noncontact coculture method described by Raub and coworkers (46). Astrocytes or C6 cells were trypsinized, resuspended in culture media, and plated at 6,000 cells/cm2 on the bottom of 24-well culture plates. After 2-3 days, when cells reached confluence, filter inserts with freshly seeded PBMEC were placed into the wells, and coculture was continued for 2-4 days or longer until PBMEC reached confluence.

In vitro hypoxia model. To induce hypoxia, confluent monolayers of PBMEC were washed once with PBS, pH 7.4. M199 medium without FCS or CM was added, and culture plates were placed into special chambers equipped with a thermostat housing that allowed us to incubate the chambers at 37°C. Chambers were incubated at 37°C either under normoxic or hypoxic conditions (2% oxygen) by gassing the special chamber with a gas mixture consisting of 95% air-5% CO2 or 93% N2-2% O2-5% CO2. For control, the concentration of oxygen in the culture incubation medium after different time periods of hypoxia was determined using a Digital O2 meter (Schott Geräte). The respiratory activity of cells did not change the oxygen content in the culture medium significantly. The pH of the medium was unchanged during up to 24 h of hypoxia. For control, cultures were incubated under normoxic conditions for the same length of time.

Assay of endothelial monolayer permeability. The tightness of the PBMEC monolayer and the effect of coculture of PBMEC with astrocytes or C6 glioma cells on monolayer integrity was determined by measurements of the transendothelial resistance (TER) as described previously (28). PBMECs showing resistance values of more than 80 Omega  · cm2 were used for the evaluation of the effects of hypoxia on the permeability of the PBMEC monolayer. For that purpose, the passage of [3H]inulin (Amersham, Buchler, Germany) across the PBMEC monolayer during normoxia and hypoxia was measured, because this method is more sensitive to detect even small changes of the permeability than measurements of the TER. The chambers consisting of the apical part containing the filter membrane inserts with the cell monolayer and the basolateral part were washed three times with PBS, and 0.146 nmol (i.e., 0.8 µCi) [3H]inulin in 400 µl M199 medium without serum or astrocyte CM or C6 cell CM were added into the apical and 600 µl of medium or CM into the basolateral chamber followed by incubation under normoxic or hypoxic conditions at 37°C. The appearance of [3H]inulin in the basolateral chamber was measured after different time periods by scintillation counting of small aliquots of the basolateral buffer. Results were expressed as the ratio of the inulin concentration determined after different incubation times in the lower chamber and the total concentration of [3H]inulin added into the upper chamber at the start of the experiment (cR/ctotal). Permeability changes were measured up to 6 h of hypoxia, because the PBMEC monolayers used always showed some leakiness to inulin and equilibrium between the basolateral and upper chamber was reached after 8 to 14 h of hypoxia.

During the course of the experiment, chambers were kept at 37°C and care was taken to ensure that fluid levels in the apical and basolateral chambers were equal (cR/ctotal). The amount of inulin that crossed the cell-free membrane was not changed during hypoxia or in the presence of astrocyte CM or C6 cell CM.

Extraction of total cellular RNA and Northern blot analysis. When cells reached confluence, plates were washed once with PBS and cultured either in M199 medium or in astrocyte CM or C6 cell CM under normoxic or hypoxic conditions for the indicated time periods. For the isolation of RNA, cells were washed once with PBS and harvested directly into guanidinium-thiocyanate buffer, and then RNA was isolated as described by Chomczynski and Sacchi (12). For Northern blot analysis, 15 µg of total RNA was denatured at 65°C in formamide and ethidium bromide containing loading buffer and subsequently electrophoresed on a 1% (wt/vol) agarose gel containing 2.2 M formaldehyde. RNA was transferred to a Hybond-N membrane by vacuum blotting and ultraviolet cross-linked. Filters were prehybridized at 42°C for 3 h in 50% (vol/vol) formamide, 6× SSC (1× SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 1 M NaCl, 1 mg/ml denatured salmon sperm DNA, and 5% (wt/vol) dextran sulfate. A fragment of the human VEGF cDNA (kindly supplied by Dr. H. Weich) was labeled with use of a multiprime labeling kit (Amersham) to a specific activity of 1 × 109 cpm/µg DNA. Denatured labeled DNA probe was added to the prehybridization buffer to a final concentration of 1 × 106 cpm/ml and incubated overnight. Filters were washed twice in 2× SSC-0.1% SDS at room temperature for 15 min each and once at 42°C for 20 min and then exposed to Kodak XAR films at -70°C for 1-4 days. An 18S cDNA probe (kindly supplied by Dr. I. Oberbäumer) was used to rehybridize membranes for reference purposes. Quantitative analysis was performed using a PhosphorImager SF (Molecular Dynamics). VEGF shows two mRNA transcripts of 3.9 and 1.7 kb. Because all the treatments used in these studies affect the expression of both VEGF transcripts to the same extent and because the expression of the lower band sometimes is very low, only the mRNA expression at 3.9 kb was used for the quantitative analysis. To correct for differences in RNA loading, the signal intensity for each sample hybridized to the VEGF cDNA probe was divided by that for each sample hybridized to the 18S cDNA probe.

