Inhibition of Neointima Hyperplasia, Inflammation, and Reactive Oxygen Species in Balloon-Injured Arteries by HVJ Envelope Vector-Mediated Delivery of Superoxide Dismutase Gene
- 532 Downloads
Extracellular superoxide dismutase (EC-SOD) has been implicated in regulation of vascular function but its underlying molecular mechanism is largely unknown. These two-step experiments investigate whether hemagglutinating virus of Japan envelope (HVJ-E) vector-mediated EC-SOD gene delivery might protect against neointima formation, vascular inflammation, and reactive oxygen species (ROS) generation, and also explore cell growth signaling pathways. The first in-vitro experiment was performed to assess the transfection efficacy and safety of HVJ-E compared to lipofectamine®. Results revealed that HVJ-E has higher transfection efficiency and lower cytotoxicity than those of lipofectamine®. Another in-vivo study initially used balloon denudation to rat carotid artery, then delivered EC-SOD cDNA through the vector of HVJ-E. Arterial section with H&E staining from the animals 14 days after balloon injury showed a significant reduction of intima-to-media area ratio in EC-SOD transfected arteries when compared with control (empty vector-transfected arteries) (p < 0.05). Arterial tissue with EC-SOD gene delivery also exhibited lower levels of ROS, as assessed by fluorescent microphotography with dihydroethidium staining. Quantitative RT-PCR revealed that EC-SOD gene delivery significantly diminished mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (p < 0.05 in all comparisons). An immunoblotting assay from vascular smooth muscle cell (VSMC) cultures showed that the EC-SOD transfected group attenuated the activation of MEK1/2, ERK1/2, and Akt signaling significantly. In conclusion, EC-SOD overexpression by HVJ-E vector inhibits neointima hyperplasia, inflammation, and ROS level triggered by balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMCs might play an important role in these inhibitory effects.
KeywordsGene therapy Reactive oxygen species Restenosis Superoxide dismutase
Extracellular signal-regulated kinase
Fetal bovine serum
Hemagglutinating virus of Japan envelope
Mitogen-activated protein kinase
Platelet-derived growth factor-BB chain
Tumor necrosis factor-α
Vascular smooth muscle cells
Increasing evidence shows that inflammation and overformation of superoxide (O2−) are important pathological components of neointima hyperplasia [1, 2]. Excessive levels of superoxide may react with nitric oxide (NO) to form reactive nitrogen species that may eventually cause depletion of endogenous vascular NO . Hence, reduction of nitric oxide generation and direct deleterious effects of superoxide may compromise vessel patency [3, 4]. Combating oxidative stress by antioxidant enzymes is important to counteract this process.
Superoxide dismutases (SODs) are a group of antioxidant enzymes that catalyze the dismutation of superoxide radicals into hydrogen peroxide and oxygen. SOD could protect cells from tissue damage associated with the inflammatory process and neutrophil-generated superoxide [5, 6]. Three SODs have been identified, including a copper/zinc containing SOD (CuZn-SOD) which is primarily cytosolic in location, a mitochondrial manganese SOD (Mn-SOD), and extracellular SOD (EC-SOD). EC-SOD is mainly expressed in blood vessels, approximately 50% of the total SOD activity in human aorta is EC-SOD-derived . EC-SOD is mainly synthesized by vascular smooth muscle cells and it is found in inflammatory cells of injured tissue and atherosclerosis [8, 9]. EC-SOD is a secreted enzyme with a long half-life (20 h) in the circulation [10, 11]. It has been implicated in the regulation of vascular function [12, 13]. Several articles have reported the beneficial impact of EC-SOD on vascular remodeling after injury. However, none of these EC-SOD gene delivery experiments [14, 15, 16, 17] have explored the cell growth signaling pathway.
Inflammation after vascular injury has been considered an important contributor to atherosclerosis .Tumor necrosis factor (TNF)-α, a proinflammatory cytokine, has been reported to be a modulator of leukocyte adhesion and migration in vascular inflammatory diseases. Tumor necrosis factor-α (TNF-α) also has an impact on the regulation of the vascular cell adhesion molecule-1 gene expression in endothelial cells . Another critical proinflammatory cytokine, interleukin (IL)-1β, is one of the known target genes of NF-κB . Previous EC-SOD gene delivery studies have not assessed the effect of EC-SOD in regulation of TNF-α and interleukin-1-beta (IL-1β) expression [14, 15, 16, 21]. The impact of EC-SOD gene transfection in regulation of TNF-α and IL-1β expression is worthy of further study.
