The balloon denudation technique to rat carotid artery was similar to our previous method [29, 30]. For anesthesia, 10-week-old male Sprague-Dawley (SD) rats, weighing 350–400 g, were anesthetized with intraperitoneal pentobarbital (50 mg/kg). For euthanasia, the rats were sacrificed by 100% carbon dioxide (CO2) inhalation in airtight chambers. The experiments were conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Veterans General Hospital.
The transfection reagent, HVJ-E, was purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan), lipofectamine® was from Thermo Fisher Scientific (Waltham, MA, USA). The human EC-SOD cDNA cloned in pcDNA3.1TOPO expression vector was obtained from the Cardiovascular Biology and Atherosclerosis laboratory of The University of Health Science Center at Houston (Houston, TX, USA). The platelet-derived growth factor-BB (PDGF-BB), and β-actin, dihydroethidum, dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, USA). EC-SOD was obtained from the Cardiovascular Biology and Atherosclerosis laboratory of The University of Health Science Center at Houston (Houston, TX, USA). Anti-rat TNF-α and anti-rat IL-1β were purchased from Novus Biologicals (Littleton, Colorado, USA) and Santa Cruz Biotechnology (Dallas, Texas, USA), respectively.
Primary rat vascular smooth muscle cells (VSMCs) were isolated from the thoracic aortas of adult male SD rats similar to our previously described method . Briefly, the isolated thoracic aorta was promptly placed in a cold PBS buffer-filled Petri dish. The fatty tissues, endothelial layer, and adventitial layers of the thoracic aortas were removed using sterile forceps and scissors. Then, the thoracic aorta was cut into small pieces of 1 × 2 mm in size. These tissues were replaced in a tissue culture dish with DMEM containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 g/mL streptomycin, and were maintained at 37 °C in a humidified 5% CO2 incubator. When the culture cells reached 70–80% confluence, they were detached using 0.05% trypsin-EDTA for sub-culturing. Cells were cultured in DMEM with 10%. When the culture cells reached 70–80% confluence, they were detached using 0.05% trypsin-EDTA for sub-culturing. Culture media was changed every 3 days and the passage numbers from three to five generations were used for experiments. After synchronization by serum deprivation for 48 h, quiescent VSMCs were stimulated with 10% FBS or 20 ng/mL PDGF-BB for 24 and 48 h, respectively. The quiescent cells cycle were progressively effective for 24 and 48 h stimulation media incubation. HeLa cell line (BCRC 60005, Hsinchu, Taiwan) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin streptomycin solution (Gibco, Life Technologies, Carlsbad, CA).
Preparation and Transfection Using HVJ-E for the In Vitro Study
For the in vitro study, VSMCs of 60% confluence were cultured in serum-free Opti-MEM overnight prior to HVJ-E transfection. Immediately prior to transfection, Opti-MEM was replaced with DMEM containing 10% FBS). The transfection reagent was prepared according to the manufacturer’s protocol. Briefly, the lyophilized HVJ-E was suspended with ice-cooled buffer. The HVJ-E solution (40 μL for each 6-well plate wells) was centrifuged at 13,000 rpm for 5 min at 4 °C. Supernatant was discarded and the pellet was resuspended in 10 μg plasmid, with or without EC-SOD (vector) solution of 1 μg/μL concentration. The plasmid without EC-SOD was regarded as a vector for control study.
Transfection Efficiency of HVJ-E Measurement
To evaluate the efficiency of HVJ-E vector gene delivery in VSMCs and HeLa cells, the plasmid-encoded green fluorescence protein (GFP) was used as the transfection reporter. In addition, transfections with or without conventional transfection reagent lipofectamine® were used as experimental control groups. The plasmids of pEGFP vector encoding the GFP and pGL3-luciferase (LUC) reporter vector encoding luciferase protein were used to measure transfection efficacy of HVJ-E. Ten micrograms of pEGFP and pGL3-LUC plasmids were transfected into VSMCs and HeLa cells by HVJ-E vector or lipofectamine® reagent, respectively. Lipofectamine® transfection was conducted according to the manufacturer’s instructions. After 24-h transfection, the cells with GFP expression were detected by fluorescence microscopy (AXIO, Zeiss, Oberkochen, Germany) and FACS analysis (BD FACSCalibur, Fitchburg, WI). LUC activity was detected by luciferase assay system (Promega) using a luminescence microplate reader (Berthold Technologies, Bad Wildbad, Germany).
Cell Viability Assay
According to the manuals from the ATCC and Roche kits, we chose MTT assay for measurement of VSMC and HeLa cells’ viability. VSMCs’ viability was measured by cell counting and by use of the MTT assay kits 24 h after transfection and incubation. Cell viability data are represented as the relative ratio to the serum-free non-transfected group.