Determination of VEGF levels in cultured media. After culturing PBMEC for different time periods either in M199 medium or in astrocyte CM or C6 cell CM under normoxic or hypoxic conditions, the medium was collected, concentrated, and desalted using Centriprep columns with a cut off of 10,000 (Amicon, Beverly, MA). Protein was determined using the Coomassie protein assay kit (Pierce, Rockford, IL). The VEGF concentrations of the CM were measured by the enzyme-linked immunosorbent assay (ELISA; Tebu, Frankfurt, Germany). The ELISA used detects all known isoforms of VEGF molecules. The protein content in each dish or well corrected all the measurements.

Measurements of cGMP concentrations. When cells reached confluence, plates were washed once with PBS and cultured in either M199 medium or in astrocyte CM or C6 cell CM under normoxic or hypoxic conditions. cGMP was extracted from the cells by rapid aspiration of medium and washed with ice-cold PBS, and ice-cold ethanol was added to 65% (vol/vol). The cells were harvested and centrifuged at 2,000 g for 10 min at 4°C. Supernatants were transferred to fresh tubes and air-dried. The cGMP concentration of the cell extracts was determined by using a cGMP 3H assay system from Amersham. cGMP concentration was normalized to protein content as determined by using the Coomassie protein assay kit (Pierce).

Immunofluorescence. PBMEC were grown either on normal Biocoat petri dishes or on plastic slides. For staining, monolayers were washed twice with PBS, fixed in 1% paraformaldehyde at 4°C for 15 min, washed with PBS, permeabilized with 0.05% Triton for 10 min, and washed again five times with PBS. Samples were blocked with 10% normal goat serum (Sigma) for 30 min at room temperature, followed by incubation with rabbit-polyclonal anti-zonula occludens-1 (ZO-1) (1:100; Dianova, Hamburg, Germany). After washing with PBS, goat anti-rat-Cy3 (1:400; Dianova) was added, and cells were incubated for 1 h at room temperature. After washing with PBS, nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) for 1 min. The samples were viewed with a fluorescent photomicroscope (Leica, Bensheim, Germany).

Statistical analysis. Results were expressed as the means ± SE. The unpaired Student's t-test or ANOVA and subsequent multiple comparisons using the Scheffé method were used for statistical analysis. Results were considered as statistically different at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Coculture of PBMEC with glia cells prevented hypoxia-induced permeability changes. The influence of astrocytes and C6 glia cells on PBMEC monolayer integrity was determined after 3-6 days of coculturing by measurements of the TER, which provides a method for the estimation of the ion permeability (13). Astrocytes induced the TER from 104 ± 7 to 219 ± 20 Omega  · cm2 after 4 days of coculture. C6 glia cells showed nearly the same effect and increased the TER to 180 ± 12 Omega  · cm2 under the same culture conditions. Hypoxia-induced permeability changes were determined by the measurement of the passage of inulin across the endothelial cell membrane after different times of hypoxia in the absence or presence of cocultured astrocytes and C6 glioma cells. Hypoxia increased the permeability to inulin of PBMEC monolayers significantly, whereas in cocultures no significant changes were measured determined up to 6 h of hypoxia. Earlier studies already revealed that viability of PBMEC is not changed during hypoxia (29). During normoxia, the paracellular passage of inulin was not changed by soluble factors released from astrocytes or C6 glia cells (Fig. 1). To evaluate whether the effect of glia cell CM on hypoxia-induced permeability changes is specific for these cells, the permeability during 6 h of normoxia and hypoxia was determined in the presence of cocultured BSMC. Under these conditions, hypoxia-induced hyperpermeability of PBMEC was unchanged.


