Male Sprague-Dawley rats (purchased from the Department of Animal, Nanchang University, Nanchang, China) weighing 300–400 g were maintained on a regular chow diet prior to the study. All procedures for the animal experiments were carried out in accordance with the National Institutes of Health Guidelines, and were approved by the Ethics Committee for Animal Axperiments of Nanchang University.

The rats were anesthetized with an intraperitoneal injection of Hydral (10%, 3.5 ml/kg; Harbin Pharmaceutical Group Co., Ltd., Harbin, China). To establish the model of vascular RS, the angioplasty balloon (1.5×20 mm; Cordis Corp., Miami, FL, USA) was inserted into the rat common carotid artery through an incision in the left external carotid artery, as previously described (21). The balloon was then sufficiently inflated in the carotid artery and was drawn 3 times consistently from the proximal area to the carotid bifurcation to produce endothelial denudation. The external carotid was ligated and blood flow in the common carotid was restored. In addition, the rats were intramuscularly injected with benzylpenicillin sodium (40×10⁴ IU/day for 3 days; Harbin Pharmaceutical Group Co., Ltd.) to prevent infection. The rats were euthanized by an overdose of Hydral at 0, 7, 14 and 28 days (n=6/group) following balloon injury. The injured common carotid arteries were collected for hematoxylin and eosin (H&E) staining or western blot analysis to evaluate vascular remodeling and the expression of Cx43 following balloon injury.

Three serial cryosections (at 5-μm-thick) were prepared from the middle portion of the rat injured common carotid arteries. The slices were stained with H&E (Sangon Biotech, Shanghai, China) and histomorphological observation was performed under a light microscope (Olympus, Tokyo, Japan) to examine the structure of the blood vessel wall following balloon injury. The intimal and medial area of the 3 serial cryosections in each sample were measured using Image-Pro Plus 5.0 software (Media Cybernetics, Inc., Houston, TX, USA).

Total RNA was extracted from the rat carotid tissue using TRIzol reagent (Tiangen, Beijing, China) and cDNA was synthesized from the extracted total RNA using the SuperScript III kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. The specific primers used for PCR were as follows: Cx43 sense, 5′-AAAGGCGTTAAGG ATCGCGTG-3′ and antisense, 5′-GTCATCAGGCCGAGG CCT-3′ [as previously described (23)]; β-actin sense, 5′-CCCA TCTATGAGGGTTACGC-3′ and antisense, 5′-TTTAATGT CACGCACGATTTC-3′. All specific primers were chemically synthesized (Generay Biotech Co. Ltd., Shanghai, China). The PCR reactions were performed using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The amplification conditions for Cx43 was as follows: 94°C for 2 min followed by 32 cycles at 94°C for 45 sec, 58°C for 45 sec, and 72°C for 90 sec, and a final extension at 72°C for 5 min. The PCR conditions for β-actin was as follows: 94°C for 2 min followed by 28 cycles at 94°C for 30 sec, 56°C for 30 sec, and 72°C for 90 sec, and a final extension at 72°C for 5 min. The amplified RT-PCR products were separated on 1.2% (w/v) agarose containing ethidium bromide (both from Sangon Biotech) for 30 min. The results of electro phoresis were photographed was using the Molecular Imager^® Chemi DOC^TXRS⁺ system, and the signal densities of the gels were analyzed using Quantity One software (both from Bio-Rad, Hercules, CA, USA).

The carotid arteries were pulverized in radioimmunoprecipitation assay (RIPA) lysis buffer (Sangon Biotech) using a homogenizer. The whole lysate was centrifuged at 12,000 rpm for 10 min at 4°C, and the pellets were resuspended in sample buffer containing 4% sodium dodecyl sulfate (SDS; Sangon Biotech). Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% skim milk for 1 h at room temperature, and were incubated with rabbit polyclonal anti-Cx43 antibody (zym-71-0700; 1:250 in 5% skim milk; Zymed Laboratories, San Francisco, CA, USA) or rabbit monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (MAB374; 1:1,000 in 5% skim milk; Chemicon, Temecula, CA, USA) overnight at 4°C. The membranes were then washed 3 times with TBST, then incubated with the relative HRP-conjugated IgG secondary antibody (ZB-5301 and ZB-2305; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 2 h at room temperature and washed in TBST 3 times. Chemiluminescence were carried out using Amersham ECL Prime Western Blotting Detection reagents, and the immunobloting signal was detected using the Molecular Imager Chemi DOC^TXRS⁺ system (Bio-Rad). The intensity of each Cx43 band was normalized to the GAPDH band, and the relative expression of Cx43 following balloon injury was normalized to the control.

