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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2015 Sep 1;8(9):9912–9921.

Inhibition of the NF-κB pathway by R65 ribozyme gene via adeno-associatedvirus serotype 9 ameliorated oxidized LDL induced human umbilical vein endothelial cell injury

Hui Zhai 1,2,*, Qing-Jie Chen 1,2,*, Xiao-Ming Gao 3,4, Yi-Tong Ma 1,2, Bang-Dang Chen 2, Zi-Xiang Yu 1,2, Xiao-Mei Li 1,2, Fen Liu 2, Yang Xiang 1,2, Jia Xie 1,2, Yi-Ning Yang 1,2
PMCID: PMC4637785  PMID: 26617700

Abstract

Objective: NF-κB signaling plays a central role in the regulation of inflammatory responses in atherosclerosis. R65 ribozyme gene suppresses activation of NF-κB pathway, therefore we studied whether R65 gene therapy can ameliorate oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) injury. Methods and results: Recombinant adeno-associated virus serotype 9 (rAVV9) vector was used to transfect the R65 ribozyme gene (rAVV9-R65) into HUVECs then following ox-LDL stimulation, expression of NF-κB p65 and p50 subunits, inflammatory mediators and cell apoptosis were examined. First, rAVV9-enhanced green fluorescent protein (eGFP)-R65 at 1×107 v.g./cell multiplicity of infection reached a long-lasting and significant increase in R65 gene expression. Second, ox-LDL treatment led to time- and dose-dependent activation of NF-κB pathway, and enhanced inflammatory response and cell death evidenced by increased expression of nuclear NF-κB p65 and p50 subunits, greater production of tumor necrosis factor α, interleukin-6 and von willebrand factor and 20.57% increasedapoptotic HUVECs. Third, over-expression ofR65 gene was 2-fold increased in HUVECs attenuated ox-LDL induced unclear accumulation and expression of p65 subunit and ameliorated inflammation and cell death (all P < 0.05). Conclusion: rAAV9-mediated R65 ribozyme gene transfection in cultured HUVECs effectively inhibits ox-LDL induced activation of NF-κB and production of inflammatory cytokines and prevents cell apoptosis.

Keywords: Ribozyme gene, adeno-associated virus, NF-κB, inflammation, endothelial

Introduction

Atherosclerosis is the most common cause of cardiovascular diseases in the world. Recent studies have suggested that inflammation plays a central role in the progression of atherosclerosis [1,2]. The nuclear transcription factor NF-κB is the key player in the development of chronic inflammatory diseases, such as atherosclerosis [3]. NF-κB signal pathway participates in the inflammatory cellular response and is a pivotal transcription factor that promotes the expression of a cascade of procoagulant and proinflammatory genes such as P-selectin, tumor necrosis factor (TNF-α), intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, monocyte chemotactic protein-1, interleukin 1 (IL-1) and tissue factor [4]. In the inactive state, the NF-κB bound to its inhibitory protein-κB (I-κB) in the cytoplasm. In the pathogenesis of atherosclerosis, endothelium injury was thought to be the initial step [5]. Upregulation of oxidized low-density lipoprotein (ox-LDL) is a hallmark of atherosclerosi [6]. Like inflammatory cytokines, accumulated of ox-LDL acted as a stress signal, releases I-κB from NF-κB, the latter then translocates to the nucleus and activates target genes involved in atherosclerosis [7,8], therefore, contributes to the development and progression of atherosclerotic endothelium injury [9,10]. Previous studies showed that ox-LDL induced the change of subcellular localization and distribution of NF-κB through modulating the activities of oxidative stress-induced NF-κB pathway [11,12].

