Systemic delivery of rAAV9 ‐ IκBα stabilized atherosclerotic plaques and attenuated intra-plaque inammation by inhibiting nuclear factor-κB activation in apolipoprotein E–decient mice

Background: The activated nuclear factor (NF)-κB pathway in atherosclerotic plaques promotes the progression of atherosclerosis. Targeting of plaque NF-κB may provide a novel strategy for limiting chronic inammation. This study was conducted to examine the effect of NF-κB inhibition by using recombinant adeno-associated virus 9 (AAV9) to deliver IκBα (AAV9 ‐ IκBα) which can be overexpressed to inuence atherosclerosis. Methods and Results: Systemic delivery of AAV9 ‐ IκBα resulted in ~2-fold overexpression of IκBα protein in the atherosclerotic plaques of apolipoprotein E -/- mice fed a high-fat diet. AAV9 ‐ IκBα delivery did not affect the body weight or lipid deposition and plaque size in the mice. During plaque formation, overexpression of IκBα suppressed intra-plaque macrophage inltration, inhibited the expression of proinammatory genes including interleukin-6, monocyte chemoattractant protein-1, tumor necrosis factor-α and matrix metalloproteinase, enhanced the contents of collagen and vascular smooth muscle cells, and decreased the plaque vulnerability index by inhibiting the phosphorylation of NF-κB subunit p65 and its nuclear translocation. Conclusions: Systemic delivery of AAV9-IκBα promotes plaque stability by inhibiting NF-κB activation and suppressing intra-plaque inammation, suggesting that inhibition of the NF-κB pathway in atherosclerotic plaques is a promising approach for treating atherosclerosis.


Background
Atherosclerosis is the main cause of coronary heart diseases and constitutes a major healthcare burden worldwide. The in ammatory response is recognized as a key factor in the progression of atherosclerosis [1,2]. Nuclear factor-κB (NF-κB) signaling has been implicated in various in ammatory pathologies including vascular diseases. The transcriptional activity of NF-κB can be induced by numerous atherogenic stimuli such as enhanced oxidized low-density lipoprotein, in ammatory cytokines, integrin/matrix signaling activation, bacterial and viral infections, hemodynamic stress, and metabolic disorders [3][4][5][6][7][8][9][10].
In the canonical NF-κB signaling pathway, NF-κB heterodimers remain in an inactive form in the cytoplasm while adhered to an inhibitor such as IκBα. Upon activation by pathological stimuli, the IκB kinase (IKK) complex rapidly phosphorylates IκBα, which interacts with the proteasome and leads to IκBα degradation [11,12]. When NF-κB heterodimers are released from IκBα, importin proteins (also known as karyopherins) guide translocation of NF-κB to the nucleus where it controls the expression of a large number of genes by combining to numerous κB elements. In the vascular endothelium and atherosclerotic plaques, NF-κB activation induces the expression of proin ammatory genes encoding cytokines, adhesion molecules, and chemoattractant proteins which jointly play important roles in atherosclerosis progression[8, 13,14]. Previous studies demonstrated that NF-κB-speci c inhibition in endothelial cells (ECs) in various genetic-modi ed mouse models (i.e. IKKγ EC knockout, IKKγ EC-inducible knockout, and dominant-negative IκBα EC transgenic) alleviated atherosclerosis [15]. Moreover, genetically inhibiting several NF-κB-targeted genes including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1, E-and P-selectins, interleukin-1 beta (IL-1β), and tumor necrosis factoralpha (TNF-α) decreases various aspects of atherosclerotic lesion formation [16][17][18][19][20]. Thus, targeting NF-κB activity shows potential for the development of novel anti-in ammatory therapies for acute and chronic in ammatory diseases.
Although several studies have evaluated the role of NF-κB in atheromatous plaque formation, the effects of IκBα activation in NF-κB inhibition and atherosclerotic plaques remain unclear. Our previous study showed that recombinant adeno-associated virus 9 (rAAV9) vectors with a human cytomegalovirus (CMV) promoter produced using the recombinant baculovirus system effectively achieved long-term expression in atherosclerotic plaques [21]. In this study, we used the same tool to investigate whether overexpression of IκBα in atherosclerotic plaques effectively suppresses in ammatory responses and therefore stabilizes plaques in apolipoprotein E-de cient (ApoE −/− ) mice, a well-established mouse model of atherosclerosis.

