Research On The Role Of AMPK Enhancing Vascular Endothelial Cells Through MTOR Under Hypoxic Conditions

Background and ObjectivesThe brain is an organ of enhanced blood flow and high comsumption of oxygen. The brain accounts for 2% of body weight,15% of the blood flow from cardiac output and 20% of whole oxygen consumption. Reserving little energy, brain uses exclusively the oxygen and glucose from blood stream and thus is highly dependent on the cerebral circulation. Irreversible brain damage occurred when hypoxic-ischemic events last more than five minutes. Cerebral circulation is essential to the normal function of the brain, especially in acute ischemia and other hypoxia conditions. Cerebral circulation forms a highly specialized vascular bed, in which vascular endothelial cells play an important role in the regulation of the cerebral circulation. In many phathological conditions involving ischemia and hypxia, vascular endothelial cells were damaged. We previously focused on the damage of neurons, glial cells, neural stem cells during hypoxia, few is known about vascular endothelial cellular damage. Ischemic hypoxia is accompany to multiple diseases. As a result, study on the molecular mechanism of damage in vascular endothelial cells during ischemic hypoxia has clinical significace.In hypoxia, functional alteration in vascular endothelia cells include apoptosis, proliferation and aotophage. In this research, we explored the molecular mechanism of apoptosis, proliferation and autophage in vascular endothelial cells during hypxia.AMP-activated protein kinase (AMPK) is the sensor of metabolism. Mammalian AMPK is formed by a,3, and Y subunits. a subunit contains a highly conserved Serine/Theonine domain. The posphoralytion of Thr-172 next to the N-terminal is crutial to the enzymatic activaty of AMPK.3 subunit is not catalytic, probably reponsible for the assembly of AMPK complex and the sensing of glucogen. Y subunit regulates the enzymatic activaty of AMPK in reponse to ADP/AMP ratio. AMPK mainteins energetic balance between ATP production and consumption. Nutrition deprivation activates AMPK while energy overload inhibits it. The net effect of AMPK includes lipid and glucose metabolism, energy expenditure, immunoreaction, cell proliferation and polarization. AMPK is widely expressed in multiple tissues. Few research focused on the AMPK in endothelial cells in brain vessels.Mammalian target of rapamycin (mTOR) is a Serine/Theonine kinase mediating cell growth. mTOR reacts to positive signals of nurition (glucose or amino acids) and growth factors. mTOR complex 1 (mTORC1) comprises four subunits:Raptor, PRAS40, mLST8 and mTOR. mTORC1 is sensitive to nuritional signal, and can be blocked rapidly by rapamycin, regulating cell growth, angiogenesis and metabolism. Conversely, mTORC2 is insensitive to nutritional signal and can not be inhibited by rapamycin. Two known substrates of mTORC1 is 4EBP1 and S6 kinase. mTOR deactivates quickly in response to various stress to avoid cells from damage. Unlike most kinase dependent on growth factors, mTOR rely mostly on nutritional signal.In hypoxia, a serious of signaling pathway are altered for cell survival, most of which are triggered by Hypoxia inducible factor 1 (HIF-1). The transcription and stability of HIF-1 is controlled by the ability of cells to use oxygen. Active HIF-1 is a heterodimer of HIF-1 a and HIF1b. HIF-1 a is unstable, tightly mediated by oxygen. HIF-la is upregulated in hypoxia. HIF-lb is irrelevent of hypoxia.Cerebrovascular endothelial cells (CVEC) go through damage under various phathological condition, such as hypoxia and ischemia. Ischemia is a common accompanied sympotom of multiple disease. Study on the adaptation of CVEC in hypoxia is clinical meaningful. AMPK has found to be essential in neural system, however, few is known about its role in CVEC. We isolated CVEC of mice to study the role of AMPK and HIF-la in the function of CVEC in hypoxia, including