Since ribozymes have enzyme activities and may be used for cutting nucleic acid sequence at the corresponding sites, it can be used as a tool to block specific gene expression. Different ribozymes can specifically combine with different RNA targets through the complementary base pairs. Therefore ribozymes have some unique advantages, for example: 1) no coding protein; 2) no immunogenicity; 3) can be repeated use. These features make ribozyme to be broadly used in gene therapy [13]. According to the mRNA nucleotide sequence of NF-κB p65 subunit, we designed a DNA coding sequence of 5’-GTGAAACTGATGAGTCCGTGAGGACGAACACCTC-3’ hammerhead ribozyme, named R65, which can combine with the cutting locus of NF-κB p65 mRNA nucleic acid sequence, then effectively restrain the NF-κB activity. The adeno-associated virus serotype 9 vector (AVV9) is a novel tool for deliver gene therapies targeting human diseases owing to its nonpathogenic capability for transducing cells and its long-term transgene expression [14]. In the present study, we used an AAV9 vector to deliver the R65 gene into human umbilical vein endothelial cells (HUVECs) and assessed whether AAV9 vector-mediated R65 gene transfection could achieve a long-term expression and inhibit activation of NF-κB thereby preventing ox-LDL-induced HUVECs injury.

Materials and methods

Efficiency of AAV9 mediated R65 gene delivery

To test the efficiency of AAV9 mediated R65 gene delivery in HUVECs, cells were seeded at the density of 1×106 onto a 60 mm dish and incubated in M199 media for 24 h, then the cells was transfected with recombinant AAV9-enhanced green fluorescent protein-R65 (rAAV9-eGFP-R65) (Virovek, USA) according to the manufacturer’s instruction. We chose AVV9 as the vival vector in the study because it is of particular interest owing to their high efficiency of gene transfection in the heart and vascular compare with other AVV types [15]. rAAV9-eGFP-R65 was transfected into HUVECs at different multiplicities of infection (MOI = 1×105, 1×106, 1×107 v.g./cell). Transfection effect was examined daily by (1) Daily cell counting. Number of eGFP-positive expression cell was counted by using an inverted fluorescence microscope (LEICA-DMI4000B, Germany) under magnificently of ×400 for 9 days. The percentage of eGFP-positive cells determined by flow cytometry, and expressed as a percentage of eGFP-positive cells in total cells under each viewing field. Five fields were randomly selected from each dish (in total 200×5 cells were counted). (2) Flow cytometry: HUVECs after 6-day transfection were collected, centrifuged at 3500 r/min for 5 min, PBS washed, and then used flow cytometry instrument (Beckman Coulter, Germany) detecting rAAV9-eGFP-R65 transfection efficiency of HUVECs.

Stimulation of HUVECs with ox-LDL

HUVECs were purchased from the American Type Culture Collection (ATCC) and plated at an appropriate density in 6-well plates and cultured according to the purpose of each experiment. HUVECs were cultured in M199 medium supplemented with 10% fetal bovine serum (Gibco, USA), 1% ampicillin and streptomycin at 37°C in a humidified incubator for 24 h, then the medium was replaced by serum-free medium for an additional 12 h before further treatments. To study the concentration- and time-dependent effects of ox-LDL on cells, HUVECs were incubated with 15, 30, 60 and 120 μg/mlox-LDL for 24 h or 30 μg/ml ox-LDL for 12, 24 and 48 h, respectively. HUVECs treated with serum-free medium only served as the control group.

NF-κB inhibition in ox-LDL treated HUVECs

To study the protective effect of inhibition of NF-κB on ox-LDL stimulated cells, the following protocol was carried out.

(1) Control.

(2) ox-LDL treatment.

(3) rAAV9-eGFP-R65 + ox-LDL.

(4) PDTC + ox-LDL.

Exposed to ox-LDL (30 μg/mL) for 24 h, then western blotting and immunofluorescence staining were prepared for p65 expression in the nucleus.

Western blotting

HUVECs were collected and washed with ice-cold phosphate-buffered saline (PBS) twice. Nuclear proteins were extracted by NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, USA) following manufacture’s instruction. The protein concentration was quantified using a BCA protein assay kit (Pierce, USA). Nuclear extract proteins were detected by Western blot for detecting the protein levels of NF-κB using specific primary antibodies for p65 and P50 (Cell Signaling Technology, USA). Extracted nucleus proteins were subjected to electrophoresis on sodium dodecyl sulfate (SDS) epolyacrylamide gels and then transferred onto immobilon-P membrane (Millipore 0.45 um, Bedford, MA). The membrane was incubated in 5% (wt/vol) dried milk protein in TBST containing Tris-base, NaCL, KCL, 0.05% (vol/vol) Tween-20 for 1 h, washed in TBST and then further reacted with primary antibodies: rabbit anti-p65 (1:1000) and rabbitanti-p50 (1:1000) at 4°C overnight. The membrane was extensively washed with TBST and then incubated with secondly anti-rabbit IgG antibody conjugated to HRP (1:5000) for 2 h at room temperature. After extensive washes, protein bands on the membrane were visualized using ECL detection kit (BioRad). The blots were then stripped using stripping buffer (100 mmol b-mercaptoethanol, 2% SDS, 62.5 mmol Tris-HCl, pH 6.7) and re-probed with GAPDH (1:1000) antibodies as loading controls.

