Programme(s) to which this project applies:
|☒ MPhil/PhD||☑ MRes[Med]||☒ URIS|
Objective and Significance:
Our recent evidence demonstrate that overexpression of SIRT1 blocks the senescence process of cultured primary endothelial cells (PAEC), at least in part through targeting LKB1 and its downstream AMP-activated protein kinase (AMPK). Both SIRT1 and AMPK are considered as essential energy regulators and redox sensors, and show stimulatory effects on endothelial nitric oxide synthase (eNOS), a key enzyme in vascular homeostasis.
The objectives of this study are to investigate whether or not endothelial over-expression of SIRT1, SIRT1(H363Y), or constitutively active AMPK affect eNOS activity in endothelial cells and the development of endothelial dysfunction associated with various pathological conditions such as obesity, insulin resistance and diabetes. The results are expected to provide important insights which will reveal the functional mechanisms underlying the vascular protective activity of SIRT1 and delineate the crosstalk with AMPK signaling pathways.
Research Plan and Methodology:
First, we will evaluate the effects of 1) overexpression of LKB1 or its dominant negative mutant LKB1(D194A) and/or 2) SIRT1 activation (by both overexpression of SIRT1 and SRT1720 treatment) or inhibition [by overexpression of SIRT1(H363Y), transfection of small silencing SIRT1 plasmid or treatment with sirtinol] on phosphorylations and activities of eNOS, Akt and IRS-1, as well as the activities of PI3K associated with IRS-1. Second, we will examine whether or not adenovirus-mediated expression of a constitutively active Akt can counteract LKB1-induced suppression of eNOS phosphorylation in PAECs co-transfected with LKB1 and SIRT1(H363Y). Third, we will test whether down-regulation of LKB1 could enhance, or inhibition of PI3K/Akt pathway could attenuate the eNOS phosphorylation observed in SIRT1 overexpressing cells.
Lastly, transgenic mice with endothelial-specific overexpression of SIRT1 or its dominant negative mutant SIRT1(H363Y), and the constitutively active AMPKα1 will be subjected to 1) high fat diet; 2) intraperitoneal injection of streptozotocin; 3) cross breeding with atherosclerosis-prone apoE knockout mice; 4) intraperitoneal injection of paraquat. Both aortic and carotid artery rings will be collected for evaluating insulin-induced aortic relaxation, acetylcholine-induced relaxation in aorta or contraction (EDCF-mediated) in carotid arteries using multi Myograph System. The aortic tissues will also be subjected to histological examination for analyzing the proportion of senescence associated β gal– positive cells. ROS will be measured with 2', 7'-dichlorodihydrofluorescein. In summary, these experiments are expected to provide important and novel information on the concordant regulations of SIRT1 and LKB1/AMPK on eNOS and endothelium-dependent vascular functions under both physiological and pathological conditions.
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