Role of endothelial cell overexpressed endothelin-1 (ET-1) in mouse model of ischemic stroke

Abstract

Endothelin-1 (ET-1) has been implicated in clinical stroke. We have demonstrated ET-1 mRNA expression is induced in astrocytes and endothelial cells after ischemic condition, suggesting that both of these cells synthesize ET-1 under this stress condition. ET-1 protected primary cultured astrocytes from ischemic stress. However, transgenic mice with over-expression of ET-1 in astrocytes displayed more severe neurological deficit and increased infract volume, suggesting that astrocytic ET-1 have neurotoxic effect on neurons. To further investigate the role of endothelial ET-1 in cerebral ischemic injury, several transgenic mouse lines (TET) were generated by microinjecting the construct, which include ET cDNA with SV40 poly A under tyrosine kinase receptor-specific for endothelial cell (Tie-1) promoter. TET mouse lines were further characterized for ET-1 over-expression. The RT-PCR analysis using the primers specific for tranagene ET-1 showed that transgene ET-1 is only expressed in the brain from TET mice. Total expression of ET-1 mRNA was also increased in brain of transgenic mice when compared to that of non-transgenic mice by semi-quantitative RT-PCR. In situ hybridization and immunocytochemical analyses showed that the increased ET-1 mRNA and peptide expressions are only in endothelial cells of cerebral vessels from the TET mice. ET-1 peptide level was also detected in the endothelial cells of cerebral vessel from TET mice. Under normal condition, no gross morphological change has been found in the brain. However, TET mice showed more severe neurological deficit, larger infarct size and volume after transient middle cerebral artery occlusion (MCAO), suggesting that overexpressing of ET-1 in endothelial cells is deleterious to neuronal survival under ischemic condition. Our present TET model will serve as an ideal model for studying the role of endothelial ET-1 in the pathogenesis of ischemic stroke.