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Fig. 1.   A: permeability of the porcine brain-derived microvascular endothelial cell (PBMEC) monolayer to [3H]inulin during 1.5 (top), 3 (middle), and 6 h (bottom) of normoxia (N) and hypoxia (H) in the absence and presence of astrocytes or C6 glioma cells. PBMEC were cultured for 1.5, 3, and 6 h at 37°C under normoxia and hypoxia in the absence (C) or in coculture with astrocytes (AS) or C6 cells (C6). B: permeability of the PBMEC monolayer to [3H]inulin during 6 h of normoxia and hypoxia in the absence and presence of bovine smooth muscle cells (BSMC). PBMEC were cultured for 6 h at 37°C under normoxia and hypoxia in the absence (C) or in coculture with BSMC (BSMC). The amount of [3H]inulin that passed across the PBMEC monolayer after 1, 5, 3, or 6 h under normoxic conditions divided by the total concentration of [3H]inulin added to the upper chamber at the start of the experiment (cR/ctotal) was set to 100% (C). Values are means ± SE; n = 15. *P < 0.05 vs. C in the absence of other cells.

Similar effects on hypoxia-induced permeability changes were obtained when hypoxia of PBMEC was performed using astrocyte CM or C6 glia cell CM instead of coculturing PBMEC with glia cells. Because a lot of growth factors and cytokines are produced during hypoxia, CM, which was collected after 24 h of hypoxia from astrocytes or C6 glia cells, was also used. No significant difference between hypoxia-induced permeability changes in the presence of CM from normoxic and hypoxic glia cells was determined (Fig. 2). Because the effects of continually conditioned medium by coculturing glia cells with PBMEC and of glia cell CM on hypoxia-induced permeability changes are not significantly different from each other, the following experiments were performed after adding glia cell CM.


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Fig. 2.   Permeability of the PBMEC monolayer to [3H]inulin during 6 h of normoxia and hypoxia in the absence and presence of astrocyte-conditioned (A) and C6 cell-conditioned (B) medium. PBMEC were cultured for 24 h at 37°C under normoxia and hypoxia in the absence (C) or presence of normoxic and hypoxic astrocyte CM or C6 cell CM [AS(N), AS(H), C6(N), and C6(H), respectively]. The amount of [3H]inulin that passed across the PBMEC monolayer after 6 h under normoxic conditions divided by the total concentration of [3H]inulin added to the upper chamber at the start of the experiment (cR/ctotal) was set to 100% (C). Values are means ± SE; n = 15. *P < 0.05 vs. C in the absence of astrocyte CM or C6 cell CM. CM, conditioned medium.

Hypoxia-induced VEGF expression is decreased by glia cell-derived CM. Earlier studies revealed that hypoxia-induced hyperpermeability is mediated by the VEGF/VEGF receptor system in an autocrine manner via the release of NO (26). Therefore, the effect of astrocyte CM and C6 cell CM on the hypoxia-induced VEGF expression was investigated. As shown in Fig. 3, the hypoxia-induced increase in VEGF mRNA expression was abolished by astrocyte CM as well as by C6 glioma cell CM. There was no difference whether glia cell CM harvested after 24 h of normoxia or hypoxia was used. During normoxia, astrocyte CM as well as C6 glioma cell CM did not change the VEGF mRNA expression significantly. Accordingly, hypoxia-induced increase in VEGF protein was abolished by glia cell CM (Fig. 4).