Ethics
Adult male ApoE -/mice (aged 8 weeks) were used in this study. All experimental and animal care procedures were performed according to the Animal Management Rule of the Chinese Ministry of Health (edition 55, 2001), and were approved by the Animal Ethics Committee of Xinjiang Medical University.

Animals and grouping
ApoE -/mice were bred and housed in a speci c pathogen-free barrier facility at 22-25°C. Animals were housed in standard cages and kept on a 12-h light/12-h dark cycle with food and water freely available. All animals were rst administered a high-fat diet (0.25% cholesterol and 21% fat) for 12 weeks followed by three different treatments for another 5 weeks: (1) control group (n = 25): mice were administered saline (100 µL saline once) via the tail vein; (2) AVV9-GFP group (n = 25): mice were injected with AVV9-GFP (5.0 × 10 11 vg/mouse/100 µL) via the tail vein, and (3) AVV9-IκBα group (n = 25): mice were injected with AVV9-IκBα (5.0 × 10 11 vg/mouse/100 µL) according to our previous study [21]. Mice were co-injected with AAV9-GFP and AVV9-IκBα via the tail vein at the doses described above. All mice were fed the highfat diet throughout the experimental period (17 weeks).
Tissue preparation and quanti cation of atheroma (plaque) size At 5 weeks after injection, the mice were starved for 12 h and then anesthetized with mixture of ketamine (100 mg/kg), xylazine (20 mg/kg), and atropine (1.2 mg/kg) via intraperitoneal injection. Blood samples were collected by cardiac puncture and cardiac palpation was used to con rm the death of mice. The chest was opened, the mice were perfused with phosphate-buffered saline through the left ventricle, and then 4% formaldehyde (pH 7.2) was perfused for 15-20 min. The aorta from the aortic arch to the iliac bifurcation and the left common carotid artery with bifurcation were isolated, immersed in 4% formaldehyde overnight (4°C), and then embedded in optical coherence tomography compound (OCT, Sakura Finetek, Inc., Torrance, CA, USA) and stored at -80°C until use [21]. Each vessel covering the entire length of the carotid artery was subjected to histological analysis.

Biochemical analysis
The serum was separated by centrifugation at 600 ×g for 10 min at 4°C. The serum levels of total cholesterol, triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein cholesterol were measured using an automatic biochemical analyzer (HITACHI-7600; Hitachi, Ltd., Tokyo, Japan) in the hospital.

Histological analysis
Conventional staining: Cross-sections (6-μm-thick) were cut at intervals of 50 μm along the carotid artery specimens. The site containing the largest plaque was selected for analysis. The aortic arches were prepared and analyzed as previously described. Sections were stained with hematoxylin and eosin for general observation and with Sirius red staining for collagen quanti cation. Lipid deposition was identi ed by Oil-red O staining. Positively stained areas in the inner-surface area of the aorta were measured and expressed as a percentage of the total aortic area.
Immuno uorescent staining: Immuno uorescent staining was performed to identify NF-κB subunit p65 localization in the carotid artery specimens. Frozen sections were incubated with 3% H 2 O 2 for 10 min, followed by blocking with sealing uid for 30 min. The sections were then incubated overnight at 4°C with an NF-κB p65 antibody (1:50, rabbit polyclonal, Thermo Fisher Scienti c), followed by incubation with goat anti-rabbit IgG, dylight TM 594 (1:100, goat anti-rabbit IgG, Thermo Fisher Scienti c). The sections were counter-stained with DAPI to identify the nuclei before mounting. The sections were used for sequential double immuno uorescence staining and then counter-stained for observation by confocal microscopy.

