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Chapter 18: Development of an in vitro cell culture system to mimic the blood-brain barrier
R. Landgraf and H.-J. Riihle (Eds.) Progress in Brain Reearch. Vol. 91 0 1992 Elsevier Science Publishers B.V. All rights reserved. A. Ermisch.
I17 CHAPTER 18
Development of an in vitro cell culture system to mimic the blood-brain barrier J. Rauh, J. Meyer, C. Beuckmann and H.-J. Galla Institut fur Biochemie, Westfalische Wilhelms-Universitat, 0-4400 Munster, Germany
Primary cultures of brain capillary endothelial cells (BCECs) cocultured together with astroglia cells were used to investigate the induction of blood-brain barrier (BBB) characteristics in vitro. By immunofluorescence, histochemical staining, twodimensional gel electrophoresis and enzyme activity tests we are able to show that BCECs in vitro loose typical blood-brain barrier properties but not their common endothelial phenotype.
Astrocytes induce the expression of the blood-brain barrier characteristic enzymes y-glutamyltranspeptidase and alkaline phosphatase but only in a coculture system with direct cell to cell contact between BCECs and astroglia cells. C,-glioma cells also re-establish the BBB phenotype but were less effective compared to astrocytes. The susceptibility of the BCECs to an astroglial stimulus depends on the proliferative state of the BCECs.
Introduction
ardt, 1986). However, very recently we were able to demonstrate clearly the loss of enzymatic properties typical for the BBB during cell culture whereas common endothelial cell markers are preserved (Mischek et al., 1989; Meyer et al., 1990). In this contribution we report further immunofluorescence, histochemical, enzymatic and electrophoretic evidence that endothelial cells in culture express their common cell phenotype. BBBcharacteristic properties are lost during culture but are induced to the in vivo level in a mixed coculture system with astrocytes.
From a pharmaceutical point of view there is widespread interest to employ the techniques of cell culture in order to study drug transport and metabolism of specific biological barriers (Kenneth et al., 1990). Especially the exchange of solutes from blood to brain extracellular fluids is regulated by cerebral endothelial cells, which form the so-called blood-brain barrier (BBB). Most techniques to study the BBB passage in vivo require experimental animals. Since the in vivo situation is rather complex due to different physiological factors and in order to avoid the use of animals many efforts have been made to establish a functional in vitro model for the blood-brain barrier based on cultured cerebrovascular endothelial cells isolated from brain microvessels (DeBault and Cancilla, 1980; Gebhardt and Goldstein, 1988). It was shown that endothelial cell monolayers exert a certain barrier function (Mischek et al., 1990) and that carrier systems are developed in vitro (Audus and Borch-
Methods Porcine brain capillary endothelial cells (BCECs) were prepared and cultured as described before (Mischek et al., 1989). Rat cerebral cortical astrocytes were prepared according to McCarthy and De Vellis (1980) with slight modifications (Meyer et al., 1991). For biochemical and histochemical enzyme tests see Meyer et al. (1991).
118 4 00
300
PB
200
3
s 100
0
2
L
6 8 days In culture
for y-glutamyltranspeptidase (y-GT) and alkaline phosphatase (ALP) which are considered to be BBB marker enzymes. y-GT activity decreases from an initial value of 105 unitslmg protein to about 10 units/mg protein during culture. An equivalent result was observed for the ALP, but enzyme activity was only decreased by a factor of four from ini-
1 0 1 2 l h
Fig. 1. Relative enzymatic activity of y-glutamyltranspeptidase (0 - 0 - O ) ,alkaline phosphatase ( - - ) and angiotensin converting enzyme ( A - A - A ) of cerebral endothelial cells in vitro at different days in culture. Activities are given with respect to the initial in vivo value. Cell protein as function of time in vitro is added to characterize the cell proliferation.
