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Angiogenesis of the blood–brain barrier in vitro and the function of cerebral pericytes¹

Department of Neuroanatomy and Molecular Brain Research, Ruhr-Universität Bochum, Bochum, Germany

²Correspondence: Department of Neuroanatomy and Molecular Brain Research, Ruhr-Universität Bochum, Universitätsstr 150, D-44801 Bochum, Germany. E-mail: rolf.dermietzel{at}ruhr-uni-bochum.de

SPECIFIC AIMS

The aim of the present study was to investigate angiogenic mechanisms of the blood–brain barrier (BBB) in vitro in order to gain better insight into the function of cerebral pericytes during morphogenesis of the BBB. We developed coculture systems that allowed us to establish heterocellular interactions which proved effective in the angiogenesis of cerebral microvasculature and the expression of BBB features.

PRINCIPAL FINDINGS

1. Cocultures of endothelial cells with astrocytes show a reorganization into capillary-like structures (CLS)
Within 2 days after addition of astrocytes to mixed endothelial pericyte cultures, endothelial cells reorganized into linear CLS. The formation of CLS is considered to reflect a reorganization of the 2-dimensional endothelial monolayer into a 3-dimensional structure. The CLS varied in thickness and length and resembled vascular networks in their microarchitecture. Immunohistochemical analysis of CLS with antibodies to anti-factor VIII-related antigen proved their endothelial origin and allowed their identification within cocultures. In the presence of astrocytes, cerebral pericytes underwent a dramatic structural change. Association of pericytes with CLS was accompanied by a switch of pericyte polygonal phenotype to a spindle-like structure. The pericyte shape changes and association with endothelial cells were not encountered in areas where endothelial cells maintained their cobblestone-like morphology. The morphological transformation of endothelial cells associated with astrocyte-induced CLS formation was accompanied by the induction of BBB features. The addition of astrocyte- or endothelial/astrocyte-conditioned media did not induce CLS in endothelial cultures.

The extent of CLS formation was quantitated by computer-assisted image analysis. The results from two independent experiments (experiment 1 and 2) are displayed in order to confirm reproducibility. To examine the kinetics of CLS modeling, the time-dependent course of astrocyte-induced CLS formation was studied (Fig. 1 a, b). Endothelium-covered area decreased continuously within 36 h as the cells differentiated into CLS (Fig. 1a ). Measuring the number and length of CLS corroborated clearly the data obtained by measuring the endothelium-covered area (Fig. 1b ). From 48 to 72 h the number and length of CLS did not significantly change. This sequence of events is suggestive of an astrocyte-induced transition of endothelial cells from a preferentially substratum-adhered to self-assembled complexes.

View larger version (48K): [in this window] [in a new window]   Figure 1. a–c) Quantitative assessment of CLS development. The effect of astrocytes (AC) or 2 ng/mL TGF-ß1 on CLS formation in mixed endothelial pericyte cultures (EPC) was measured every 12 h for a total of 72 h. Each data point represents the mean (a) and the sum (b, c) of 20 randomly selected fields per sample. For each time point, a different sample was used. a) Endothelium-covered area (ECA) declined with time in coculture with AC or treatment with TGF-ß1. After 72 h, ECA was reduced 5% in coculture of EPC with AC and to almost 0% in the presence of TGF-ß1. b) The number and length of CLS in coculture with astrocytes increased within 36 h and remained constant for up to 72 h (total number of CLS depicted in the z axis). c) In TGF-ß1-treated EPC the highest level of CLS was observed after 48 h; at longer incubation times the ratio of CLS was significantly reduced.

2. Effect of TGF-ß1 on CLS formation
We further studied whether TGF-ß1 was responsible for the observed reduction of endothelium-covered area in coculture with astrocytes and the formation of CLS.

Treatment of mixed endothelial pericyte cultures with 2 ng/mL TGF-ß1 for 2 days led to morphological changes similar to those seen in mixed endothelial pericyte cultures cocultured with astrocytes. In contrast to astrocyte-induced CLS, pericytes were never found to associate with CLS, but instead kept their polygonal morphology and distance from endothelial cords. Apparently, the presence of astrocytes is essential for the association of pericytes with CLS.

Quantitative evaluation of the CLS formation was done within the identical time frame as for coculture experiments with astrocytes. In contrast to astrocyte-induced CLS formation, TGF-ß1 resulted in a time-dependent reduction of endothelial cells, reaching minimal levels at 72 h (Fig. 1a ). Reduction of endothelial cells was due to a decrease in number and length of CLS after the 48 h peak (Fig. 1c ).

3. Coculture-induced apoptotic cell death
The decrease in endothelial cell numbers after coculturing with astrocytes or exposure to TGF-ß1 was suspected to be due to cell death since endothelial cells frequently exhibited morphological features of apoptosis. After coculturing with astrocytes or treatment with 2 ng/mL TGF-ß1, a considerable number of endothelial cells exhibited shrinkage and nuclear and cytoplasmic condensation. TUNEL technique data provide evidence that apoptosis is an essential morphogenetic mechanism in CLS modeling. Since an anti-apoptotic effect on endothelial cells seems to be locally restricted in cocultures with astrocytes where pericytes associate with CLS, we compared mixed cultures of endothelial cells and pericytes with pure endothelial cultures with respect to their behavior under astrocyte and TGF-ß1 conditions.