Immunofluorescence staining

HUVECs were incubated with primary antibody p65 (1:100 dilutions, Cell Signaling Technology, USA) at 4°C overnight. After PBS wash for 3 times, cells were incubated with DyLight 594 Affinity Pure donkey anti-rabbit IgG-labeled secondary antibody (1:100 dilutions; EaythOx, LLC, USA) for 30 min at room temperature then followed by incubation with 4’,6-diamidino-2-phenylindole (DAPI) staining solution (20 mg/mL, Roche, Germany) for 15 min. Multiple images were acquired digitally using fluorescence microscope (LEICA, Germany). And analysis software (LASAF Application). The expression of p65 after secondary antibody colored red, the color of DAPI staining nuclei is blue, if the blue nucleus area appears red, prove that the p65 intranuclear expression. Experiments were repeated at least 3 times and results were averaged.

Measurement of inflammatory factors and endothelial cell function markers in cell culture media

Cell culture media was collected, centrifuged at 3,000 g for 15 min at 4°C, then transferred to an eppendorf tube. TNF-α, IL-6, endothelial nitric oxide ribozymes (eNOS) and von willebrand factor (vWF) protein concentrations were quantified by using enzyme-linked immunosorbent assay (ELISA) kits (all from Ray Biotech, USA) in accordance with the manufacturer’s instructions. The optical density was read on a Dynatech MicroElisa Reader (Chantilly, VA, USA) at a wavelength of 450 nm. Each sample was measured in duplicate.

Flow cytometry to detect cell apoptosis

Apoptotic HUVECs were detected by using the Annexin-V-FIOUS Staining Kit (Roche, USA). Annexin V has a strong Ca2+-dependent affinity for phosphatidylserine, which translocates from the internal to the external surface of the plasma membrane as a probe for detecting early apoptosis. Cells that have the loss of membrane integrity will show red staining (propidium iodide, PI) throughout the nucleus for detecting late apoptosis cells. And therefore will be easily distinguished between the early and the late apoptotic cells/necrotic cells. Samples were incubated at room temperature for 15 min in the dark with Annexin V and PI. Then quantitatively analyzed by a Fluorescence Activated Cell Sorter low cytometer (Beckman Coulter, Germany).

Statistical analysis

Results were expressed as mean ± SE. Comparison of data in each group was undertaken by one-way ANOVA. P < 0.05 was deemed statistically significant. All statistical analysis was performed using SPSS 17.0 software.

Results

rAAV9 mediated R65 gene expression

We examined the different MOI efficiency of eGFP expression as the indicating gene and found that transfection for 1 day rAAV9-mediated eGFP expression was significantly higher in HUVECs at MOI of 1×107 v.g./cell (from 12.3% increased to 52.8%) compare to MOI of 1×106 v.g./cell and it was almost undetectable (1.4%) in 1×105 v.g./cell MOI (Figure 1). Further, we found that the maximal transfection efficiency was achieved at day 6 of eGFP expression, and then declined. In 1×107 MOI, the transfection efficiency was increased from 30% at day-1 to 52.8% at day-6. In 1×106 MOI, the transfection efficiency was increased from 6.7% at day-1 to 12.8% at day-6. While, in 1×105 MOI, there was no much increased eGFP expression (Figure 2).

Figure 1.