In order to examine the role of Cx43 in the development of vascular RS, the lentivirus expressing shRNA targeting Cx43, Cx43-RNAi-LV, was constructed (GeneChem, Shanghai, China). In brief, the shRNA sequence for Cx43 (5′-AGAGCACGGCAAGGTGAAA-3′) was designed using the manufacturer’s RNA interference (RNAi) Designer program, and the negative control construct (control shRNA) was created using a scrambled sequence (5′-TTCTCCGAACGTGTCACGT-3′), as previously described (22). DNA oligos were chemically synthesized (GeneChem, Shanghai, China), annealed and inserted into the expression vector by double digestion with Age I and Eco RI (New England Biolabs, Ipswich, MA, USA), and ligated with T4 DNA ligase (Takara, Dalian, China) in accordance to the manufacture’s instructions. The ligation was transformed into competent E. coli cells and then confirmed by restriction enzyme analysis and DNA sequencing. The sequences were then cloned into pGCSIL-GFP to generate lentiviral vectors. The expression vectors and package vectors were then transfected into HEK293T cells (ATCC, Manassas, VA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Following 48 h of culture, the supernatants containing the lentiviruses, such as Cx43-RNAi-LV and NC-GFP-LV (negative control) were harvested. Purification was then performed using ultracentrifugation and the lentiviral titer was determined.

Firstly, the knockdown efficiency of Cx43 by the lentiviral vector, Cx43-RNAi-LV, was confirmed in NRK-52E cells (ATCC). In brief, NRK-52E cells at 30% confluence were infected with Cx43-RNAi-LV and NC-GFP-LV, and the cells were then harvested 7 days following infection to obtain whole cell lysate. Following electrophoresis of the cell lysate, the expression level of Cx43 in each group was examined by western blot analysis. To determine the effect of Cx43-RNAi-LV on the balloon injury-induced expression of Cx43 in the arteries, the rats were randomly divided into 4 groups (n=6 in each group) as follows: i) the control group (no balloon injury); ii) the untreated group with ballon injury (balloon injury only); iii) the group with ballon injury treated with Cx43-RNAi-LV (balloon injury + Cx43-RNAi-LV); and iv) the group with ballon injury treated with NC-GFP-LV (balloon injury + NC-GFP-LV). In order to knockdown Cx43 in the VSMCs, the lentivirus (5×10⁸ TU/ml, 100 μl) expressing shRNA or the scrambled RNA was injected into the balloon-injured rat carotid arteries using a polyethylene catheter. The rats were euthanized by an overdose of Hydral 28 days following balloon injury, and the injured carotid arteries were collected for H&E staining or western blot analysis to evaluate vascular remodeling and the knockdown efficiency of Cx43.

The quantitative data are presented as the means ± standard deviation (SD). Data were analyzed using either the Student’s t-test to compare 2 conditions or ANOVA followed by planned comparisons of multiple conditions. A value of P<0.05 was considered to indicate a statistically significant difference.

Balloon injury was inflicted to the rat common carotid arteries for the establishment of a model of vascular RS. At 0, 7, 14 and 28 days following balloon injury, the rats were sacrificed by an overdose of Hydral. The carotid arteries were removed and subjected to cryosection at 5 μm. A histomorphological observation of the arterial sections with H&E staining was carried tou to evaluate RS at different time points following balloon injury. Neointima formation occurred in the injured arteries at 7 days (Fig. 1A). Intimal hyperplasia and RS were particularly evident at 14 and 28 days following balloon injury. Compared with the control arteries, the intimal area of the arteries was significantly increased at 14 and 28 days following balloon injury (Fig. 1B). These histomorphological changes in the carotid arteries indicated that the model of vascular RS induced by balloon injury was successful established.

To investigate the role of the GJ protein, Cx43, in the development of vascular RS, the mRNA and protein expression of Cx43 in the arteries following balloon injury was examined by RT-PCR and western blot analysis, respectively. Compared with the controls, the mRNA expression of Cx43 was temporarily decreased at 7 days, and was subsequently increased at 14 and 28 days following balloon injury (Fig. 2A and B). Similarly, the protein expression of Cx43 was significantly upregulated at 14 and 28 days following balloon injury, although a transient decrease in Cx43 expression was detected at 7 days (Fig. 2C and D). These results suggest that the GJ protein, Cx43, is involved in the development of intimal hyperplasia and vascular RS following balloon injury.

In order to directly demonstrate the role of Cx43 in the development of vascular RS following balloon injury, the lentiviral vector expressing shRNA targeting Cx43 (Cx43-RNAi-LV) and the negative control lentiviral vector (NC-GFP-LV) were constructed. In vitro experiments revealed that the expression of Cx43 was specifically decreased in the NRK-52E cells infected with Cx43-RNAi-LV, but not in the NC-GFP-LV-infected cells (Fig. 3A). These results indicated that the lentiviral vector, Cx43-RNAi-LV, was effective in silencing Cx43. For the knockdown of Cx43 in vivo, the injured rat arteries were infected with Cx43-RNAi-LV or NC-GFP-LV, and the infection efficiency in vivo was examined by measuring the fluorescent signal of GFP. The green fluorescence was strongly observed in the neointima and media in the Cx43-RNAi-LV-treated and NC-GFP-LV-treated arteries, but not in the controls (Fig. 3B). This indicated that the balloon-injured rat carotid arteries were successfully infected with the lentivirus.

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.