Preparation and Transfection Using HVJ-E for the In Vivo Study
EC-SOD cDNA was inserted into the mammalian expression vector pcDNA3.1D/V5-His-TOPO, and the construct was delivered using the HVJ-E vector. Non-transfected and pcDNA 3.1-TOPO vector-transfected specimens were used as experimental controls. The transfection procedure was performed following manufacturer’s protocol. Briefly, lyophilized HVJ-E powder provided by the manufacturer was suspended with ice-cooled buffer. The 200 μL of HVJ-E solution used for each rat was centrifuged at 13,000 rpm for 5 min at 4 °C. Supernatant was discarded and the pellet was resuspended in 50 μg of either vector or EC-SOD plasmid solutions. Reagent B was added into the DNA-HVJ-E mixture and centrifuged at 13,000 rpm for 5 min at 4 °C to enhance the adhesion of the DNA to the HVJ-E membrane. Supernatant was discarded and the pellet resuspended in buffer combined with Reagent C to increase affinity between the EC-SOD bearing HVJ-E and the cells. The final mixture of 125 μL was injected into the carotid artery and maintained there for 15 min.
Balloon Injury Model
This study was conducted in accordance with the guidelines of “Kaohsiung Veterans General Hospital Animal Care and Use Committee” under the approved animal study protocol (VGHKS-101-A008). The balloon denudation technique was performed to rat left carotid artery; the right side carotid artery without balloon injury was used as the control. The detail process of surgical method was similar to the method used in our previously reports [29, 30]. Briefly, male Sprague-Dawley (SD) rats weighing 350–400 g were anesthetized with intraperitoneal pentobarbital (50 mg/kg, Sigma-Aldrich, Inc. Missouri, USA), then the left carotid artery was exposed. A Fogarty 2F embolectomy balloon catheter was inserted into the left external carotid artery via arteriotomy and advanced to the aortic arch. The balloon was inflated and withdrawn three times with rotation at the same pressure. The injured segment was clamped with two hemostatic clips on both ends and washed three times with normal saline to remove all residual blood. Plasmid with EC-SOD (n = 12) or without EC-SOD (n = 12) bearing HVJ-E mixture of 125 μL was injected into the carotid artery and maintained there for 15 min. In other words, the SOD-treated group was the rat treated with EC-SOD gene transfer through HVJ-E (or balloon injury + EC-SOD group). The control groups included (1) the right carotid artery without balloon injury and (2) the left carotid artery received balloon denudation technique and transfected with pcDNA 3.1-TOPO vector (balloon injury + HVJ E vector group) but without SOD gene. Fourteen days after balloon injury, the rats were sacrificed using 100% CO2 inhalation in airtight chambers, and sections from both the right and left carotid arteries were excised. The lesion length of balloon injury was about 2.0~2.5 cm. Before preparation, the carotid artery specimen, the portion of each carotid artery with length of 5 mm near the aortic arch, was removed. The reason to discard this most proximal portion was being avoiding the measurement variation due to balloon injury-induced local reaction. The remaining specimen was separated into two parts: the portion close to the aortic arch was prepared for quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and ROS detection studies; the distal portion was fixed with 6% formalin for subsequent analysis. The paraffin-embedded samples were sectioned into 6-μm thickness and used for hematoxylin and eosin (H&E) stain, dihydroethidium (DHE) stain, and immunohistochemistry analysis. The extent of neointimal formation was quantified by computed planimetry of histologically stained sections. The intima-to-media (I/M) area ratio was measured using ImageJ software (NIH, version 1.45).
Detection of Reactive Oxygen Species
Reactive oxygen species (ROS), such as superoxide, have been proposed to be important signaling molecules in the pathogenesis of intimal thickening in atherosclerosis. To evaluate the effect of HVJ-E-EC EC-SOD in detecting the in situ ROS radical in balloon-injured carotid arteries, the sections were stained with DHE. Carotid artery tissue section from the experimental SD rat was placed on a glass slide. The DHE (10 μM) was topically applied to each tissue section, and covered with a cover slip. Slides were incubated in a dark chamber at 37 °C for 30 min. The tissue section was washed three times in PBS. Fluorescence was detected with a fluorescent microscope (Olympus BX51, Tokyo, Japan) under an excitation wavelength of 535 nm. Fluorescence intensity was measured using ImagingJ software. The extent of fluorescence intensity of the DHE stained cross sections of carotid arteries was expressed as “mean gray levels.” The DHE fluorescence stained area was also measured. It was displayed as the ratio of the cross-sectional area of aorta tissue (including intima, media, and adventitia) to that of outside border of entire aorta (%). The intensities of the DHE fluorescence of the intima and media layers of carotid arteries were also measured.