proliferation, apoptosis. Autophage is also an important way for cells to keep homeostasis. We also observed the effect of AMPK-mTOR in the autophage of CVEC.Part1 In hypoxia,the role of AMPK in the proliferation and mitichondria related apoptosis in cerebral vascular endothelial cellsAim:To study the expression of AMPK in CVEC during hypoxia and whether HIF-1a participates in this process and how AMPK mediating the proliferation and apoptosis of CVECMethods:CVEC were isolated and cultured in hypoxia (1% oxygen). The expression of AMPK was detected by real time qPCR in 0 hr,4 hr,12 hr and 24 hr respectively after exposed to hypoxia. To explore whether HIF-1a was involved in this process we treated CVEC with HIF-1a inhibitor during CVEC exposed to hypoxia and then detected the activation of AMPK. To study the role of AMPK in the proliferation and apoptosis of CVEC, MTT was applied to monitor the proliferation of CVEC in hypoxia treated with or without AMPK shRNA or AMPK inhibitor. The apoptosis of CVEC in hypoxia in the presence or absence of AMPK shRNA was determined by flow cytometry. And the expression of Bcl-2 and Cleaved Caspase 3 in this process were detected by Western blot. Ros production was also tested accodringly.Results:AMPK2a was mainly expressed in mice CVEC and the phosphorylation of AMPK was enhanced in hypoxia. When HIF-a was inhibited, the expression of AMPK induced by hypoxia was decreased and the phosphorylaiton of AMPK was reduced accordingly, suggesting AMPK was activated by HIF1a in hypoxia. AMPK promoted cell proliferation and inhibited apoptosis by enhancing Bcl2 and depressing Caspase 3 cleavage. Inhibition of AMPK led to over produciton of ROS in mitochondria.Part2 In hypoxia,AMPK-mTOR regulated autophage in cerebral vascular endothelial cellsAim:To obeserve the autophage of CVEC in hypoxia and whether HIF-1 participated in this process and the relationship between AMPK and apoptosis. To study whether the effect of mTOR on CVEC is under the control of AMPK and explore interaction of apoptosis and autophage in CVEC induced by hypoxia.Methods:After exposed to hypoxia, we detect the distribution of LC3 by immunofluorescence, and the expression of LC3I, LC3II and the situation after AMPK downregulation. To understand hypoxia induced autophage through HIF-1, we treated CVEC with HIF-1 inhibitor, and then observe the expression of HIF-1 a, phosphorylated AMPK, LC3 I, LC3 II by western blot and the binding of Bcl-2 with Beclin through immunoprecipitation. To study the relationship between AMPK and apoptosis, we detected the level of cleaved caspase 8 and Bid. To study whether AMPK mediated mTOR in CVEC, we block AMPK in anoxic CVEC, and detect the expression of AMPK, mTOR phosphorylation and S6K phosphorylation. To further observe mTOR, AMPK and autophage, we inhibited AMPK with shRNA, and then detect the expression of mTOR, LC3, the distribution of LC3 and the binding of Bcl-2 and Beclin.Results:During hypoxia, LC3 redislocated in cells as a indicator of autophage. Western blot showed that LC3 II increased in hypoxia, yet decreased after AMPK was blocked, indicating hypoxia leading to autophage of CVEC. Decrease of HIF-1 caused low phosphorylation of AMPK, showing that HIF-1 induced autophage through activation of AMPK. In hypoxia, inhibition of AMPK led to caspase-8 cleavage and higher expression of Bid, suggesting that AMPK has a protective role on CVEC in hypoxia. Inactivation of AMPK enhanced activation of mTOR signaling, while inhibition of mTOR resulted in increased autophage, illustrating by higher level of LC3 II. mTOR inhibited autophage in hypoxia. Decrease of mTOR enhanced the expression of Bcl-2, implying autophage antagonized apoptosis in hypoxia.ConclusionsTaken together, we found in hypoxia, AMPK-mTOR promoted the proliferation of CVEC, decreased apoptosis and increased autophage. And we explored the molecular mechanism behind this phenomenon to lay theoretical foundation for relevent clinical application.