Two-dimensional gel electrophoresis was performed according to Celis et al. (1990). Cells were treated with a denaturing lysis buffer and stored at - 70°C. Two-dimensional gels were silver-stained (Ansorge, 1985). Sample preparation for immunofluorescence is described elsewhere (Meyer, 1991). Results
We investigated the enzymatic properties of BCECs with time in culture. Fig. 1 summarizes the results. Angiotensin converting enzyme (ACE), a common endothelial but not a BBB-specific marker is expressed in proliferating BCECs. The ACE activity of freshly isolated cells with a value of 16 units/mg protein drops to about 30% after seeding but recovers until cells have reached confluency around day in which may be deduced from the increasing cell Protein as indicator for cell Proliferation. Completely different behavior was observed
Fig. 2. Two-dimensional gel electrophoresis. Horizontal separation by isoelectric focusing, vertical separation with respect to molecular weight.
Fig. 3. Brain capillary endothelial cells and astrocytes in primary culture. u,b,c. Confluent monolayers stained by indirect immunofluorescence for ACE (a), factor VlII related antigen (b)and DiI-Ac-LDL (c).d,e. Cells are stained by immunofluorescence for 3 days (g) and 5 y-GT after 1 day (d)and after 8 days in vitro (e).f,g,h. Corresponding histochemical stain for y-GT after 1 day 0, days (h) in culture. j. Type 1 astrocytes stained for GFAP by immunofluorescence. k,i. Histochemical stain for ALP; BCECs after 6 days in culture including 2 days in coculture with astrocytes (k)or 2 days in coculture with C6-glioma cells (0.Magnification: 200 x (a-.Lj,0; 100 x (g.h,k).
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tially 2 units/mg protein. Thus we can state that BBB characteristics are lost in pure BCEC cultures whereas the common endothelial marker ACE is conserved. To endorse the interpretation of our enzymatic results we performed two-dimensional gel electrophoresis of BCEC proteins. The horizontal direction of the gels shown in Fig. 2 is an isoelectricfocusing whereas in the vertical direction proteins are separated with respect to their molecular weight by SDS gel electrophoresis. A molecular weight standard is included in Fig. 2. Quantitatively we can state that the electrophoresis pattern of freshly isolated BCECs is very similar to the pattern of BCECs in culture. From the molecular weight and the corresponding isoelectric point we may preliminary assign some of the spots. The analysis shows that the common expression pattern of proteins like actin (spot a), tubulin (spot b), the group of tropomyosins (spots around c and d)or Golgi-land Golgi-Zproteins (spots e and fi are conserved, which is in agreement with our assumption of an undisturbed expression of common proteins in BCECs in culture. Corresponding results were obtained with immunofluorescence. Fig. 3a- c clearly shows that ACE (Fig. 3a) and factor VIII related antigen (Fig. 3b), another common endothelial marker, are stained by their corresponding antibodies. The uptake of fluorescence labeled acetylated LDL (Fig. 3c) is another proof for the endothelial character of our cells in culture. The y-GT is present in freshly isolated cells, but not in proliferating cells (Fig. 3d - h). Obviously cultured BCECs do not fulfill the requirement of a BBB in vitro model. Since mammalian brain microvessels are surrounded by astroglial endfeet in vivo, which are probably responsible for the induction and the maintenance of the BBB-specific BCEC phenotype (Stewart and Coomber, 1986), it was quite obvious to scrutinize whether cerebral astrocytes are able to induce the differentiated BBB phenotype in BCECs in culture. Type 1 astrocytes (Fig. 351 are
characterized by their positive response to an antibody against glial fibrillary acidic protein (GFAP). We cocultured BCECs and astrocytes in a mixed cell monolayer. BCECs had to be cultured for some days in advance since astrocytes inhibit the proliferation of BCECs. Histochemical tests for ALP and y-GT were performed in order to obtain a local assignment of the possible induction of enzyme activity. Fig. 3k gives an example of a histochemical ALP stain after 6 days in vitro including 2 days in coculture. The BCEC colony which does not show ALP stain without astrocyte coculture exhibits an outer belt of increased ALP activity. If the coculture time is expanded, colonies become homogeneously stained. However, the induction effect was reduced if BCECs were kept for a longer time in culture before astrocytes were added. ALP or y-GT activity is not induced in BCEC monolayers grown to confluence before astrocyte addition. The induction of BBB markers can, however, only be observed if astrocytes and BCECs are able to form direct cell to cell contact. No induction was observed if cells were separated during coculture by a medium filled cleft or by a filter. Astrocyte conditioned media had no effect. A comparable but weaker effect was observed in a coculture with C,-glioma cells (Fig. 31). BCECs in culture did not respond to L-929 fibroblastoma cells. We conclude that astroglia cells induce the BBB phenotype in BCECs in culture only under conditions with direct cell to cell contact. The susceptibility of BCECs to astroglial induction depends on the proliferative state of the BCECs. The evoked spatial induction is transferred to BCECs that are not in direct contact with the astroglial cells.
Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft and from the Fonds der Chemischen Industrie. We like to thank H. Elverich for her expert help with the manuscript.
121
References Ansorge, W. (1985) Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J. Biochem. Biophys. Methods, 11: 13 - 20. Audus, K.L. and Borchardt, R.T. (1986) Characterization of the large neutral amino acid transport system of bovine brain microvessel endothelial cell monolayers. J. Neurochem., 47: 484 - 488. DeBault, L.E. and Cancilla, P.A. (1980) y-Glutamyltranspeptidase in isolated brain endothelial cells; induction by glial cells in vitro. Science, 207: 653 -655. Celis, J.E., Gesser, B., Rasmussen, H.H., Madsen, P., Leffers, H., Dejgaard, K., Honore, B., Olsen, E., Ratz, F., Lauridsen, J.B., Basse, B., Mouritzen, S., Hellerup, M., Andersen, A., Walbum, E., Celis, A., Bauw, G., Puype, M., Van Damme, J. and Vandekerckhove, J. (1990) Comprehensive twodimensional gel protein databases offer a global approach to the analysis of human cells: the transformed amnion cells (AMA) master database and its link to genome DNA sequence data. Electrophoresis, 11: 989 - 1071. Gebhardt, A.M. and Goldstein, G.W. (1988) Use of an in vitro system to study the effects of lead on astrocyte-endothelial cell interactions; a model for studying toxic injury to the bloodbrain barrier. Toxicol, Appl. Pharmacoi., 94: 191 -206. Kenneth, L.A., Bartel, R.L., Hidalgo, I.J. and Borchardt, T. (1990) The use of cultured epithelial and endothelial cells for drug transport and metabolism studies. Pharm. Res., 7: 435 - 451.
McCarthy, D. and De Vellis, J. (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Ceil Biol., 85: 890 - 902. Meyer, J. (1991) Untersuchungen von Endothelzell-AstrogliaWechselbeziehungenin Kultur zur Entwicklung ekes in vitroModells der Blut-Hirn-Schranke. Inaugural Dissertation, Darmstadt. Meyer, J., Mischek, U., VeyN, M., Henzel, K. and Galla, H.-J. (1990) Blood-brain barrier characteristic enzymatic properties in cultured brain capillary endothelial cells. Brain Res., 514: 305 - 309. Meyer, J., Rauh, J. and Galla, H.-J. (1991) The susceptibility of cerebral endothelial cells to astroglial induction depends on their proliferative state. J. Neurochem., 57: 1971- 1977. Mischek, U., Meyer, J. andGalla, H.-J. (1989)Characterization of y-glutamyl-transpeptidaseactivity of cultured endothelial cells from porcine brain capillaries. Cell Tissue Res., 256: 221 - 226. Mischek, U., Hein, M., Weske, M., Baethman, M., Qin, Y., Meyermann, R. and Galla, H.-J. (1990) Resistance modulation of endothelial cell monolayers by activated Tlymphocytes. In: B.B. Johansson and C. Owman (Eds.), Proceedings of the Fernstrom Symposium Series, Vol. 14: Pathophysiology of the Blood-Brain Barrier, Elsevier, Amsterdam, pp. 447-451. Stewart, P.A. and Coomber, B.L. (1986) Astrocytes and the blood-brain barrier. In: S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. I , Academic Press, Orlando, FL, pp. 31 1 - 328.