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Fig. 3.   A: Northern blot analysis (top) of total RNA extracted from PBMEC after 24 h of normoxia and hypoxia in the absence and presence of astrocyte CM. Cultures of PBMEC were cultured for 24 h at 37°C under normoxia and hypoxia in the absence (C) or presence of normoxic and hypoxic astrocyte CM [AS(N) and AS(H)], followed by RNA isolation and Northern blotting as described in MATERIALS AND METHODS. Human vascular endothelial growth factor (VEGF) cDNA fragment was used as probe. Same blot was stripped and hybridized with an 18S cDNA probe. Bottom: quantitative analysis of VEGF mRNA expression after 24 h of normoxia and hypoxia in the absence and presence of astrocyte CM. Quantitative analysis of Northern blots was performed using a PhosphorImager. VEGF mRNA expression was normalized to corresponding expression of 18S mRNA. Value determined after 24 h of normoxia in the absence of astrocyte CM was set to 100% (C). Values are means ± SE; n = 5. *P < 0.05 vs. C in the absence of astrocyte CM. B: Northern blot analysis (top) of total RNA extracted from PBMEC after 24 h of normoxia and hypoxia in the absence and presence of C6 cell CM. Cultures of PBMEC were cultured for 24 h at 37°C under normoxia and hypoxia in the absence (C) or presence of normoxic and hypoxic astrocyte CM [C6 (N), C6 (H)]. Northern blotting and quantitative analysis (bottom) was performed as described in A. Value determined after 24 h of normoxia in the absence of C6 cell CM was set to 100% (C). Values are means ± SE; n = 5. *P < 0.05 vs. C in the absence of C6 cell CM.



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Fig. 4.   Quantitative analysis of the amount of VEGF released into the culture medium after culturing PBMEC for 24 h of normoxia and hypoxia in the absence and presence of astrocyte-conditioned (A) and C6 cell-conditioned (B) medium. PBMEC were cultured for 24 h at 37°C under normoxia and hypoxia in the absence (C) or presence of normoxic and hypoxic astrocyte CM or C6 cell CM [AS(N), AS(H), C6(N), and C6(H), respectively], followed by collecting the culture medium. The amount of VEGF released into the culture medium was determined using ELISA techniques as described in MATERIALS AND METHODS. Value determined after 24 h of normoxia in the absence of astrocyte CM or C6 cell CM was set to 100% (C). Values are means ± SE; n = 5. *P < 0.05 vs. C in the absence of astrocyte CM or C6 cell CM.

To evaluate whether the effect of glia cell CM on the VEGF expression is specific for these cells, the same experiments were performed using CM from BSMCs. Quantitative analysis of Northern blots revealed that BSMC CM did not show any effect on the VEGF mRNA expression during normoxia and hypoxia. Accordingly, the protein expression was not changed by BSMC CM (Fig. 5).


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Fig. 5.   Quantitative analysis of VEGF mRNA (A) and VEGF protein (B) expression after 24 h of normoxia and hypoxia in the absence and presence of BSMC CM. PBMEC were cultured for 24 h under normoxia or hypoxia in the absence (C) or presence of BSMC CM (BSMC). Northern blotting and determination of the amount of VEGF released into the culture medium were performed as described in MATERIALS AND METHODS and according to Fig. 3 and 4. Value determined after 24 h of normoxia in the absence of BSMC CM was set to 100% (C). Values are means ± SE (n = 5). *P < 0.05 vs. C in the absence of BSMC CM.

Hypoxia-induced NO release is decreased by glia cell-derived CM. Previous studies revealed that VEGF induces permeability in PBMEC via a signaling cascade involving NO synthesis (26). To confirm the effect of glia cell CM on the VEGF expression during hypoxia, the release of NO should be inhibited to the same extent. NO was measured by the activity of the guanylate cyclase. The amount of cGMP during hypoxia was reduced by astrocyte CM as well as by C6 glioma cell CM, harvested after 24 h of normoxia and hypoxia, to values determined during normoxia (Fig. 6).


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Fig. 6.   cGMP concentration in PBMEC cultures after culturing PBMEC for 24 h of normoxia and hypoxia in the absence and presence of astrocyte CM (A) and C6 cell CM (B). PBMEC were cultured for 24 h at 37°C under normoxia and hypoxia in the absence (C) or presence of normoxic and hypoxic astrocyte CM or C6 cell CM [AS(N), AS(H), C6(N), and C6(H), respectively]. cGMP was determined and normalized to the corresponding protein value as described in MATERIALS AND METHODS. Value determined after 24 h of normoxia in the absence of astrocyte CM or C6 cell CM was set to 100% (C). Values are means ± SE; n = 9. *P < 0.05 vs. C in the absence of astrocyte CM or C6 cell CM.

Exogenous VEGF did not change hypoxia-induced VEGF mRNA expression. Because glia cells are known to produce VEGF, the effect of exogenous VEGF on VEGF expression in PBMEC was investigated. Exogenous VEGF was added to PBMEC cultures during normoxia as well as during hypoxia, and VEGF mRNA expression was determined. Quantitative analysis of Northern blots revealed that exogenous VEGF did not alter the VEGF mRNA expression during normoxia as well as during hypoxia (Fig. 7).


