CFTR Suppresses Neointimal Formation Through Attenuating Proliferation and Migration of Aortic Smooth Muscle Cells

Figures

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  FIGURE 1.. CFTR expression decreased as neointimal formation progressed in balloon-injured rat carotid arteries. A, Representative western blot images and plot graphs show the protein expression levels of CFTR in VSMC media of carotid arteries isolated from sham-operated (Sham) and balloon-injured (Injury) rats at 14 days after surgery. n = 5 per group, *P < 0.05 versus Sham. B, Representative images show immunohistochemical staining for CFTR (yellow brown) in sections from sham-operated (Sham) or balloon-injured carotid arteries at 14 days after surgery (scale bar = 20 μm). Plot graphs show relative CFTR expression. n = 5 per group, **P < 0.01 versus Sham. (C, D), Rat carotid arteries were injured with a balloon and locally infected with adenovirus expressing green fluorescent protein (GFP) alone (Ad-GFP) or Ad-CFTR (1 × 109 PFU) for 30 minutes. C, Injured arteries were isolated for staining after 14 days. Western blot analysis showed the adenovirus transfection were effective in VSMC media of carotid arteries. D, Representative H&E-stained carotid arterial sections and the intima/media ratio of rat injured arteries (scale bar = 100 μm). n = 6 per group, #P < 0.05, ####P < 0.0001 versus…

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  FIGURE 2.. CFTR suppresses balloon injury–induced and PDGF-BB–stimulated VSMC proliferation. A, Protein expression of PCNA in injured arteries transfected with Ad-CFTR or Ad-GFP alone. n = 5, #P < 0.01 versus Sham, ***P < 0.001 versus Injury. B, VSMCs were treated with Ad-CFTR, Ad-GFP, or Ad-shCFTR and then exposed to PDGF-BB (20 ng/mL) for 24 hours. Cell viability was quantified by the MTT assay. n = 6, ####P < 0.0001 versus control cells. **P < 0.01, ****P < 0.0001 versus PDGF-BB–stimulated cells. (C, D), Protein expression of PCNA in VSMCs infected with Ad-GFP, Ad-shCFTR, or Ad-CFTR, respectively. n = 6, ###P < 0.001, ####P < 0.0001 versus control cells. *P < 0.05 versus PDGF-BB–stimulated cells. (E, F), Expression of Ki67 in PDGF-BB–stimulated VSMCs infected Ad-GFP, Ad-shCFTR, or Ad-CFTR, respectively. n = 5, *P < 0.05 versus PDGF-BB–stimulated cells. n = 5, ###P < 0.001, ####P < 0.0001 versus control cells,*P < 0.05 versus PDGF-BB–stimulated cells.

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  FIGURE 3.. CFTR inhibits the activation of PDGFRβ and SGK-1. (A, B), Ad-shCFTR- or Ad-CFTR-infected VSMCs were treated with or without 20 ng/mL PDGF-BB for 2 minutes, and then, PDGFRβ protein and p-PDGFRβ expression were analyzed by western blotting. n = 6, ###P < 0.001, ####P < 0.0001 versus control cells, *P < 0.05, **P < 0.01 versus PDGF-BB–stimulated cells. C, Representative images show immunohistochemical staining for p-PDGFRβ in sections from sham-operated, balloon-injured carotid, Ad-CFTR-treated, or Ad-GFP-treated injured arteries at 14 days after surgery (scale bar = 50 μm). Plot graphs show relative p-PDGFRβ expression. n = 5 per group, ####P < 0.0001 versus Sham, **P < 0.01 versus Injury. (D, E), Protein expression of SGK1 and p-SGK1. n = 6, ##P < 0.01 versus control cells. *P < 0.05, **P < 0.01 versus PDGF-BB–stimulated cells.

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  FIGURE 4.. CFTR inhibits the activation of p38, JNK, and ERK. (A, B), Ad-shCFTR- or Ad-CFTR-infected VSMCs were treated with or without 20 ng/mL PDGF-BB for 2 minutes, and p38 expression was analyzed by western blotting. n = 6, ###P < 0.001, ####P < 0.0001 versus control cells, *P < 0.05 versus PDGF-BB–stimulated cells. (C, D), Protein expression of JNK and p-JNK in VSMCs treated with 20 ng/mL PDGF-BB for 15 minutes n = 6, ##P < 0.01, ####P < 0.0001 versus control cells, *P < 0.05, **P < 0.01 versus PDGF-BB–stimulated cells. (E, F) Protein expression of ERK and p-ERK in VSMCs treated with 20 ng/mL PDGF-BB for 1 minute n = 6, ####P < 0.0001 versus control cells, **P < 0.01 versus PDGF-BB–stimulated cells.

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  FIGURE 5.. CFTR suppresses PDGF-BB–induced VSMC migration. VSMC migration was evaluated by a scratch assay (A and C) and transwell assay (B and D). (A and C) Representative images of the scratch assay and the quantification results for the wound closure assay n = 5, ###P < 0.001 versus control cells, *P < 0.05, ****P < 0.0001 versus PDGF-BB–stimulated cells. (B and D). Ad-shCFTR- or Ad-CFTR-infected VSMCs were transferred to transwell filters, and the migration ability of VSMCs in response to 20 ng/mL PDGF-BB was determined. Representative transwell filters are shown (100×) from 5 independent experiments. n = 5, ####P < 0.0001versus control cells, *P < 0.05, ****P < 0.0001 versus PDGF-BB–stimulated cells.

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  FIGURE 6.. CFTR downregulated MMP2/9 expression in VSMCs. Ad-shCFTR- or Ad-CFTR-infected VSMCs were transferred to VSMCs and stimulated with 20 ng/mL PDGF-BB for 24 hours. Quantitative real-time RT-PCR analysis of mRNA levels of MMP2 (A) and MMP9 (B). n = 5, ##P < 0.01, ####P < 0.0001versus control cells, *P < 0.05, ***P < 0.001, ****P < 0.0001 versus PDGF-BB–stimulated cells.