Quantitative real-time PCR
Real-time quantitative PCR was performed to assess the expression of various genes. Total RNAs were extracted from the total aorta of mice using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Next, 1 μg DNase I-treated (Thermo Fisher Scienti c) total RNA was reversetranscribed using the Primer Script Strand cDNA Synthesis Kit (Promega, Madison, WI, USA). Reverse transcription and real-time uorescent quantization were performed according to the manufacturer's instructions. The PCR device (Bio-Rad MyCycler), electrophoresis apparatus trophoresis (DYY-6D, Liuyi Brand, Japan), and high-speed freezing centrifuge (HC-3018R) were used. Samples were evaluated in triplicate. The housekeeping GAPDH gene was used for internal normalization. The mean SQ values of the target gene primers were compared to those of GAPDH-speci c primers using the double standard curve method. The data are presented as fold-changes of transcripts for the target gene normalized to GAPDH compared to in control mice. Primers were designed to amplify mouse IκBα, IL-6, TNF-α, MCP-1, MMP-2, and GAPDH. All primers (Table 1) were obtained from Shanghai Sangong Company (Shanghai, China). The reaction was performed under the following conditions: 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. A DNA puri cation kit was used (Tiangen Biotech Co., Ltd., Beijing, China), cDNA was ampli ed using a Quanti Fast SYBR® Green Real-time PCR Master Mix (Qiagen, Hilden, Germany) in 96-well optical reaction plates on an uorescent quantization PCR device (CFX96, Bio-Rad) according to the manufacturer's protocol. The cycling parameters were as follows: 95°C for 5 min and then 40 cycles of 95°C for 10 s and 60°C for 30 s, followed by melting curve analysis. Quantitative values were obtained from the threshold cycle value (Ct) and the data were analyzed by the 2 -∆∆CT method. The housekeeping gene GAPDH was ampli ed and quanti ed as an internal control.

Western blot analysis
Protein extracted from fresh aorta plaques were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene uoride membranes. The membranes were blocked with 5% non-fat milk, and then washed with phosphate-buffered saline containing 0.1% Tween 20 and incubated with an appropriate primary antibody at 4°C overnight. The blots were probed with antibodies against GAPDH Signaling Technology (Danvers, MA, USA). After overnight incubation, the blots were washed with Trisbuffered saline-Tween-20 and incubated with secondary antibodies at room temperature for 2 h, followed by three washes for 10 min each membranes and detection using the ECL Western blot detection system. Quantity One software (Bio-Rad) was used for analysis; the integral optical density value was calculated as the target protein divided by the internal reference GAPDH protein. The relative values of protein bands in each group were expressed as the mean ± standard deviation.

Statistics analysis
Statistical analysis was performed with SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA). After the numeric variable data were tested for normality and homogeneity of variance, all parameters determined in this study were presented as the mean ± standard deviation. Differences among groups were compared by general linear model-univariate or one-way analysis of variance. P < 0.05 was considered to indicate a statistically signi cant difference.

Results
Systemic delivery of AAV9-IκBα resulted in overexpression of IκBα in atherosclerotic plaques First, we examined IκBα expression in aortic plaques in three different groups (control, AAV9-GFP, and AAV9-IκBα groups) at 5 weeks after tail vein injection. There were no differences in IκBα protein or gene expression between the control and AAV9-GFP groups. In the AAV9-IκBα group, the protein and mRNA levels of IκBα were increased by 1-and 4.7-fold compared to control values, respectively ( Figure 1, both P < 0.05). These results indicate that single injection of the AAV9 delivery system can achieve stable and effective overexpression of a targeted gene at a speci c site.
Systemic delivery of IκBα did not in uence animal body weight or lipid pro le Five weeks after transfection of IκBα, body weight and circulating lipid levels were comparable among the three groups ( Table 2), suggesting that IκBα gene transfection did not signi cantly affect body weight and the circulating lipid pro le in atherosclerotic mice.
Overexpression of IκBα stabilized atherosclerotic plaques without in uencing plaque size Histological and immunohistochemical studies revealed the accumulation of macrophages as identi ed by MOMA-2, lipids as detected by oil-red O staining, collagen as detected by Sirius red staining, and SMCs detected by evaluating α-smooth muscle actin in atherosclerotic plaques of the carotid artery. In macrophages, although lipids, collagen, and SMCs showed comparable contents between the control and AAV9-GFP groups, AAV9-IκBα gene transfection signi cantly reduced the accumulation of macrophages and increased collagen deposition and SMCs in the plaques (Figure 2, all P < 0.05). These changes reduced by plaque vulnerability index by more than 50% in the AAV9-IκBα group versus in the control and AAV9-GFP groups ( Figure 2). Unexpectedly, AAV9-IκBα gene transfection did not in uence the size of atherosclerotic plaques in the whole aorta, as evidenced by similar lesion areas stained with oil-red O ( Figure 3); comparable lipid accumulation was also observed in the plaques (Figure 2).