The effect of HVJ-E-EC-SOD on balloon injury-mediated inflammatory response was evaluated by inflammatory cytokines TNF-α and IL-1β detection with immunohistochemistry (IHC) analysis in carotid artery sections. Formalin-fixed, paraffin-embedded core biopsies were sectioned into 6-μm sections and mounted on slides. Following deparaffinization in xylene, slides were dehydrated in an alcohol graded series and placed in running water. The Novolink Polymer Detection System (Leica) was used for immunohistochemistry. The antigen was retrieved by heating in 10 mM citrate buffer (pH 6.0). Slides were then incubated with peroxidase block to neutralize endogenous peroxidase activity, followed by anti-TNF-α (Abcam, Cambidge, MA, USA) and anti-IL-1β (Abcam, Cambidge, MA, USA) antibody (1:100, H00011065-M01, Abnova). Slides were activated with Novolink polymer followed by diaminobenzidine hydrochloride (DAB) chromogen solution to develop peroxidase activity to facilitate visualization of the antibody–DAB complex. Slides were then counterstained with hematoxylin, and the intensities of TNF-α and anti-IL-1β staining measured by ImageJ software (version 1.45). The method for quantifying the intensity of the immunohistochemistry (IHC) staining was similar to the commonly used method of the ImageJ software . The image intensity was defined as “gray level.” The mean gray level was the ratio of the integrated gray levels of total pixels in the region of interest (ROI) divided by the total pixel numbers in same ROI area. Firstly, the ROI of specific area for evaluation was selected by using the ROI Manager. The background image intensity of the cavity of carotid artery was initially determined by measuring the mean gray levels of the cavity area, or “Intensity-1.” Afterwards, we determined the mean gray level of the selected ROI of the outside border of the entire carotid artery tissue of the IHC images, which included IHC staining particles in the carotid artery tissue and the carotid artery cavity (without IHC staining). The resultant mean gray level of the entire ROI was the “Intensity-2.” Thus, the Intensity-2 minus the Intensity-1 was the actual intensity of the IHC staining. The quantifying of the TNF-alpha and IL-1beta staining intensities for individual intima and media layers were also studied.
Total RNA of the rat carotid arteries was extracted with Trizol reagent (Invitrogen, USA), and the first-strand cDNA synthesized at 42 °C for 60 min using SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). The mRNA expression of IL-1β or TNF-α in the rat carotid arteries was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) with SYBR Green PCR master Mix (Applied Biosystems, Carlsbad, CA, USA). The sequences of IL-1β primers: 5′-TCT TTGAGGCTGACAGAC-3′ and 5′-CTTGGGTCCTCATCCTGGAA-3′; TNF-α primers: 5′-CCAGGCGGTGTCTGTGCCTC-3′ and 5′-CGACGTGGGCTACGGGCTTG-3′. The temperature conditions of 40 thermal cycles were denaturated at 95 °C for 1 s, with annealing and extension at 60 °C for 20 s. The relative expression levels of TNF-α and IL-1β in rat carotid arteries were calculated by internal control GAPDH. The sequences of GAPDH primers were 5′-GACATGCCGCCTGGAGAAAC-3′ and 5′-AGCCCAGGATGCCCTTTAGT-3′, respectively.
Western Blot Analysis
After synchronization by serum deprivation for 48 h, quiescent VSMCs were incubated in the absence or presence of EC-SOD gene for 48 h and subsequently divided into three groups: VSMCs without HVJ-E transfection (non-transfected group), VSMCs with HVJ-E but without EC-SOD gene transfection (vector transfected group), and VSMCs with EC-SOD gene transfer through HVJ-E (EC-SOD transfected group). Afterwards, VSMCs were stimulated with 10% FBS (10 min), or 20 ng/mL PDGF-BB (20 min). Reactions were terminated by washing twice with PBS and then homogenized with lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM DTT, 2.5 mM sodium fluoride, 50 μM sodium orthovanadate, 0.5 mM sodium pyrophosphate, 0.5 mM β-glycerophosphate, 1 mM AEBSF, 0.8 μM aprotinin, 50 μM bestatin, 15 μM E-64, 20 μM leupeptin, and 10 μM pepstatin A. Protein concentration was determined using the Bio-Rad protein assay kit and bovine serum albumin (BSA) as a standard. Twenty micrograms of protein were separated on a 10% sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) gel and transferred onto a poly vinylidene fluoride (PVDF) membrane. Western blot analysis was conducted following the manufacturer’s instructions and using specific antibodies against p-Akt, total Akt, p-ERK, total ERK, p-MEK, total MEK (Cell Signaling Technology, Beverly, MA, USA), EC-SOD (Abcam, Cambidge, MA, USA), and β-actin, respectively. Protein was detected with horseradish peroxidase conjugated secondary antibody (Chemicon, USA). At the end of incubation, the membranes were extensively washed with TBS. The immunoreactive bands were detected by chemiluminescence (ECL) reagents and developed by Hyperfilm (GE Healthcare, USA).
Continuous variable data were expressed as mean ± SD. The statistical significance of the inter-group differences was determined initially by one-way ANOVA with Bonferroni correction for multiple comparisons, followed by unpaired Student’s t test. A p value < 0.05 was considered statistically significant.