·NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier

Ingolf E. Blasig, Helga Giese, Matthias L. Schroeter, Anje Sporbert, Darkhan I. Utepbergenov, Igor B. Buchwalow, Katrin Neubert, Gilbert Schönfelder, Dorette Freyer, Ingolf Schimke, Wolf Eberhard Siems, Martin Paul, Reiner F. Haseloff, Rosel Blasig

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

To investigate the relevance of ·NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (γ-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced γ-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 μM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in ·NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature.

Original languageEnglish
Pages (from-to)114-127
Number of pages14
JournalMicrovascular Research
Volume62
Issue number2
DOIs
Publication statusPublished - Jan 1 2001

Keywords

  • Blood-brain barrier
  • Brain capillary endothelial cells
  • Immortalization
  • Nitric oxide
  • Reactive oxygen species

ASJC Scopus subject areas

  • Biochemistry
  • Cardiology and Cardiovascular Medicine
  • Cell Biology

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  • Cite this

    Blasig, I. E., Giese, H., Schroeter, M. L., Sporbert, A., Utepbergenov, D. I., Buchwalow, I. B., Neubert, K., Schönfelder, G., Freyer, D., Schimke, I., Siems, W. E., Paul, M., Haseloff, R. F., & Blasig, R. (2001). ·NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier. Microvascular Research, 62(2), 114-127. https://doi.org/10.1006/mvre.2001.2318