Abstract
Atherosclerosis is an inflammatory disorder of arteries and reactive oxygen species (ROS) have been implicated as a major etiology. Various anti-ROS genes, such as superoxide dismutates and catalase, have been studied by gene transfer for their abilities to limit a variety of ROS-related cardiovascular injuries such as balloon-induced and ischemia-reperfusion injury. However the thiol-ROS compartment has never been explored by gene therapy/ gene delivery for potential therapeutic intervention. Human peroxiredoxin 6 (hPRDX6, AOL2) is fully unique in that in addition to its thiol-specific anti-oxidant ability it has a coupled phospholipase A2 catalytic domain. Here we delivered hPRDX6, expressed by the cytomegalovirus immediate early promoter (CMV), by systemic pseudotyped adeno-associated virus 2/8 (AAV2/8) gene transfer into low density lipoprotein receptor knockout mice on high cholesterol diet (LDLR KO HCD). It was found that AAV2/8/hPRDX6 gene delivery and expression inhibited systolic blood velocity, aortic cross sectional area, and aortic wall thickness compared to Neo-HCD control, consistent with reduced atherosclerosis. Markers of macrophages/foam cells, CD68, ITGAM, EMR, verified lower atherosclerosis, as all were lower in the AAV8/hPRDX6-HCD-treated animals compared to Neo-HCD controls, analyzed by either quantitative reverse transcriptase polymerase chain reaction amplification or by immuno-histochemistry, or both. Analysis of the immune state of the aortas (Th1 or Th2 cytokine expression) revealed nothing significant, with only IL-10 expression being lower in PRDX6-treated animals. This study, for the first time, demonstrated that hPRDX6 gene delivery, augmenting endogenous mouse (m)PRDX6 expression, giving therapeutic benefit against HCDdriven atherosclerosis and suggests further studies of PRDX6, and related anti-thiol-ROS approaches, is warranted.
Highlights
Reactive oxygen species (ROS) are present in all cells and are eliminated by antioxidants
To investigate the use of peroxiredoxin 6 (PRDX6) we utilized the LDLR KO mouse which develops atherosclerosis when placed on high cholesterol diet (HCD)
HPRDX6 gene expression is not pro-fibrotic. These data demonstrate that hPRDX6 gene delivery by associated virus 2/8 (AAV2/8) using systemic tail vein injection is effective in both delivery and therapeutic effect
Summary
Reactive oxygen species (ROS) are present in all cells and are eliminated by antioxidants. Sometimes ROS acts as intracellular signaling molecules [2], but ROS is very important because of the widespread damage it can cause to cellular proteins [3], DNA [4], and lipids [5]. ROS appears to be a factor in the earliest stage of atherosclerosis, the activation of endothelial cells, leading to their dysfunction [8,9,10]. Adhesion molecules are up-regulated which leads to the recruitment of monocytes, macrophages and other inflammatory cells. These recruited cells promote the generation of lipid engorged foam cells and the proliferation of vascular smooth muscle cells, both hallmarks of atherosclerosis. Clinical studies support ROS being a significant contributor to atherosclerosis [11,12]
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