Regulation of brain capillary endothelial thrombomodulin mRNA expression.
OBJECTIVE
Endothelial cells regulate hemostasis in part via expression of thrombomodulin, a potent anticoagulant protein. The purpose of this study was to analyze brain capillary endothelial cell expression of thrombomodulin mRNA.


METHODS
Bovine brain capillary endothelial cells were grown in a blood-brain barrier model in which endothelial cells form capillary-like structures. In situ hybridization and polymerase chain reaction (PCR) were used to examine thrombomodulin expression. Endothelial cells were then cocultured with astrocytes. We examined both coculture and monoculture preparations for gamma-glutamyl transpeptidase (GGTP), a marker of the blood-brain barrier. We then used 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 studies demonstrated thrombomodulin mRNA expression by endothelial cells. During 1 week of astrocyte-endothelial coculture, there was (1) progressive 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 thrombomodulin mRNA compared with monocultures (9 +/- 2 versus 189 +/- 62 pg/mL; P < .025). This thrombomodulin mRNA decrease thus occurred when elements of the 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 astrocyte regulation of thrombomodulin mRNA expression in vitro and suggest an important role for the blood-brain barrier in the regulation of thrombomodulin.
Tran
N D
ND
Department of Neurology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Wong
V L
VL
Schreiber
S S
SS
Bready
J V
JV
Fisher
M
M
D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D001812:Blood-Brain Barrier / Q000502:physiology*; D001921:Brain / Q000098:blood supply*; D002196:Capillaries / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D018920:Coculture Techniques; D005786:Gene Expression Regulation; D017403:In Situ Hybridization; D051379:Mice; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis*; D018180:Thrombomodulin / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D018180:Thrombomodulin