Figure 1

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A. Flow cytometry analysis of eGFP protein expression in different MOI (MOI = 1×105, 1×106, 1×107 v.g./cell). The transfection efficiency of eGFP can be calculated and presented in the frame. The green indicate positive transfection. B. eGFP expression in HUVECs observed by fluorescence microscopy (MOI = 1×105, 1×106, 1×107 v.g./cell). eGFPis almost undetectable in MOI = 1×105 group, but is detected in MOI = 1×106 and 1×107 group, and expressed most in 1×107 group. Green spots indicate positive staining. Image is 400×.

Figure 2.

Figure 2

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A. eGFP expression observed by inverted fluorescence microscopic in MOI = 1×107 v.g./cell under magnificently of ×400 for 9 days. Changes of eGFP expression of day 1, 3, 6 and 9 were selected. The peak level of eGFP expression was at the sixth day, and was lasted for 9 days after transfection. B. Transfection efficiency of rAAV9-eGFP-R65 in different MOI groups (MOI = 1×105, 1×106, 1×107 v.g./cell) were recorded from 1st to 9th days by inverted fluorescence microscopic.

Effect of ox-LDL stimulation on activation of NF-κB in HUVECs

HUVECs have been widely used as a cell model to study the endothelial cell injury and mechanisms in atherosclerosis. In the present study, we investigated changes in expression of nuclear p65 and p50 subunits of NF-κB induced by different doses of ox-LDL for 24 h and by 30 μg/ml of ox-LDL for different time in HUVECs. Western blotting showed that unclear expression of p65 and p50 subunits were stepwise increased upon ox-LDL stimulation in a dose- and time-dependent manner (Figure 3A). Compared to p65 subunit, a dramatically time-dependent increase in p50 subunit expression was detected (Figure 3B).

Figure 3.

Figure 3

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ox-LDL stimulated NF-κB activation. p65 and p50 were examined using Western blotting after that HUVECs were treated with different concentrations of ox-LDL for 24 h (A) and 30 μg/ml of ox-LDL for indicated times (B). Extracts were blotted with anti-p65, p50 and GAPDH (loading control) antibodies. Western Blot shown is representative.

rAAV9-eGFP-R65 transfection attenuated ox-LDL induced NF-κB activation

Nuclear translocation/accumulation of NF-κB p65 subunitis an indication of NF-κB activation upon stimulations. In our cell model, using immunofluorescence staining we found that p65 subunit was located primarily in the cytoplasm before ox-LDL stimulation, but translocated to the nucleus after ox-LDL treatment. And rAAV9-eGFP-R65 pre-transfection significantly reduced the ox-LDL-induced nuclear translocation of p65 subunit in HUVECs (Figure 4). Further, by Western blot, we confirmed that ox-LDL stimulation induced expression of p65 in HUVECs, which were markedly inhibited by rAAV9-eGFP-R65 transfection. Similarly, pre-treatment of a NF-κB inhibitor, PDTC, also downregulated the expression of p65 in ox-LDL stimulated HUVECs (Figure 5).

Figure 4.

Figure 4

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Effects of rAAV9-eGFP-R65 on nuclear accumulation of p65 induced by ox-LDL. Representative images acquired by a fluorescence microscope from HUVECs 30 μg/ml ox-LDL for 24 h. Nuclei were stained by DAPI (dark blue, left panel) and HUVECs were stained by a p65 antibody (red, middle panel). The overlay of dark blue and red colors indicates a positive inflammatory cell staining (purple, bottom panel).

Figure 5.

Figure 5

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Expression of nuclear protein p65 after transfection with rAAV9-eGFP-R65. Expression of p65 in the nucleus significantly increased in ox-LDL-induced group compared to their corresponding control levels, rAAV9-eGFP-R65 or NF-κB inhibitor PDTC attenuated the up-regulation of p65 (*P < 0.05 vs. control; #P < 0.05 vs. ox-LDL induced group).

rAAV9-eGFP-R65 transfection via inhibition of NF-κB prevented ox-LDL induced inflammatory response and endothelial cell dysfunction