I17 CHAPTER 18
Development of an in vitro cell culture system to mimic the blood-brain barrier J. Rauh, J. Meyer, C. Beuckmann and H.-J. Galla Institut fur Biochemie, Westfalische Wilhelms-Universitat, 0-4400 Munster, Germany
Primary cultures of brain capillary endothelial cells (BCECs) cocultured together with astroglia cells were used to investigate the induction of blood-brain barrier (BBB) characteristics in vitro. By immunofluorescence, histochemical staining, twodimensional gel electrophoresis and enzyme activity tests we are able to show that BCECs in vitro loose typical blood-brain barrier properties but not their common endothelial phenotype.
Astrocytes induce the expression of the blood-brain barrier characteristic enzymes y-glutamyltranspeptidase and alkaline phosphatase but only in a coculture system with direct cell to cell contact between BCECs and astroglia cells. C,-glioma cells also re-establish the BBB phenotype but were less effective compared to astrocytes. The susceptibility of the BCECs to an astroglial stimulus depends on the proliferative state of the BCECs.
Introduction
ardt, 1986). However, very recently we were able to demonstrate clearly the loss of enzymatic properties typical for the BBB during cell culture whereas common endothelial cell markers are preserved (Mischek et al., 1989; Meyer et al., 1990). In this contribution we report further immunofluorescence, histochemical, enzymatic and electrophoretic evidence that endothelial cells in culture express their common cell phenotype. BBBcharacteristic properties are lost during culture but are induced to the in vivo level in a mixed coculture system with astrocytes.
From a pharmaceutical point of view there is widespread interest to employ the techniques of cell culture in order to study drug transport and metabolism of specific biological barriers (Kenneth et al., 1990). Especially the exchange of solutes from blood to brain extracellular fluids is regulated by cerebral endothelial cells, which form the so-called blood-brain barrier (BBB). Most techniques to study the BBB passage in vivo require experimental animals. Since the in vivo situation is rather complex due to different physiological factors and in order to avoid the use of animals many efforts have been made to establish a functional in vitro model for the blood-brain barrier based on cultured cerebrovascular endothelial cells isolated from brain microvessels (DeBault and Cancilla, 1980; Gebhardt and Goldstein, 1988). It was shown that endothelial cell monolayers exert a certain barrier function (Mischek et al., 1990) and that carrier systems are developed in vitro (Audus and Borch-
Methods Porcine brain capillary endothelial cells (BCECs) were prepared and cultured as described before (Mischek et al., 1989). Rat cerebral cortical astrocytes were prepared according to McCarthy and De Vellis (1980) with slight modifications (Meyer et al., 1991). For biochemical and histochemical enzyme tests see Meyer et al. (1991).
118 4 00
300
PB
200
3
s 100
0
2
L
6 8 days In culture
for y-glutamyltranspeptidase (y-GT) and alkaline phosphatase (ALP) which are considered to be BBB marker enzymes. y-GT activity decreases from an initial value of 105 unitslmg protein to about 10 units/mg protein during culture. An equivalent result was observed for the ALP, but enzyme activity was only decreased by a factor of four from ini-
1 0 1 2 l h
Fig. 1. Relative enzymatic activity of y-glutamyltranspeptidase (0 - 0 - O ) ,alkaline phosphatase ( - - ) and angiotensin converting enzyme ( A - A - A ) of cerebral endothelial cells in vitro at different days in culture. Activities are given with respect to the initial in vivo value. Cell protein as function of time in vitro is added to characterize the cell proliferation.