Hemagglutinating virus of Japan (HVJ) envelope (HVJ-E) is a purified product prepared through complete inactivation of Sendai virus. The HVJ-E vector kit (named GenomOne) has been claimed to be very useful and safe for transfection of molecules into cells and animal tissue by means of membrane fusion.  Thus, two-step experiments were performed to investigate (1) the transfection efficacy and safety issue of HVJ-E and (2) to evaluate the impacts of EC-SOD gene delivery in inhibiting neointima hyperplasia, reactive oxygen species (ROS), and vascular inflammation especially on TNF-α and IL-1β expression, as well as the fundamental cell growth signaling pathways.
All forms of reconstruction, whether directly attack the occluding lesion (endarterectomy, angioplasty) or bypass it (vein or prosthetic bypass), cause injury and a wound healing response [23, 24, 25]. This wound healing response may lead to luminal narrowing and ultimate failure of the reconstruction . Luminal narrowing or restenosis was the result of excessive intimal hyperplasia, which was a particular problem after carotid endarterectomy [26, 27] or carotid angioplasty and stenting . The aim of this study was to test the hypothesis that gene therapy using EC-SOD gene may have the potential in inhibiting the neointima hyperplasia, which might decrease restenosis after carotid artery reconstruction in patients with severe carotid artery stenosis.
Materials and Methods
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.
Comparison of Transfection Efficacy Between HVJ-E and Lipofectamine®
To determine the safety issue of HVJ-E vector in the gene transfection, the MTT assay was conducted on VSMCs and HeLa cells with HVJ-E and lipofectamine® treatment. HVJ-E treatment revealed significantly greater cell counts compared to those of lipofectamine® treatment (p < 0.005 between HVJ-E and lipofectamine treatment in VSMCs; and p < 0.001 between HVJ-E and lipofectamine® treatment in HeLa cells, respectively) (Fig. 1e). This result suggests that HVJ-E has a lower cytotoxicity than lipofectamine®.
HVJ-E-EC-SOD Gene Delivery Inhibits Neointima Hyperplasia
HVJ-E-EC-SOD Gene Delivery Inhibits ROS
HVJ-E-EC-SOD Gene Delivery Attenuates TNF-α and IL-1β Protein and mRNA Expression
Effect of EC-SOD on Phosphorylation of ERK, MEK, and Akt
Intravenous administration of EC-SOD has been shown to possess no inhibitory effect on the development of neointima hyperplasia . The present study investigated the inhibiting effect of neointima hyperplasia of rat carotid artery after balloon injury by local gene delivery of EC-SOD using HVJ-E vector. We demonstrated that HVJ-E-mediated EC-SOD expression led to a significant reduction of neointima growth, superoxide production, inflammatory response of the TNF-α, and IL-1β mRNA expression in rats with balloon-injured arteries. A mechanistic study in primary VSMCs showed that HVJ-E-EC-SOD downregulated cell growth signaling of ERK1/2, MEK1/2, and Akt activation. Our data suggest that local gene delivery of EC-SOD with HVJ-E vector may be a useful approach to protect against balloon injury-induced artery disease.
Data on the first experiment of this study has verified the transfection efficiency and safety of HVJ-E. It demonstrated that HVJ-E had a better transfection efficacy than that of the conventional vector-lipofectamine® (Fig. 1). The higher cell viability in HVJ-E transfected group might possibly contribute to the increased transfection rate compared to the lipofectamine-transfected group. The fundamental difference between HVJ envelope vector and lipofectamine vector is that HVJ-E vector transfection reagent is a novel and unique transfection material, which employs the membrane fusion ability of the envelope of Sendai virus (Hemagglutinating virus of Japan: HVJ). The genome RNA of HVJ is completely inactivated, so it can be considered to be a “non-viral” transfection reagent with safe and efficiency for transfection use. According to manufactory Manu, HVJ-E vector can be used in vitro and vivo, which was also supported by our results (Fig. 1). Conventional non-viral transfection tools, including lipofectamine or other cationic lipids, are incorporated into cells through endocytosis which results in degradation of most parts of the transferred DNA by lysosomes. On the other hand, HVJ-E vector resists degradation by lysosomes, making it easy to transfer the specified DNA. Therefore, HVJ-E vector yields highly efficient gene expression . Furthermore, sialic acid receptors, which are needed to trigger binding with HN protein, exist in almost all animal cells. Thus, HVJ-E vector is useful for a wide range of targets . After the first in-vitro experiment, we felt more confident about using HVJ-E in the in-vivo study.