NF-κB signal pathway participates in the inflammatory cellular response and regulates the transcription of down-stream targets genes involved in atherosclerosis. We therefore examined the influence of rAAV9-mediated R65 gene transfection on expression of inflammatory (TNF-α, IL-6) and endothelial cell functional markers (vWF and eNOS) in ox-LDL stimulated HUVECs. Using ELISA, we found the levels of TNF-α, IL-6 and vWF in cultured media had 51.5%, 36.3% and 27.0% increase over the control values, respectively, after 24 h ox-LDL (30 μg/ml) stimulation (all P < 0.05, Figure 6), while such increase was diminished by rAAV9-eGFP-R65 transfection and by the treatment of NF-κB inhibitor, PDTC (Figure 6). In contrast, ox-LDL stimulation induced a 25% decrease in the level of eNOS compared to the control value. rAAV9-eGFP-R65 transfection or PDTC treatment restored the level of eNOS (Figure 6). These results indicate that ox-LDL induced cell injury is through the activation of NF-κB.

Figure 6.

Figure 6

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Changes in expression and production of various proinflammatory mediators and cytokines of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), endothelial nitric oxide ribozymes (eNOS) and von willebrand factor (vWF) as determined by ELISA from the HUVECs supernatant subjected to control or ox-LDL-induced or rAAV9-eGFP-R65 + ox-LDL or PDTC + ox-LDL. *P < 0.05 compared with control, #P < 0.05 compared with the group treated with ox-LDL alone.

rAAV9-eGFP-R65 transfection reduced ox-LDL induced HUVECs apoptosis

Flow cytometry analysis showed that ox-LDL (30 μg/ml) stimulation for 24 h induced 20.3% increase in the percentage of apoptotic HUVECs versus control levels (Figure 7). rAAV9-eGFP-R65 transfection and treatment with a NF-κB inhibitor, PDTC, prevented ox-LDL induced HUVECs apoptosis. Notably, reduction in apoptotic cell percentage by rAAV9-eGFP-R65 transfection was more evident than that by PDTC treatment (Figure 7), suggesting a potent anti-apoptotic effect of R65 ribozyme targeted on NF-κB.

Figure 7.

Figure 7

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Protective anti-apoptotic effects of rAAV9-eGFP-R65 examined by flow cytometry in HUVECs. A: Control group; B: ox-LDL-induced group; C: rAAV9-eGFP-R65 transfected + ox-LDL-induced group; D: PDTC + ox-LDL-induced group.

Discussion

In the present study, first, we established rAAV9-mediated NF-κB R65 gene transfection in cultured HUVECs. Second, we examined activation of NF-κB in ox-LDL stimulated HUVECs. Third, we assessed effects of NF-κB R65 transfection on preventing ox-LDL-induced cell inflammation and apoptosis. We have made several findings: (1) rAAV9-mediated gene expression in cultured HUVECs was stable, efficient and dose-dependent; (2) There was a time- and dose-dependent effect on ox-LDL-induced activation of NF-κB in HUVECs evidenced by increasing expression of p65 and p50 subunits; (3) rAAV9-mediated R65 gene transfection suppressed ox-LDL-induced expression of NF-κB p65 subunits and inflammatory mediators and prevented cell.

Because of the effective and stable transduction in target organs, rAAV vector-mediated gene transfer has emerged as a novel method for the treatment of human disease [16]. We previously showed that rAAV9-eGFP-R65 provides efficient gene transfer in H9C2 in vitro, using eGFP as the indicator, rAAV9-eGFP-R65 transfection at MOI 1×105, 1×106, 1×107 v.g./cell for 4 days reached (14.1±0.2)%, (35.1±4.8)%, (56.8±0.1)% in eGFP expression respectively [17]. In rat cardiac fibroblasts cells from our previous study, the transduction efficiency of rAAV9-eGFP was (2.6±0.2)%, (7.3±1.4)% and (45.1±2.7)% at MOI 1×105, 1×106, 1×107 v.g./cell for 5 days respectively [18]. In the present study, using the same system, we achieved superior direct gene transfer in HUVECs which is dose and time-dependent. After transfection, rAAV9 vector mediated robust and early onset gene expression in HUVECs, peaked at day-6 and lasted for 9 days during the study period. Compared to fibroblasts and H9C2 cells from our previous observation, HUVECs showed a prolonged phase and high levels of gene expression after rAAV9 vector administration.