Overexpression of IκBα suppressed expression of in ammatory mediators in the plaques
Considering the crucial role of in ammation in the pathogenesis of atherosclerosis, we further examined whether IκBα gene delivery regulates vascular in ammatory responses in the atherosclerotic lesions. There was no difference in the expression of IL-6, TNF-α, MCP-1, and MMP-2 between the control and AAV9-GFP groups according to immunohistochemistry analysis and real-time PCR to examine mRNA levels ( Figure 4). However, AAV9-IκBα gene delivery signi cantly decreased the positively stained areas of IL-6, TNF-α, MCP-1, and MMP-2 (approximately 50% reduction) compared to control levels, showing similar decreases in the mRNA levels of these genes (Figure 4). These results suggest that IκBα overexpression suppressed in ammatory responses in atherosclerotic lesions.

Overexpression of IκBα suppressed NF-κB pathway in atherosclerotic plaques
We next examined the effect of IκBα gene delivery on NF-κB inhibition. As expected, corresponding to increased expression of IκBα in aortic plaques (Figure 1), phosphorylation of the NF-κB subunit p65 was decreased in the AAV9-IκBα group compared to in the control and AAV9-GFP groups ( Figure 5A and B, P < 0.05). Our immuno uorescence staining results showed that the density of p65 overlapping with the nuclei (stained by DAPI) was decreased by 30% compared to the control level ( Figure 5C and D). However, there were no differences in these parameters between the control and AAV9-GFP groups. There was no signi cant difference in p65 and p50 protein expression, whereas AAV9-IκBα effectively inhibited the expression of NF-κB nuclear protein p-p65 ( Figure 5A-D).