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Fig. 7.   Quantitative analysis of VEGF mRNA expression after 24 h of normoxia and hypoxia in the absence and presence of exogenous VEGF. PBMEC were cultured for 24 h under normoxia and hypoxia in the absence (C) or presence of VEGF (5 ng/ml). Quantitative analysis of Northern blots was performed using a PhosphorImager. VEGF mRNA expression was normalized to corresponding expression of 18S mRNA. Value determined after 24 h of normoxia in the absence of VEGF was set to 100% (C). Values are means ± SE; n = 5. *P < 0.05 vs. C in the absence of VEGF.

Hypoxia-induced changes of the distribution pattern of tight junction protein ZO-1 were reversed by glia cell-derived CM. Because inulin uses the paracellular route to cross the PBMEC monolayer, we evaluated whether hypoxia is changing the expression or distribution of tight junction proteins. Normoxic cells showed a regular, linear labeling of ZO-1 at the cell periphery, which was not changed by C6 cell CM. Hypoxic cells have a less regular ZO-1 immunoreactivity at regions of cell-cell contact, which became regular again in the presence of C6 cell CM (Fig. 8).


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Fig. 8.   Immunolocalization of zonula occludens (ZO-1) in confluent PBMEC. Cells were grown for 24 h under normoxia in the absence (A), or presence of C6 cell CM (B), or under 24 h of hypoxia in the absence (C), or in the presence of C6 CM (D). Bar = 10 µm. Results are representative of 4 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is now well established that many of the unique features that are characteristic of PBMEC are induced by astrocytes (31, 49). Among these astrocyte-induced characteristics of PBMECs are changes in the permeability of endothelial cell monolayers, which are attributed primarily to structural changes in tight junctions (10, 46, 52, 64). However, there are still controversial reports of whether astrocyte-secreted factors are involved or whether cell-cell contact is required. Some studies show that these changes require PBMEC-astrocyte contact (4, 41), whereas others found that a secreted factor or factors enhance the junction formation between brain-derived endothelial cells (46, 52). Although in vivo astrocyte foot processes are intimately associated with PBMEC, they are separated by a basement membrane, and no direct cell-cell contact has been described, which suggests that a soluble signal(s) is required to induce special features of the brain endothelium. Therefore, in the present study, either a coculture model is used, where glia cells and PBMEC have no direct contact, or glia cell CM is added to PBMEC cultures. Coculture of astrocytes or C6 glioma cells with PBMEC increased the transcellular resistance across the PBMEC monolayer about twofold, which is in agreement with results of Rubin et al. (52). Furthermore, hypoxia-induced permeability changes of PBMEC were decreased significantly in the presence of astrocytes and C6 glioma cells to the same extent. The finding that astrocytes and glioma cells evoke similar effects is in agreement with several other studies (40, 58). The same effect on hypoxia-induced permeability changes was determined when normoxic, but also hypoxic, glia cell CM was used. This suggests that probably glia cell-secreted factors, whose expression is induced during hypoxia, are not involved in decreasing hypoxia-induced hyperpermeability. Until now, only few studies are known investigating the effect of glia cells on hypoxia-induced permeability changes, like that of Kondo et al. (36), who demonstrated that astroglial cells inhibit the increasing permeability of brain endothelial cell monolayer following hypoxia/reoxygenation. They suggest that the astrocytes may eliminate free radicals released from endothelial cells following hypoxia/reoxygenation. Oxygen radicals are preferentially generated during the phase of reoxygenation, and, since earlier studies revealed that no radicals are released during hypoxia of PBMEC (28), the protective role of glia cells in our model probably is not related to a radical scavenger function.

