Adenovirus-mediated prothymosin α gene transfer inhibits the development of atherosclerosis in ApoE-deficient mice

Abstract

Prothymosin α (ProT) is involved in regulating expression of the oxidative stress-protective genes and it also exerts immunomodulatory activities. In this study, we investigated the therapeutic effects of ProT gene transfer on atherosclerosis in endothelial cells and in ApoE-deficient mice. Adenoviruses encoding mouse ProT (AdProT) were used for the management of atherosclerosis. In vitro, the effects of ProT on antioxidant gene expressions and the protection effect against oxidant-mediated injury in endothelial cells were examined. In vivo, AdProT were administered intraventricularly into the heart of ApoE(-/-) mice. Histopathological and immunohistochemical assessments of the aortic tissues were performed. Expressions of HO-1 and antioxidant genes in the aortic tissues were also determined. Our results demonstrated that ProT gene transfer increased antioxidant gene expressions, eNOS expression and NO release, as well as reduced the reactive oxygen species production in endothelial cells. Intraventricular administration of AdProT reduced the lesion formation, increased expressions of HO-1 and SOD genes, and reduced infiltrating macrophages in the aorta of ApoE(-/-) mice. This study suggests that ProT gene transfer may have the therapeutic potential for the management of atherosclerosis via inducing antioxidant gene expressions, eNOS expression and NO release, reducing ROS production and macrophage infiltration in endothelium.

Keywords: antioxidant gene expressions; atherosclerosis; gene transfer; prothymosin α..

Figure 1

Expressions of ProT and HO-1 in HUVECs after ProT gene transfer. HUVECs were infected with AdLacZ or AdProT at a MOI of 100 for 48 h. (A) ProT and HO-1 mRNA expressions were detected by RT-PCR. (B) HO-1 expression was detected by Western blotting.

Figure 2

Reductions of ROS formation and H₂O₂-induced cell damage in HUVECs after ProT gene transfer. (A) HUVECs were treated with AdLacZ or AdProT at a MOI of 100 for 72 h, and incubated with H₂DCFDA for 30min. The intracellular ROS was measured by flow cytometry. (B) HUVECs were treated AdLacZ or AdProT at a MOI of 100 for 48 h prior to a 4 h H₂O₂ (250 μM) incubation. Cell viability was determined by MTT assay. Results are expressed as mean ± SEM (n = 4). *, p<0.05; ***, p<0.001.

Figure 3

Effects of ProT overexpression on the antioxidant gene expressions, eNOS expression and NO production in HUVECs. HUVECs were infected with AdLacZ or AdProT at a MOI of 100 for 48 h. The SOD expressions were detected with RT-PCR (A), eNOS expression was detected by Western blotting (B) and the accumulation of nitrite in the supernatants was measured with Griess reagent (C). Data are expressed as mean ± SEM. *, p< 0.05.

Figure 4

Effects of ProT overexpression on iNOS expression and NO production in RAW264.7 cells. RAW264.7 cells were infected with AdLacZ or AdProT at a MOI of 400. (A) 48 h post-infection, the iNOS expression was detected by Western blotting, and (B) the accumulation of nitrite in the supernatants was measured with Griess reagent.

Figure 5

AdLuc expression and effects of ProT overexpression on HO-1 and SOD expressions in the aorta of ApoE^-/- mice intraventricularly receiving AdLuc or AdProT. 9-week-old ApoE^-/- mice were received 2 × 10⁸ PFU AdLuc (n=2) or AdProT (n=2). Tissues were collected 3 days after injection. (A) Luciferase activity in the heart and aorta of AdLuc-treated mice determined by luciferase assay. (B) Effects of ProT overexpression on HO-1 and SOD mRNA expressions in the aortic tissues of ApoE^-/- mice detected by RT-PCR.

Figure 6

AdProT reduced the atherosclerotic lesion formation in ApoE^-/- mice. 9-week-old ApoE^-/- mice were received intraventricularly 2 × 10⁸ PFU AdLacZ (n=6) or AdProT (n=6). Tissues were fixed, dehydrated, and embedded in paraffin. Serial sections (n=8) of 5 μm thickness were collected. (A) Histological assessment of lesion size in the aortic sinus. Sections were stained with H&E and observed under a microscopy (magnification × 200). The quantitative data were shown in (B). (C) Immunostaining for macrophages in the lesion (magnification × 400). (D) Quantitative comparison of macrophage composition in AdLacZ- and AdProT-treated groups (n = 6). Data are expressed as mean ± SEM. *, p<0.05.