Selective astrocytic endothelin-1 overexpression contributes to dementia associated with ischemic stroke by exaggerating astrocyte-derived amyloid secretion.

Endothelin-1 (ET-1) is synthesized by endothelial cells and astrocytes in stroke and in brains of Alzheimer's disease patients. Our transgenic mice with ET-1 overexpression in the endothelial cells (TET-1) showed more severe blood-brain barrier (BBB) breakdown, neuronal apoptosis, and glial reactivity after 2-hour transient middle cerebral artery occlusion (tMCAO) with 22-hour reperfusion and more severe cognitive deficits after 30 minutes tMCAO with 5 months reperfusion. However, the role of astrocytic ET-1 in contributing to poststroke cognitive deficits after tMCAO is largely unknown. Therefore, GET-1 mice were challenged with tMCAO to determine its effect on neurologic and cognitive deficit. The GET-1 mice transiently displayed a sensorimotor deficit after reperfusion that recovered shortly, then more severe deficit in spatial learning and memory was observed at 3 months after ischemia compared with that of the controls. Upregulation of TNF-α, cleaved caspase-3, and Thioflavin-S-positive aggregates was observed in the ipsilateral hemispheres of the GET-1 brains as early as 3 days after ischemia. In an in vitro study, ET-1 overexpressing astrocytic cells showed amyloid secretion after hypoxia/ischemia insult, which activated endothelin A (ETA) and endothelin B (ETB) receptors in a PI3K/AKT-dependent manner, suggesting role of astrocytic ET-1 in dementia associated with stroke by astrocyte-derived amyloid production.

Endothelin-1 overexpression leads to further water accumulation and brain edema after middle cerebral artery occlusion via aquaporin 4 expression in astrocytic end-feet.

Stroke patients have increased levels of endothelin-1 (ET-1), a strong vasoconstrictor, in their plasma or cerebrospinal fluid. Previously, we showed high level of ET-1 mRNA expression in astrocytes after hypoxia/ischemia. It is unclear whether the contribution of ET-1 induction in astrocytes is protective or destructive in cerebral ischemia. Here, we generated a transgenic mouse model that overexpress ET-1 in astrocytes (GET-1) using the glial fibrillary acidic protein promoter to examine the role of astrocytic ET-1 in ischemic stroke by challenging these mice with transient middle cerebral artery occlusion (MCAO). Under normal condition, GET-1 mice showed no abnormality in brain morphology, cerebrovasculature, absolute cerebral blood flow, blood-brain barrier (BBB) integrity, and mean arterial blood pressure. Yet, GET-1 mice subjected to transient MCAO showed more severe neurologic deficits and increased infarct, which were partially normalized by administration of ABT-627 (ET(A) antagonist) 5 mins after MCAO. In addition, GET-1 brains exhibited more Evans blue extravasation and showed decreased endothelial occludin expression after MCAO, correlating with higher brain water content and increased cerebral edema. Aquaporin 4 expression was also more pronounced in astrocytic end-feet on blood vessels in GET-1 ipsilateral brains. Our current data suggest that astrocytic ET-1 has deleterious effects on water homeostasis, cerebral edema and BBB integrity, which contribute to more severe ischemic brain injury.

Endothelial cell-specific over-expression of endothelin-1 leads to more severe cerebral damage following transient middle cerebral artery occlusion.

Previously, we have demonstrated that mRNA expression of endothelin-1 (ET-1), a potent vasoconstrictor, is induced in astrocytes and endothelial cells after ischemic conditions, suggesting that both of these cells synthesize ET-1 under this stress condition. Furthermore, ET-1 protected primary cultured astrocytes from ischemic stress. In order to further investigate the role of endothelial ET-1 in cerebral ischemic injury, transgenic mouse lines (TET) with a transgene that included ET cDNA with SV40 polyA under tyrosine kinase with immunoglobulin and epidermal growth factor homology domain (Tie-1) promoter were used. TET mouse lines were further characterized for ET-1 over-expression in the brain. The reverse transcription-polymerase chain reaction (RT-PCR) analysis using the primers specific for transgene 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 the transgenic brain compared with the non-transgenic brain by semi-quantitative RT-PCR. In situ hybridization and immunocytochemical analyses showed that the increased ET-1 mRNA and peptide expressions were detected in endothelial cells of cerebral vessels of TET mice. Under normal conditions, the TET mice that have a slightly increased blood pressure compared with that of non-transgenic mice showed no gross morphological abnormalities in the brain. However, after transient middle cerebral artery occlusion, TET mice showed a more severe neurological deficit, and larger infarct size and volume, suggesting that over-expressing ET-1 in endothelial cells is deleterious to neuronal survival under ischemic conditions. Our present TET model will serve as an ideal model for studying the role of endothelial ET- 1 in the pathogenesis of ischemic stroke.