Lentivirus-mediated RNAi knockdown of the gap junction protein, Cx43, attenuates the development of vascular restenosis following balloon injury

Abstract

Percutaneous coronary intervention [PCI or percutaneous transluminal coronary angioplasty (PTCA)] has been developed into a mature interventional treatment for atherosclerotic cardiovascular disease. However, the long-term therapeutic effect is compromised by the high incidence of vascular restenosis following angioplasty, and the underlying mechanisms of vascular restenosis have not yet been fully elucidated. In the present study, we investigated the role of the gap junction (GJ) protein, connexin 43 (Cx43), in the development of vascular restenosis. To establish vascular restenosis, rat carotid arteries were subjected to balloon angioplasty injury. At 0, 7, 14 and 2 days following balloon injury, the arteries were removed, and the intimal/medial area of the vessels was measured to evaluate the degree of restenosis. We found that the intimal area gradually increased following balloon injury. Intimal hyperplasia and restenosis were particularly evident at 14 and 28 days after injury. In addition, the mRNA and protein expression of Cx43 was temporarily decreased at 7 days, and subsequently increased at 14 and 28 days following balloon injury, as shown by RT-PCR and western blot analysis. To determine the involvement of Cx43 in vascular restenosis, the lentivirus vector expressing shRNA targeting Cx43, Cx43-RNAi-LV, was used to silence Cx43 in the rat carotid arteries. The knockdown of Cx43 effectively attenuated the development of intimal hyperplasia and vascular restenosis following balloon injury. Thus, our data indicate the vital role of the GJ protein, Cx43, in the development of vascular restenosis, and provide new insight into the pathogenesis of vascular restenosis. Cx43 may prove to be a novel potential pharmacological target for the prevention of vascular restenosis following PCI.

Figure 1

Intimal hyperplasia and restenosis (RS) were induced in rat carotid arteries following balloon injury. (A) Histomorphological observation of control (no injury) and balloon-injured arterial sections. Carotid arteries were removed from the control or balloon-injured rats at the indicated time points. Following hematoxylin and eosin (H&E) staining, the general histological features of the arterial sections at 5 μm were examined under a microscope. Images at low and high magnification are shown in the upper and lower panel, respectively (upper panel, x100 magnification; bottom panel, x400 magnification). I, intima; M, media. (B) The intimal and medial area of the arterial sections was measured using Image-Pro Plus 5.0 software. Data represent the means of 6 independent experiments. ^*P<0.01 vs. control.

Figure 2

mRNA and protein expression of connexin 43 (Cx43) during the development of restenosis (RS) following balloon injury. (A) Electrophoresis of the RT-PCR product of Cx43 and β-actin on an agarose gel. Lanes 1–5 represent samples from the control, and days 0, 7, 14 and 28, respectively. Lane M represents DNA Marker. β-actin was used as an endogenous reference. DNA ladder was 2,000, 1,000, 750, 500, 250 and 100 bp from top to bottom, respectively. (B) Quantification of relative Cx43 mRNA level was indicated as the normalization of ratio of Cx43/β-actin in each sample relative to the control. Data represent the means of at least 3 independent experiments. ^*P<0.05 and ^**P<0.01 vs. control. (C) Western blot analysis of Cx43 protein expression during the development of RS following balloon injury. Lanes 1–5 represent samples from the control, and days 0, 7, 14 and 28, respectively. GAPDH was used as an endogenous reference. (D) Quantification of relative Cx43 expression was indicated as the normalization of ratio of Cx43/GAPDH in each sample to relative to the control. Data represent the means of at least 3 independent experiments. ^*P<0.05 and ^**P<0.01 vs. control.

Figure 3

Lentivirus-mediated RNAi knockdown of connexin 43 (Cx43) in vitro and the infection efficiency of the lentivirus vector in injured arteries. (A) Knockdown of Cx43 in NRK-52E cells by Cx43-RNAi-LV. Following infection with the lentiviral vectors, Cx43-RNAi-LV or NC-GFP-LV, the cells were harvested and the expression of Cx43 in each group was examined by western blot analysis. GAPDH was used as an endogenous reference. Lanes 1–4 represent samples from the control, 2.5×10⁸ TU/ml Cx43-RNAi-LV-treated cells, 5×10⁸ TU/ml Cx43-RNAi-LV-treated cells and NC-GFP-LV-treated cells, respectively. (B) Infection with Cx43-RNAi-LV and NC-GFP-LV in arteries. Following infection with lentivirus, the fluorescence signal of GFP in the arteries was examined under a fluorescence microscope to show the infection of Cx43-RNAi-LV or NC-GFP-LV. Scale bar, 100 μm.

Figure 4

Connexin 43 (Cx43)-RNAi-LV significantly inhibits the balloon injury-induced expression of Cx43 in arteries. (A) Expression of Cx43 in arteries. Lanes 1–4 represent samples from the control, balloon-injured arteries, Cx43-RNAi-LV + balloon injury and NC-GFP-LV + balloon injury, respectively. GAPDH was used as an endogenous reference. (B) Quantification of Cx43 expression was indicated as the normalization of ratio of Cx43/GAPDH in each sample relative to the control. Data represent the means of at least 3 independent experiments. ^**P<0.01 vs. control (no injury); ^*P<0.05 vs. untreated group.

Figure 5

Connexin 43 (Cx43)-RNAi-LV effectively attenuates the development of intimal hyperplasia and restenosis (RS) in arteries following balloon injury. (A) Histomorphological observation of arteries after the indicated treatments. Carotid arteries were removed after the indicated treatments, and were subjected to cryosections at 5 μm. Following hematoxylin and eosin (H&E) staining, the general histological features of the arterial sections were examined under a microscope. Images at low and high magnification are shown in upper and lower panels, respectively (upper panel, x100 magnification; bottom panel, x400 magnification). N, neointima; M, muscle. (B) The intimal and medial area of the arterial sections was measured using Image-Pro Plus 5.0 software. Data represent the means of 6 independent experiments. ^**P<0.01 vs. control (no injury); ^*P<0.05 vs. untreated group.