Because hypoxia-induced hyperpermeability is decreased by glia cell CM, hypoxia-induced expression of one or more permeability-increasing agents may be reduced by glia cell-secreted factors. Recently, it was demonstrated that hypoxia-induced permeability changes in vitro are mediated by VEGF in an autocrine manner via the release of NO (26). NO only increases the permeability under conditions stabilizing the second messenger NO, like those during hypoxia or in the presence of antioxidants. Our results demonstrate that astrocyte CM as well as C6 glia cell CM decreased the hypoxia-induced VEGF mRNA and protein expression significantly. During normoxia, the VEGF expression was unchanged by glia cell CM. There are several reports showing hypoxia-induced VEGF expression in astrocytes and C6 glia cells (34, 38, 56). However, normoxic glia cell CM decreased the hypoxia-induced VEGF expression to the same extent as hypoxic CM, suggesting that a self-regulated mechanism to reduce hypoxia-induced VEGF expression is not involved. This was confirmed by our result that the addition of exogenous VEGF during hypoxia did not reduce hypoxia-induced VEGF mRNA expression in PBMEC. The effect on the hypoxia-induced VEGF expression was specific for astrocytes and C6 glioma cells, because BSMC CM did not change the VEGF expression. There are studies in progress to evaluate more precisely the mechanism of glia cell CM on the VEGF expression. The previous studies suggest that glia cells decrease hypoxia-induced permeability changes by inhibiting VEGF expression. Earlier studies demonstrated that hypoxia up to 3 h upregulates VEGF mRNA expression posttranscriptionally by enhancing the stability of the VEGF mRNA (27). Nevertheless, the mechanism of hypoxia to induce VEGF expression may be changed during longer times of hypoxia. Therefore, studies will be performed to evaluate whether the half-life of VEGF mRNA or the transcriptional activation of the VEGF gene during longer times of hypoxia is changed in the presence of astroglial cell CM. Astroglial factors may induce the synthesis of nuclear transacting factors, which would regulate the gene expression of VEGF. Because glia cell-secreted factors did not change VEGF expression during normoxia, it is possible that factors induced by hypoxia and substances secreted by astrocytes interact cooperatively with the VEGF gene at the transcriptional level. More studies will be necessary to identify these factors. Some of the factors secreted by astroglia cells were identified as interleukin-6 and transforming growth factor (59, 62). However, both of these seem unlikely to be responsible for the reducing effect of astroglia cell CM on the VEGF expression during hypoxia, because they were shown to increase VEGF expression (7, 14).

VEGF in vitro has been shown to increase the release of NO, which, under reducing conditions, increases the permeability of the PBMEC monolayer (26). Therefore, the amount of NO released during hypoxia should be abolished by glia cell CM in the same way as determined for the VEGF expression. NO released during hypoxia was determined indirectly by measurements of the cGMP level, which is increased by activation of the guanylate cyclase (19). Our data demonstrate that hypoxia-elevated cGMP levels were decreased by glia cell CM to the same extent as the hypoxia-induced VEGF expression, suggesting that the effect of glia cells on the VEGF expression causes the reduced permeability during hypoxia.

Hypoxia-induced permeability changes were measured by the passage of inulin, which crosses the PBMEC monolayer via the paracellular route, suggesting that glia cells may contribute to the maintenance of tight junctions between endothelial cells during hypoxia. To support this suggestion, it was evaluated by immunocytochemistry whether hypoxia is changing the expression or distribution of tight junction proteins. Several molecules are associated with tight junctions, such as ZO-1, cingulin, ZO-2, and 7H6 (11). Recently, two other proteins, occludin, an integral membrane protein localized at tight junctions (30), and rab13, a GTP-binding protein codistributed with ZO-1 (68), have been described. In this study, the expression of ZO-1 was determined, and it was shown that hypoxia disrupted the continuity of the ZO-1 expression along the cell border, which was prevented when hypoxia was performed in the presence of C6 CM. This suggests that glia cells prevent hypoxia-induced paracellular hyperpermeability.

Conclusively, glia cells probably secrete one or more factors, which decrease hypoxia-induced VEGF expression and permeability. During normoxia, glia cell CM did not change VEGF expression, suggesting that hypoxia is necessary to activate this (or these) factor(s). The described effect may be one of the mechanisms by which astrocytes protect the BBB from hypoxia-induced endothelial paracellular permeability and which might delay the development of the vasogenic brain edema.


    ACKNOWLEDGEMENTS

We thank M. Clauss for helpful discussions, M. Wiesnet for technical assistance, and A. Möbs for preparing photographs.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Fischer, Max-Planck Institute for Physiological and Clinical Research, Dept. of Anesthesiology and Intensive Care, 61231 Bad Nauheim, Germany (E-mail: s.fischer{at}kerckhoff.mpg.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 March 2000; accepted in final form 19 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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