There are considerable amounts of literatures targeting neointimal hyperplasia in multiple diverse animal models of balloon angioplasty, which have demonstrated that significant neointimal formation occurred at 10–14 days after balloon angioplasty [33, 34, 35]. In these references, neointima was undetectable in the first and third day specimens . Uniform layers of neointima cells first appeared at 7 days and neointima compartment gradually increased thickness from days 7 to 14 . Another animal study found that the relative expression of α-smooth muscle actin in injured segments dramatically decreased from 2 to 4 days after injury and slowly recovered at day 7 reaching the normal level at day 14. As expected, histological examination revealed a significant increase in intimal thickness associated with a slight increase in medial thickness at day 30 post injury. It means that at day 14 post injury, the neointimal formation can be visualized because there are main structural changes in the intimal layer and internal elastic laminae . Since the structural changes of neointimal formation can be well visualized at 14 days post injury and our previous experience found that significant reduction of neointima growth occurred at 2 weeks following carotid artery injury [29, 30]. Thus, this study decided to evaluate the effects of locally administered HVJ-E-SOD 2 weeks after carotid artery injury. We found that HVJ-E-EC-SOD-mediated reduction of neointima growth could be measured at 14 days following carotid artery injury.
ROS are important signaling molecules in the pathogenesis of atherosclerosis and restenosis . ROS production in vascular smooth muscle may contribute to oxidative stress and impaired relaxation in atherosclerotic vessels . Since EC-SOD acts as a superoxide scavenger, which is crucial in maintaining the physical function of cardiovascular system , this study provided evidence that EC-SOD gene therapy significantly reduces ROS radicals, as seen in the differences of fluorescence intensities in the arterial tissue sections in comparison with the groups without EC-SOD transfection (Fig. 3). This anti-oxidative effect may be one of the important mechanisms in inhibiting neointima hyperplasia after balloon injury.
Interleukin-1-beta (IL-1β) and tumor necrosis factor alpha (TNF-α) are key cytokines produced by monocytes and activated macrophages. They are the most important mediators of inflammation . This study assesses the effect of EC-SOD overexpression to the balloon injury-induced TNF-α and IL-1β expression using the immunohistochemistry assay. This study found that HVJ-E-EC-SOD gene delivery had significant impact on balloon injury-mediated reduction of the carotid artery inflammation (Fig. 4). This study also assessed the inflammatory response to balloon injury using a quantitative RT-PCR method. We demonstrated that HVJ-E-EC-SOD transfection remarkably inhibits TNF-α and IL-1β mRNA expression (p < 0.01) after balloon injury (Fig. 5). These findings indicate that overexpression of EC-SOD by HVJ-E vector has an anti-inflammatory effect on the arterial wall after balloon injury.
This study also evaluated the anti-inflammation effect of EC-SOD transfection after FBS- and PDGF-BB-stimulation using immunoblotting assay. PDGF was released from platelet alpha-granules in response to aggregation and inflammatory stimuli. Secretion of PDGF occurs after adhesion to the injured vessel wall, which is one of the key stimuli for smooth muscle cell migration and subsequent atherosclerosis [40, 41]. The anti-inflammation effect of EC-SOD transfection after PDGF BB-stimulation had not been investigated in previous in-vivo EC-SOD gene transfection experiments [14, 15, 16, 21]. The results of this study clearly demonstrate that transfection with EC-SOD is able to reduce FBS- and PDGF BB-stimulated inflammatory response (Fig. 6).
It has been reported that the fetal bovine serum (FBS) is the ideal medium growth supplement and generally preferred over other types of sera. Its high levels of nutrients and optimal combination of growth factors make FBS the most effective growth promoter for virtually any cell culture system . While PDGF-BB, a tyrosine kinase receptor agonist, may induce the proliferation in human smooth muscle cells [43, 44]. Both FBS and PDGF have been used as medium growth supplements for the in-vitro cell culture study. Since our previous experiments have experience in using the 10% FBS and 20 ng/mL PDGF-BB in stimulating the cell growth [29, 30], this study also used similar concentrations, i.e., 10% FBS and 20 ng/mL PDGF-BB.
To the best of our knowledge, most in-vivo cardiovascular EC-SOD gene therapy studies used adenovirus as the vector for gene transfer [14, 15, 16, 21, 45, 46]. Using a viral vector for gene therapy may have several disadvantages such as strong immunogenicity , more serious side effects, or even death [48, 49]. Here, we used a novel vector—the HVJ-E—as the transfection vector. HVJ-E has been developed as a non-viral vector, consisting of a HVJ envelope but its viral genome is removed . The HVJ-E vector can efficiently deliver therapeutic molecules such as genes, siRNA, decoy oligonucleotides, proteins, and anti-cancer drugs to various tissues in animals and humans with high degree of efficiency . As the viral genome is eliminated in the HVJ-E vector, replication does not occur and viral genes are not expressed in transfected cells [22, 51]. So far, no significant side effects have been reported. Furthermore, it has been claimed that the HVJ-E vector may be superior to presently available vectors for the treatment of intractable human diseases such as bladder carcinoma or melanoma .