Two-dimensional gel electrophoresis was performed according to Celis et al. (1990). Cells were treated with a denaturing lysis buffer and stored at - 70°C. Two-dimensional gels were silver-stained (Ansorge, 1985). Sample preparation for immunofluorescence is described elsewhere (Meyer, 1991). Results
We investigated the enzymatic properties of BCECs with time in culture. Fig. 1 summarizes the results. Angiotensin converting enzyme (ACE), a common endothelial but not a BBB-specific marker is expressed in proliferating BCECs. The ACE activity of freshly isolated cells with a value of 16 units/mg protein drops to about 30% after seeding but recovers until cells have reached confluency around day in which may be deduced from the increasing cell Protein as indicator for cell Proliferation. Completely different behavior was observed
Fig. 2. Two-dimensional gel electrophoresis. Horizontal separation by isoelectric focusing, vertical separation with respect to molecular weight.
Fig. 3. Brain capillary endothelial cells and astrocytes in primary culture. u,b,c. Confluent monolayers stained by indirect immunofluorescence for ACE (a), factor VlII related antigen (b)and DiI-Ac-LDL (c).d,e. Cells are stained by immunofluorescence for 3 days (g) and 5 y-GT after 1 day (d)and after 8 days in vitro (e).f,g,h. Corresponding histochemical stain for y-GT after 1 day 0, days (h) in culture. j. Type 1 astrocytes stained for GFAP by immunofluorescence. k,i. Histochemical stain for ALP; BCECs after 6 days in culture including 2 days in coculture with astrocytes (k)or 2 days in coculture with C6-glioma cells (0.Magnification: 200 x (a-.Lj,0; 100 x (g.h,k).
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120
tially 2 units/mg protein. Thus we can state that BBB characteristics are lost in pure BCEC cultures whereas the common endothelial marker ACE is conserved. To endorse the interpretation of our enzymatic results we performed two-dimensional gel electrophoresis of BCEC proteins. The horizontal direction of the gels shown in Fig. 2 is an isoelectricfocusing whereas in the vertical direction proteins are separated with respect to their molecular weight by SDS gel electrophoresis. A molecular weight standard is included in Fig. 2. Quantitatively we can state that the electrophoresis pattern of freshly isolated BCECs is very similar to the pattern of BCECs in culture. From the molecular weight and the corresponding isoelectric point we may preliminary assign some of the spots. The analysis shows that the common expression pattern of proteins like actin (spot a), tubulin (spot b), the group of tropomyosins (spots around c and d)or Golgi-land Golgi-Zproteins (spots e and fi are conserved, which is in agreement with our assumption of an undisturbed expression of common proteins in BCECs in culture. Corresponding results were obtained with immunofluorescence. Fig. 3a- c clearly shows that ACE (Fig. 3a) and factor VIII related antigen (Fig. 3b), another common endothelial marker, are stained by their corresponding antibodies. The uptake of fluorescence labeled acetylated LDL (Fig. 3c) is another proof for the endothelial character of our cells in culture. The y-GT is present in freshly isolated cells, but not in proliferating cells (Fig. 3d - h). Obviously cultured BCECs do not fulfill the requirement of a BBB in vitro model. Since mammalian brain microvessels are surrounded by astroglial endfeet in vivo, which are probably responsible for the induction and the maintenance of the BBB-specific BCEC phenotype (Stewart and Coomber, 1986), it was quite obvious to scrutinize whether cerebral astrocytes are able to induce the differentiated BBB phenotype in BCECs in culture. Type 1 astrocytes (Fig. 351 are
characterized by their positive response to an antibody against glial fibrillary acidic protein (GFAP). We cocultured BCECs and astrocytes in a mixed cell monolayer. BCECs had to be cultured for some days in advance since astrocytes inhibit the proliferation of BCECs. Histochemical tests for ALP and y-GT were performed in order to obtain a local assignment of the possible induction of enzyme activity. Fig. 3k gives an example of a histochemical ALP stain after 6 days in vitro including 2 days in coculture. The BCEC colony which does not show ALP stain without astrocyte coculture exhibits an outer belt of increased ALP activity. If the coculture time is expanded, colonies become homogeneously stained. However, the induction effect was reduced if BCECs were kept for a longer time in culture before astrocytes were added. ALP or y-GT activity is not induced in BCEC monolayers grown to confluence before astrocyte addition. The induction of BBB markers can, however, only be observed if astrocytes and BCECs are able to form direct cell to cell contact. No induction was observed if cells were separated during coculture by a medium filled cleft or by a filter. Astrocyte conditioned media had no effect. A comparable but weaker effect was observed in a coculture with C,-glioma cells (Fig. 31). BCECs in culture did not respond to L-929 fibroblastoma cells. We conclude that astroglia cells induce the BBB phenotype in BCECs in culture only under conditions with direct cell to cell contact. The susceptibility of BCECs to astroglial induction depends on the proliferative state of the BCECs. The evoked spatial induction is transferred to BCECs that are not in direct contact with the astroglial cells.
Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft and from the Fonds der Chemischen Industrie. We like to thank H. Elverich for her expert help with the manuscript.
121
References Ansorge, W. (1985) Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J. Biochem. Biophys. Methods, 11: 13 - 20. Audus, K.L. and Borchardt, R.T. (1986) Characterization of the large neutral amino acid transport system of bovine brain microvessel endothelial cell monolayers. J. Neurochem., 47: 484 - 488. DeBault, L.E. and Cancilla, P.A. (1980) y-Glutamyltranspeptidase in isolated brain endothelial cells; induction by glial cells in vitro. Science, 207: 653 -655. Celis, J.E., Gesser, B., Rasmussen, H.H., Madsen, P., Leffers, H., Dejgaard, K., Honore, B., Olsen, E., Ratz, F., Lauridsen, J.B., Basse, B., Mouritzen, S., Hellerup, M., Andersen, A., Walbum, E., Celis, A., Bauw, G., Puype, M., Van Damme, J. and Vandekerckhove, J. (1990) Comprehensive twodimensional gel protein databases offer a global approach to the analysis of human cells: the transformed amnion cells (AMA) master database and its link to genome DNA sequence data. Electrophoresis, 11: 989 - 1071. Gebhardt, A.M. and Goldstein, G.W. (1988) Use of an in vitro system to study the effects of lead on astrocyte-endothelial cell interactions; a model for studying toxic injury to the bloodbrain barrier. Toxicol, Appl. Pharmacoi., 94: 191 -206. Kenneth, L.A., Bartel, R.L., Hidalgo, I.J. and Borchardt, T. (1990) The use of cultured epithelial and endothelial cells for drug transport and metabolism studies. Pharm. Res., 7: 435 - 451.
McCarthy, D. and De Vellis, J. (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Ceil Biol., 85: 890 - 902. Meyer, J. (1991) Untersuchungen von Endothelzell-AstrogliaWechselbeziehungenin Kultur zur Entwicklung ekes in vitroModells der Blut-Hirn-Schranke. Inaugural Dissertation, Darmstadt. Meyer, J., Mischek, U., VeyN, M., Henzel, K. and Galla, H.-J. (1990) Blood-brain barrier characteristic enzymatic properties in cultured brain capillary endothelial cells. Brain Res., 514: 305 - 309. Meyer, J., Rauh, J. and Galla, H.-J. (1991) The susceptibility of cerebral endothelial cells to astroglial induction depends on their proliferative state. J. Neurochem., 57: 1971- 1977. Mischek, U., Meyer, J. andGalla, H.-J. (1989)Characterization of y-glutamyl-transpeptidaseactivity of cultured endothelial cells from porcine brain capillaries. Cell Tissue Res., 256: 221 - 226. Mischek, U., Hein, M., Weske, M., Baethman, M., Qin, Y., Meyermann, R. and Galla, H.-J. (1990) Resistance modulation of endothelial cell monolayers by activated Tlymphocytes. In: B.B. Johansson and C. Owman (Eds.), Proceedings of the Fernstrom Symposium Series, Vol. 14: Pathophysiology of the Blood-Brain Barrier, Elsevier, Amsterdam, pp. 447-451. Stewart, P.A. and Coomber, B.L. (1986) Astrocytes and the blood-brain barrier. In: S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Vol. I , Academic Press, Orlando, FL, pp. 31 1 - 328.