4. Pericytes promote CLS formation
In mixed endothelial pericyte cultures (EPC), the endothelium-covered area approached a maximum of 63% within 6 days of culture (Fig. 2 a, section 1). Pure endothelial cultures (EC) reached complete confluency (100%) during the same period. Apparently the fraction of pericytes in mixed cultures provides an inhibitory effect on endothelial cell proliferation. After 2 days in culture in the presence of 2 ng/mL TGF-ß1 (Fig. 2a , section 2), a decrease of 80% in endothelium-covered area in mixed endothelial pericyte cultures and 70% in pure endothelial cultures occurred compared to controls without TGF-ß1 treatment. Parallel experiments with exploiting both endothelial cultures to astrocyte coculture conditions showed a different pattern of behavior (Fig. 2a , section 3). In this case, the endothelium-covered area in mixed endothelial pericyte cultures cocultured with astrocytes decreased 86% in experiment 1 and 73% in experiment 2, respectively. In pure endothelial cultures, the addition of astrocytes elicited an even more dramatic decrease in endothelium-covered area of 96%. Addition of exogenous pericytes and astrocytes to mixed endothelial pericyte cultures and pure endothelial cultures (Fig. 2a , section 4) yielded a relative increase of endothelium-covered area compared to the coculture with astrocytes alone (Fig. 2a , section 3). To further investigate this effect, the role of pericytes on CLS formation was elucidated.

View larger version (34K): [in this window] [in a new window]   Figure 2. a–d) Quantitative assessment of CLS formation in mixed endothelial pericyte cultures (EPC) and pure endothelial cultures (EC) under the indicated coculture conditions (6 days, the latter 2 days in coculture). Each data point represents the mean (a) and the sum (b, c, d) of 20 randomly selected fields per sample. a) In controls section 1 and in the presence of TGF-ß1 section 2, endothelium-covered area was lower in EPC than in EC. However, after the addition of astrocytes, the endothelium-covered area of EPC was higher than that of EC section 3. In tricultures consisting of astrocytes and added exogenous pericytes (ePC), endothelium-covered area was increased in both endothelial cultures (EPC and EC, section 4). b) Effect of coculture with astrocytes (AC) on the number and length of CLS in EPC and EC. In the presence of EPC, the angiogenic effect of astrocytes () is profoundly higher than in cultures with pure endothelial cells •. c) Effects after the addition of exogenous pericytes (ePC) to EPC. Number and length of CLS is profoundly increased under triculture conditions () vs. EPC plus ePC () and EPC alone (). This effect becomes even more pronounced when pure endothelial cells (EC) are subjected to triculture condition () (d).

First, we compared the number and length of CLS in mixed endothelial pericyte cultures and pure endothelial cultures cocultured with astrocytes. Again the presence of pericytes in the mixed endothelial pericyte/astrocyte cultures provided an advantage in CLS formation over pure endothelial/astrocyte cultures (Fig. 2b ). Second, when mixed endothelial pericyte cultures were confronted with astrocytes or/and exogenous pericytes (ePC), the tricultures (EPC and ePC and AC in Fig. 2c ) yielded the highest number of CLS. Finally, pure endothelial cultures subjected to the same coculture conditions corroborated the profound effect of pericytes on CLS formation with the highest number and a shift to longer CLS assemblies under the triculture conditions (Fig. 2d ).

CONCLUSIONS AND SIGNIFICANCE

The formation of capillaries from existing blood vessels (angiogenesis) requires a complex series of sequential events in cellular behavior. The in vitro model of cerebral angiogenesis used in this study indicates that astrocytes induce endothelial cell and pericyte differentiation and thus provides a useful model with which to study microvessel morphogenesis and vascular cell interactions.

A close endothelial-astroglial association appeared to be required for the induction and organization of CLS. Alternatively, diffusible factors secreted by astrocytes might be responsible for CLS formation. We also found that TGF-ß1 had a profound effect on CLS formation by mixed endothelial pericyte cultures lacking astrocytes. In contrast to the effects of astrocytes, however, TGF-ß1 did not induce association of pericytes with CLS and led to a reduction in number and length of CLS over time.

A feasible explanation for the latter phenomenon is the association of endothelial cells with pericytes in astrocyte cocultures, which may increase not only the number of CLS, as shown by addition of pericytes in tricultures, but also their viability. Pericytes appear to be engaged in the regulation of endothelial differentiation by inducing termination and vessel maturation. They may also be involved in regulating anti-apoptotic mechanisms, which help CLS to survive under tissue culture conditions. The fact that endothelial cells assembled in CLS together with pericytes are more resistant to apoptotic stimuli than isolated endothelial cells lends some credit to the idea that cooperativity between both cell entities exists to prevent them from entering the apoptotic cascade.

View larger version (52K): [in this window] [in a new window]   Figure 3. Schematic presentation of the angiogenic effects in our coculture system. A) Addition of astrocytes to a mixed culture of cerebral endothelial cells and pericytes (EPC) leads to the formation of capillary-like structures (CLS) where pericytes associate with CLS. Additional supplementation of exogenous pericytes increases the number and viability of CLS presumably by exerting an anti-apoptotic effect (stabilized). B) TGF-ß1 also induces formation of CLS but without association of pericytes to the endothelial cords. CLS lacking associated pericytes prove less viable under tissue culture conditions (unstabilized).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0814fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.01-0814fje

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