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

Han XJ, Chen M, Hong T, Zhu LY, He D, Feng JG, Jiang LP - Int. J. Mol. Med. (2015)

Bottom Line: To establish vascular restenosis, rat carotid arteries were subjected to balloon angioplasty 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Pharmaceutical Science, Nanchang University, Nanchang, Jiangxi 330006, P.R. China.

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.

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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.
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f4-ijmm-35-04-0885: 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.

Mentions: Since the mRNA and protein expression of Cx43 was upregulated during the development of vascular RS (Fig. 2), we firstly examined the effect of Cx43-RNAi-LV on the balloon injury-induced expression of Cx43. Compared with the untreated or NC-GFP-LV-treated arteries, infection with Cx43-RNAi-LV significantly decreased the balloon injury-induced expression of Cx43 (Fig. 4). In addition, the histomorphological observation of the arterial sections (Fig. 5A) and statistical analysis of the intimal/medial areas (Fig. 5B) revealed that the lentivirus-mediated RNAi knockdown of Cx43 significantly attenuated balloon injury-induced intimal hyperplasia and RS. These results directly demonstrate that the GJ protein, Cx43, contributes to the development of vascular RS following balloon injury, although the underlying mechanisms require further investigation.


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

Han XJ, Chen M, Hong T, Zhu LY, He D, Feng JG, Jiang LP - Int. J. Mol. Med. (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356439&req=5

f4-ijmm-35-04-0885: 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.
Mentions: Since the mRNA and protein expression of Cx43 was upregulated during the development of vascular RS (Fig. 2), we firstly examined the effect of Cx43-RNAi-LV on the balloon injury-induced expression of Cx43. Compared with the untreated or NC-GFP-LV-treated arteries, infection with Cx43-RNAi-LV significantly decreased the balloon injury-induced expression of Cx43 (Fig. 4). In addition, the histomorphological observation of the arterial sections (Fig. 5A) and statistical analysis of the intimal/medial areas (Fig. 5B) revealed that the lentivirus-mediated RNAi knockdown of Cx43 significantly attenuated balloon injury-induced intimal hyperplasia and RS. These results directly demonstrate that the GJ protein, Cx43, contributes to the development of vascular RS following balloon injury, although the underlying mechanisms require further investigation.

Bottom Line: To establish vascular restenosis, rat carotid arteries were subjected to balloon angioplasty 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Pharmaceutical Science, Nanchang University, Nanchang, Jiangxi 330006, P.R. China.

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.

Show MeSH
Related in: MedlinePlus