Functional role of endothelin-1 on astrocytes and neurons under hypoxia/ischemia by using ET-1 transgenic and knockout mice
The Functional Role of Endothelin-l on Astrocytes and Neurons under
HypoxiaJIschemia by Using ET-l Transgenic and Knockout Mice Submitted by
Yaw Lai Ping
For the degree of Master of Philosophy in Molecular Biology The University of Hong Kong
In August 2003
Endothelin (ET)-I, originally isolated from procine aortic endothelial cells, has recently been referred to as a survival factor for human smooth muscle cells and endothelial cells. ET-I and its receptors (ETA and ETB), distributed throughout the CNS and ET -I, has been implicated in numerous physiological and pathological conditions, such as hypertension, stroke, and Alzheimer's disease. Under such pathological conditions of the CNS, intense ET-like immunoreactivity was significantly increased in astrocytes of the brain. Our laboratory has previously reported that astrocytes of mouse embryo brain express elevated level of ET -I mRNA upon hypoxia/ischemia (H/I) condition.
In addition, induction of ET -I in astrocytes under H/I condition appears to be essential for their survival since astrocytes from ET -I knockout mice died more readily under H/I condition and addition of ET-I protect them from H/I stress. To better understand the exact signaling mechanisms of ET -I survival effect on astrocytes under
H/I condition, we made use of astrocytes cultured from transgenic mice (GET tg) with astrocytic ET -lover-expression. During normal culture condition, proliferation of this GET tg astrocytes was double to those of nontransgenic (ntg). Under H/I condition, astrocytes from the ntg mice released a higher amount of lactate dehydrogenase indicating more cell death than those from GET tg mice. Furthermore, we also demonstrated that the induction of ET -1 under H/I condition, activated Erk-2 signaling and may play an important role in astrocyte survival and induction of GF AP expression. However, astrocytic ET -1 had detrimental effects on neuronal survival since culturing of neuroblastoma cells with ET -lover-expressing astrocytes or astrocyte-conditioned media (ACM) from these astrocytes showed more neuronal death as compared with those cultured with either normal astrocytes or ACM from non-transfected astrocytes.
In order to further understand the role of ET -1 and ET -I-mediated signals for the proliferation of astrocytes and neuronal death, proteomics approach were used. Brains from embryonic day 17.5 (EI7.5d) ET-I deficient and wild type mice were isolated for global scanning for the expression profile of proteins in the brain that may be involved in the ET -I-mediated cell proliferation and death has shown to playa major role in relation to cell proliferation, differentiation and development or transducing upstream signal to downstream effectors. Several proteins implicated in development and proliferation. Seven spots from 2-DE were identified and further characterized. Mass spectrometric and database analyses revealed that these proteins are Nucleoside Diphosphate Kinase (NDPK)-A, NDPK-B, Cyclophilin (CyP)-A, Adenylate Kinase (AK)-I Rho GDPdissociation inhibitor (RhoGDI-I), brain-lipid-binding protein (BLBP) and TI-225
(Ubiquitin C). Among them NDPK-A, NDPK-B, and CyP-A showed significantly different levels of expression in the brain from ET+/+ compared to that from ET/-. These proteins are known to be involved in cell proliferation, differentiation and development, suggesting that ET -1 plays an important role in proliferation, survival and differentiation of astrocytes and neurons.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:endothelins astrocytes neurons ischemia anoxemia transgenic mice
Date of Publication:01/01/2004