Our preliminary data demonstrated that, in an atherosclerosis prone experimental model (hypercholesterolemic LDL receptor knockout mice - LDLR0), mitochondria are relevant sources of cellular oxidative stress due to a low antioxidant capacity of NADPH-dependent enzymes when compared to control mice. Moreover, LDLR0 mice showed an increased lipogenesis and steroidogenesis suggesting that these biosynthetic processes depleted the reducing equivalents from NADPH generating the oxidative stress observed in these knockout mice. A pharmacological inhibition of steroidogenesis could not be used in favor of our hypothesis due to direct toxic effects of statins on mitochondria. Therefore, our hypothesis that mitochondrial oxidative stress will be reduced in parallel with the reduction of cholesterol synthesis requires a molecular approach (antisense oligonucleotides and/or RNAi for knockdown in vivo and in vitro of HMGCoA). Considering that these mice have a C57BL6/J background, and that they also present a genetic deficiency in the gene that encodes for the mitochondrial NADP-transidrogenase (NNT), an enzyme essential for maintaining the content of NADPH in the mitochondria, the role of this enzyme will also be investigated. We will use the NNT knockdown in wild mice and transfection of NNT into LDLR0 cells. Therefore, in this proposal we will investigate the origin of mitochondrial oxidative stress observed in tissues and cells of hypercholesterolemic mice. Our hypothesis is that depletion of NADPH demonstrated in these mitochondria is a consequence of two possibilities, not mutually exclusive: increased steroidogenesis and deficiency of NNT. Therefore, using molecular tools in vivo and in vitro studies, in genetically engineered cells, we intend to quantify the relative contribution of each of these processes.
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