Discussion
We previously showed that recombinant adeno-associated virus 9 (rAAV9) vectors with a human cytomegalovirus promoter produced with the recombinant baculovirus system effectively achieved longterm expression in atherosclerotic plaques. Our study is the rst study, we used this system to investigate overexpression of IκBα in atherosclerotic plaques effectively suppresses in ammatory responses and stabilizes plaques in apolipoprotein E-de cient (ApoE-/-) mice. In this study, we investigated the effectiveness of systemic delivery of AAV9-IκBα in inhibiting NF-κB activation and in uencing atherosclerotic plaque stability and related in ammation. We believe that our study makes a signi cant contribution to the literature because we found that systemic delivery of IκBα by AAV9 resulted in stable and effectively enhanced expression of IκBα in atherosclerotic lesion, resulting in stabilization of plaques and attenuating the expression of in ammatory mediators. Thus, inhibiting NF-κB activation in atherosclerotic plaques is a potential therapeutic approach for treating atherosclerosis. AAV9-IκBα also had no effect on atherosclerotic lesion size or the lipid component in plaques (Fig. 3), which is a limitation of the current study. A higher dose may lead to higher expression levels of the targeted gene, which may yield greater protective effects and explain why the lesion size was not reduced. The enhanced expression of IκBα in the present study resulted in plaque stabilization, as evidenced by decreased macrophage accumulation and increased collagen deposition and SMC numbers. The protective role of IκBα in atherosclerosis has been documented in numerous studies. For example, myeloid IκBα de ciency promoted atherogenesis by enhancing leukocyte recruitment to developing plaques [28]. Overexpression of DNIκBα resulted in more dramatic, nearly complete inhibition of atherosclerotic plaque growth and vulnerability in ApoE -/mice, which may be because of incomplete ablation of NEMO in the endothelial lining of the arterial wall [15]. However, in contrast to the ndings showing that IκBα overexpression inhibited early aortic lesion formation, our study revealed that systemic transgenic overexpression of IκBα did not affect the size of advanced carotid plaques in mice. This may be because relatively short-term manipulation of IκBα expression in an already established plaque is not adequate for inducing atherosclerotic regression. Further studies are needed to address this important issue.
The NF-κB signaling pathway plays a critical role in regulating the expression of multiple genes involved in the progression of atherosclerosis including in ammatory-related genes/mediators. Previous studies indicated that inhibition of NF-κB in speci c cell types has discrepant effects. Although EC-speci c inhibition of NF-κB resulted in reduced lesion formation in ApoE −/− mice [15,29,30], the effect of altering NF-κB activation in myeloid cells is more complicated. For example, LDL receptor-de cient mice transplanted with IKK2/IKKβ-de cient macrophages showed increased atherosclerosis lesions, which was associated with increased numbers of apoptotic cells in the plaque [31]. In contrast, myeloid-speci c IKKβ de ciency reduced atherosclerosis in LDL receptor-de cient mice [32]. Another study showed that myeloid IκBα de ciency increased atherogenesis by promoting leukocyte aggregation to advanced plaques [28]. Moreover, bone marrow de ciency of NF-κB1 led to reduced atherosclerotic plaque sizes and macrophage foam cells but resulted in enhanced lesion in ammation [33]. These data illustrate that celltype-speci c inhibition upstream of the NF-κB pathway may have diverse effects on the plaque composition and atherosclerotic lesion formation.
Our results suggest that systemic delivery of IκBα mediated the inhibition of NF-κB activation by attenuating the phosphorylation and nuclear translocation of NF-κB p65 subunit. The atheroprotective role of NF-κB inhibition has been reported in different mouse models. NEMO ablation interferes with IKK activation or transgenic expression of degradation-resistant DNIκBα blocks nuclear translocation of NF-κB [15]. Pharmacological inhibition of NF-κB in human vascular SMCs maintained cytosolic levels of IκBα which paralleled the decreased p65 nuclear translocation [34]. These results agree with our current ndings showing that enhanced IκBα expression in the lesion inhibited NF-κB activation, thereby alleviating atherosclerosis. Moreover, our recent study revealed that downregulation of the NF-κB p50 subunit led to endothelial dysfunction and enhanced in ammatory and apoptosis, which was associated with the risk and severity of coronary heart disease [35]. Taken together, these results demonstrate a critical role for NF-κB in the development of atherosclerosis and indicate that inhibition of NF-κB by gene therapy is a promising approach for limiting atherosclerosis. Two distinct pools of IκBα are known to exist in cells; the larger IκBα pool is associated with NF-κB and the minor pool remains as 'free' protein [36]. IκBα is an unstable protein whose rapid degradation is slowed upon binding to NF-κB; bound IκBα then requires IKK phosphorylation and ubiquitination for slow basal degradation. Altering free IκBα degradation dampens NF-κB activation [37]. This steady-state level of total IκBα may partially explain how overexpression of IκBα inhibits NF-κB activation.
The current study has several limitations. IκBα mediates NF-κB to regulate in ammatory factors, whether there is a feedback regulation between in ammatory factors and NF-κB, and the effect of overexpression of IκBα on the corresponding tissues other than blood vessels need further investigation. There are a variety of cells in atherosclerotic plaques in this study, and it is still necessary to further clarify the types of cells targeted by the virus and the speci c functions of the cells affected by the intervention. Macrophages play a key role in the occurrence and development of AS. The study on the effect of IκBα on macrophages and the effect on liver glucose and lipid metabolism will also be an important target of anti-in ammatory and anti-AS, which is worth further exploration.

Conclusions
In summary, systemic delivery of IκBα by AAV9 achieved stably and effectively enhanced expression of IκBα in atherosclerotic lesion, resulting in stabilization of plaques and attenuating the expression of in ammatory mediators. These results suggest that inhibition of NF-κB activation in atherosclerotic plaques is a potential therapeutic approach for treating atherosclerosis.

Declarations
Ethics approval and consent to participate All experimental and animal care procedures were performed according to the Animal Management Rule of the Chinese Ministry of Health (edition 55, 2001), and were approved by the Animal Ethics Committee of Xinjiang Medical University.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.
Funding Tables   Table 1. Sequences of examined primers.

Gene
Forward Reverse