Abstract
Extracellular superoxide dismutase (EC-SOD) has been implicated in regulation of vascular function but its underlying molecular mechanism is largely unknown. These two-step experiments investigate whether hemagglutinating virus of Japan envelope (HVJ-E) vector-mediated EC-SOD gene delivery might protect against neointima formation, vascular inflammation, and reactive oxygen species (ROS) generation, and also explore cell growth signaling pathways. The first in-vitro experiment was performed to assess the transfection efficacy and safety of HVJ-E compared to lipofectamine®. Results revealed that HVJ-E has higher transfection efficiency and lower cytotoxicity than those of lipofectamine®. Another in-vivo study initially used balloon denudation to rat carotid artery, then delivered EC-SOD cDNA through the vector of HVJ-E. Arterial section with H&E staining from the animals 14 days after balloon injury showed a significant reduction of intima-to-media area ratio in EC-SOD transfected arteries when compared with control (empty vector-transfected arteries) (p < 0.05). Arterial tissue with EC-SOD gene delivery also exhibited lower levels of ROS, as assessed by fluorescent microphotography with dihydroethidium staining. Quantitative RT-PCR revealed that EC-SOD gene delivery significantly diminished mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (p < 0.05 in all comparisons). An immunoblotting assay from vascular smooth muscle cell (VSMC) cultures showed that the EC-SOD transfected group attenuated the activation of MEK1/2, ERK1/2, and Akt signaling significantly. In conclusion, EC-SOD overexpression by HVJ-E vector inhibits neointima hyperplasia, inflammation, and ROS level triggered by balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMCs might play an important role in these inhibitory effects.
Highlights
Increasing evidence shows that inflammation and overformation of superoxide (O2−) are important pathological components of neointima hyperplasia [1, 2]
From the fluorescence microscopic images, we found that the numbers of green fluorescent cells were much more in the hemagglutinating virus of Japan envelope (HVJ-E) group than that of the lipofectamine® group in both vascular smooth muscle cell (VSMC) and HeLa cells
Hemagglutinating virus of Japan (HVJ)-E and lipofectamine®-mediated green fluorescence protein (GFP) transfection efficacy were determined by fluorescenceactivated cell sorting (FACS) analysis, which revealed that HVJ-E had significantly higher transfection rates than those
Summary
Increasing evidence shows that inflammation and overformation of superoxide (O2−) are important pathological components of neointima hyperplasia [1, 2]. SOD could protect cells from tissue damage associated with the inflammatory process and neutrophil-generated superoxide [5, 6]. EC-SOD is a secreted enzyme with a long half-life (20 h) in the circulation [10, 11]. It has been implicated in the regulation of vascular function [12, 13]. Several articles have reported the beneficial impact of EC-SOD on vascular remodeling after injury. None of these EC-SOD gene delivery experiments [14,15,16,17] have explored the cell growth signaling pathway
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