REGULATION OF BRAIN CAPILLARY ENDOTHELIAL THROMBOMODULIN MESSENGER-RNA EXPRESSION

Background and Purpose Endothelial cells regulate hemostasis in part v ia expression of thrombomodulin, a potent anticoagulant protein. The p urpose of this study was to analyze brain capillary endothelial cell e xpression of thrombomodulin mRNA. Methods Bovine brain capillary endot helial cells were grown in a blood-brain barrier model in which endoth elial cells form capillary-like structures. In situ hybridization and polymerase chain reaction (PCR) were used to examine thrombomodulin ex pression. Endothelial cells were then cocultured with astrocytes. We e xamined both coculture and monoculture preparations for gamma-glutamyl transpeptidase (GGTP), a marker of the blood-brain barrier. We then u sed quantitative-competitive PCR to compare thrombomodulin expression in endothelial monocultures and astrocyte-endothelial cocultures after 1 and 7 days of culture. Results Both in situ hybridization and PCR s tudies demonstrated thrombomodulin mRNA expression by endothelial cell s. During 1 week of astrocyte-endothelial coculture, there was (1) pro gressive association of astrocytes with capillary-like structures and (2) expression of GGTP; endothelial monocultures did not express GGTP. There was no significant difference in thrombomodulin mRNA expression for cocultures versus monocultures after 1 day. After 1 week, however , astrocyte-endothelial cocultures had markedly decreased thrombomodul in mRNA compared with monocultures (9+/-2 versus 189+/-62 pg/mL; P<.02 5). This thrombomodulin mRNA decrease thus occurred when elements of t he blood-brain barrier phenotype were demonstrable, ie, when astrocyte association with capillary-like structures was maximal and when GGTP was expressed in cocultures. Conclusions These findings indicate astro cyte regulation of thrombomodulin mRNA expression in vitro and suggest an important role for the blood-brain barrier in the regulation of th rombomodulin.