The current European Society for Vascular Surgery (ESVS) 2017 Clinical Practice Guidelines of management of atherosclerotic carotid disease was published in January, 2018. This report has recommended that in “average surgical risk” patients with an asymptomatic 60–99% carotid artery stenosis, carotid endarterectomy should be considered in the presence of one or more imaging characteristics that may be associated with an increased risk of late ipsilateral stroke, provided documented perioperative stroke/death rates are < 3% and the patient’s life expectancy exceeds 5 years. The carotid stenting may be an alternative treatment to carotid endarterectomy in such patients . The response to the vessel wall injury subsequently triggers platelet aggregation, inflammatory cell infiltration, and proliferation of smooth muscle cells, resulting in restenosis, characterized by neointima hyperplasia . Our study shows that local delivery of EC-SOD gene may contribute to the inhibition of neointima hyperplasia, which might have the potential in decreasing the restenosis after carotid artery reconstruction.
There are several limitations that should be mentioned. Firstly, although this study demonstrated that local gene delivery of EC-SOD using HCJ-E vector was capable of inhibiting the neointima hyperplasia and inflammation, whether this method can be applied to clinical use still needs further investigations. Secondly, this study has observed the effect of local gene delivery of EC-SOD at 14 days after balloon injury, but has not assessed whether the inhibiting effect can sustain for a longer period of time. Further investigation for assessing the data after 14 days may be needed. Thirdly, investigation of the ROS and inflammation as well as the TNF-alpha and IL-1beta activities at much earlier time points (before 14 days) may be helpful in understanding the dynamic changes after balloon injury and EC-SOD treatment. However, since the rats’ aortic tissues were small, we only had limited quantity for immunohistochemistry, qRT-PCR, and ROS assay and therefore did not have adequate tissue to study the ROS and inflammation at early stages. It is the limitation of this study. Further investigations are needed to explore the early changes after balloon injury and EC-SOD treatment. Fourthly, although the in-vitro study indirectly demonstrated that EC-SOD gene delivery could significantly attenuate the activation of MEK1/2, ERK1/2, and Akt signaling. It would be interesting to perform further in-vivo study to demonstrate the direct evidence regarding how EC-SOD affecting these signaling activities. However, since the rats’ aorta tissue was limited, we did not have adequate tissue to study these protein signaling. Further in-vivo study is needed.
This study demonstrates that EC-SOD overexpression using HVJ-E gene delivery has significant effects in reducing the inflammatory response and ROS, and inhibiting the expression of TNF-α and IL-1β cytokines. All of these effects may contribute to the inhibition of neointima hyperplasia after balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMC might play an important role in these inhibitory effects. Local gene therapy using EC-SOD with HVJ-E vector might have potential clinical applications for the prevention of restenosis after carotid artery reconstruction.
The authors thank Miss Chen, Li-Ying for her hard work and technical assistance.
S.L. Lin conceived and designed the experiments, manuscript writing, and editing; P.C. Tsai performed experiments; T.H. Chang provided technical assistance; T.H. Chang, J.L.Yeh, W.C. Huang, and E. Sulistyowati analyzed the data; S.T. Lee and M. Wassler contributed reagents/material/analytical tools; Y.J. Geng and J.L.Yeh manuscript writing and editing.
This research was supported by the Kaohsiung Veterans General Hospital (Grant No.VGHKS-101-043), the Ministry of Science and Technology, Taiwan (Grant No. MOST 106-2320-B-037-010-MY3), and the Yuan’s General Hospital (Grant No. RG 15-010).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This study was approved by the Animal Study Committee of the Kaohsiung Veterans General Hospital (Approval No. VGHKS-101-A008). This article does not contain any studies with human participants performed by any of the authors.
- 33.GenomONE™ - Neo EX HVJ Envelope Transfection Kit / Cosmo Bio. https://www.cosmobio.com/contents/products_isk_20070531.html. Accessed 17 May 2018.
- 40.Arango Duque G, Descoteaux A. Macrophage Cytokines: Involvement in Immunity and Infectious Diseases. Front Immunol. 2014; 5. https://doi.org/10.3389/fimmu.2014.00491.
- 42.Fetal bovine serum product line- Cell culture. Atlanta Biologicals. https://www.atlanta-biologicals.com/wp-content/uploads/2017/07/FBS-Brochure.pdf. Accessed 15 June 2018.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.