Atherosclerosis is characterized by the accumulation of lipids and inflammatory cells in the arterial wall. ox-LDL induces endothelial dysfunction and endothelial cell apoptosis thereby it is an important pro-atherogenic factor [19]. NF-κB is a transcription factor belonging to ‘Rel’ family, consisting of 5 members: p65 (RelA), c-Rel, RelB, NF-κB1 (p50 and its precursor p105) and NF-κB2 (p52 and its precursor p100), that represents a crucial intracellular signal transduction system involved in several inflammatory diseases including atherosclerosis. The most abundant complex, often referred to as being “NF-κB”, is p65/p50. Activation of NF-κB mediated signal transduction has been established at different stages of atherosclerosis, beginning from plaque formation to its destabilization and rupture. The NF-κB pathway is also involved in apoptotic [20]. To investigate the role but not relevance of the NF-κB system, especially p65 subunit, in ox-LDL-induced HUVECs injury, we first examined ox-LDL treated HUVECs and found that ox-LDL stimulation induced significant increase in both NF-κB p65 and p50 subunits in a dose- and time-dependent manner which was consistent with other studies [8]. In resting situation, the NF-κB p65/p50 dimer is kept inactive by I-κB. Association with I-κB keeps NF-κB predominantly sequestered in the cytoplasm. A wide range of extra cellular stimuli will mediate the phosphorylation of I-κB, resulting in its ubiquitination and degradation, leaving the NF-κB dimer free to translocate to the nucleus, where it can activate specific target genes through selective binding to the NF-κB consensus sequence [21]. Increased expression of p65 and p50 in the nucleus upon ox-LDL treatment indicates that activation of NF-κB pathway is the ox-LDL-induced cell injury.

NF-κB, a nuclear transcription factor, is known to be the key regulator for multiple functional genes expression including adhesion molecules, inflammatory factors, endothelial cells functional factors [22,23]. As aforementioned the role of NF-κB in atherogenesis, activation of NF-κB pathway enhances regional inflammatory responses, damages endothelial cell function and promotes endothelial cell apoptosis during lipid-induced injury [24]. Our study using ox-LDL-induced cell injury model demonstrated that ox-LDL treatment resulted in increased production of TNFα, IL-6 and vWF but decreased eNOS production in HUVECs, which mimic the pathological responses occurred during atherosclerotic process. Endothelial cell injury is the initiating factor in atherosclerosis, vWF and eNOS are both key proteins in endothelial cell function. Numerous clinical and experimental reports suggest that high vWF levels reflect damage to the endothelium or endothelial dysfunction. The close association between vWF and atherogenesis also suggests that high vWF levels may be a useful indirect indicator of atherosclerosis [25]. And it is evident that decreased bioavailability of NO produced by eNOS plays a crucial role in the development and progression of atherosclerosis [26]. rAAV9 mediated R65 expression significantly suppressed productions of TNFα, IL-6 and vWF but restored eNOS levels in ox-LDL treated HUVECs. Further AAV9 mediated R65 expression prevented ox-LDL-induced HUVECs apoptosis. The efficacy of these protections mediated by R65 overexpression was similar as that by the selective NF-κB inhibitor, PDTC. These results demonstrated that rAAV mediated R65 gene expression is an effective and useful tool for gene therapy targeting on inhibition of NF-κB pathway. However, it must be aware that optimized dose and duration of rAAV9-mediated R65 expression need to be obtained in different systems and on different targets as NF-κB pathway possessing a broad regulatory action on cell biology. Especially, the efficacy and safety issue need to be assessed in vivo system.

In conclusion, the present study demonstrated that rAAV9 mediated NF-κB p65 ribozyme (R65) gene expression effectively inhibited ox-LDL-induced activation of NF-κB. Inhibition of NF-κB in turn to ameliorate cellular inflammatory response and endothelial cell dysfunction by suppressing expression of inflammatory mediators and restoring expression of endothelial cell function markers thereby preventing HUVECs apoptosis. Our work provided evidence to support rAAV9 as a useful tool to deliver a gene therapy, and intracellular p65 ribozyme expression is able to effectively inhibit activation of NF-κB pathway therefore experts a protection against ox-LDL-induced cell injury.

Acknowledgements

This work was supported by a project grant of Natural Science Fund of China (ID: 81160042).

Disclosure of conflict of interest

None.

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