Silencing of hypoxia inducible factor-1α gene attenuated angiotensin Ⅱ-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice

Atherosclerosis 252 (2016) 40e49

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Silencing of hypoxia inducible factor-1a gene attenuated angiotensin Ⅱ-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice Le Yang a, Lin Shen b, Gang Li a, Hai Yuan a, Xing Jin a, *, Xuejun Wu a, ** a b

Department of Vascular Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China Department of Ophthalmology, QiLu Hospital to Shandong University, Jinan, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 March 2016 Received in revised form 2 July 2016 Accepted 7 July 2016 Available online 29 July 2016

Background and aims: We aimed to determine the effect of HIF-1a, the main regulatory subunit of the hypoxia inducible factor 1 (HIF-1), on the development of the abdominal aortic aneurysm (AAA). Methods: AAA was induced in ApoE
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mice by angiotensinⅡ (AngⅡ) infusion. In vivo silencing of HIF-1a was achieved by transfection of lentivirus expressing HIF-1a shRNA. Results: Time course analysis of the AngⅡ infusion model revealed that HIF-1a was persistently upregulated during a 28-day period of AAA development. Silencing of the HIF-1a gene reduced the aneurysm size (2.84 ± 1.96 mm vs. 1.41 ± 0.85 mm respectively at day 28, p ¼ 0.0002). Silencing of HIF-1a also alleviated infiltration of macrophages (38.8 ± 14.7 vs. 11.4 ± 4.4 macrophages/0.1 mm2, p ¼ 0.0006) and neovascularity (5.56 ± 2.14 vs. 1.27 ± 1.05 microvessels/0.1 mm2, p ¼ 0.0008) in the AngⅡ infusion model, at day 28. The activity of MMP-2 and MMP-9 was also decreased by knockdown of HIF-1a. The early increased expression of pro-inflammatory factors, angiogenic factors, and MMPs during AAA induction was alleviated by HIF-1a silencing. Conclusions: Activation of HIF-1 signaling pathway participates in the Ang Ⅱ-induced AAA formation in mice. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Abdominal aortic aneurysm HIF-1a Inflammation MMP Neovascularity

1. Introduction Abdominal aortic aneurysm (AAA) is the major disease of adult aorta [1]. Open surgery and endovascular therapy are the only two established managements of AAA nowadays. These two therapies are recommended when the aortic diameter is greater than 5 cm [2]. Despite the rapid development of the screening program and endovascular therapy in the last 10 years, the rate of morbidity and mortality from aortic rupture remains high [3]. Physicians remain incapable of modifying the progression of AAA. Investigating the specific cellular mechanisms underlying the formation and progression of aneurysm may open a new chapter for AAA management.

* Corresponding author. Department of Vascular Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250012, China. ** Corresponding author. Department of Vascular Surgery, Shandong Provincial Hospital Affilaiated to Shandong University, Jinan, 250012, China. E-mail addresses: [email protected] (X. Jin), [email protected] (X. Wu). http://dx.doi.org/10.1016/j.atherosclerosis.2016.07.010 0021-9150/© 2016 Elsevier Ireland Ltd. All rights reserved.

Hypoxia inducible factor-1 (HIF-1) is a major transcriptional factor in the cellular response to hypoxia. HIF-1 is a heterodimer consisting of an unstable alpha subunit and a stable beta subunit [4]. The hypoxia inducible factor-1a (HIF-1a) is rapidly degraded under normoxic conditions, but stabilized when oxygen supply is limited. Accumulation of HIF-1a is commonly an acute and beneficial response to hypoxia. However, numerous recent studies have shown that HIF-1a levels can be regulated in a transcriptional manner under normoxia condition. For example, angiotensinⅡ (AngⅡ) and several inflammatory cytokines, such as tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6), may induce HIF-1a expression in vascular smooth muscle cells (VSMC) [5]. This transcriptional regulation may have shear effect on the amount of HIF1a protein. Both transcriptional and post-translational regulation mechanisms work together to modulate the doses of HIF-1a protein in eukaryotic organisms. Several previous studies showed a casual correlation between hypoxia and AAA development [6e8]. Hypoxia conditions caused by intraluminal thrombus (ILT) may stimulate smooth muscle cells and macrophages to secrete matrix metalloproteinases (MMPs),

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and contribute to the development of AAA [6]. However, in vivo studies of HIF-1 in the pathogenesis of AAA are lacking. On the other hand, the inhibition of prolyl-hydroxylase-2 (PHD-2), which is the main enzyme degrading HIF-1a under normoxia conditions, shows a protective role in the development of AAA [9]. These controversial results call for investigation to shed light into the role of HIF-1a in the pathogenesis of the aneurysm.

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sample of total RNA of the aorta was reverse-transcribed to cDNA with the Strand cDNA Synthesis Kit (Takara). qRT-PCR analysis was performed using the Power SYBR Green PCR Mastermix (Takara) and the Applied Roche 480 Real-Time PCR system according to the manufacturer's protocol. Primers used in the PCR are described in the Supplemental Table 1. 2.5. Western blot

2. Materials and methods 2.1. Time course study of murine AAA model All procedures were approved by the Animal Care and Use Committee of Shandong Provincial Hospital and were conducted following the institutional guidelines. Apolipoprotein E (ApoE
/
) deficient mice (C57Bl6/J background) were originally purchased from the Beijing Vital River Laboratory Animal Technology Corporation and fed a normal chow. Male mice (8 weeks of age) were divided into 2 groups (n ¼ 30 for each group), and implanted with Alzet osmotic minipumps (Model 1004, Durect Corporation), filled either with PBS (PBS group) or AngII solutions (Ang group, Abcam, Cambridge, United Kingdom. Ab120183, 1000 ng/kg/min) under anesthesia, by intraperitoneal injection of chloral hydrate (30 g/kg of body mass). The infusion persisted up to 4 weeks. At indicated time points (0d, 1d, 3d, 7d, 14d, 28d) after the implantation of osmotic pumps, 5 mice of each group were sacrificed for image acquisition and aortic harvest. Aortic diameter was measured by computer-assisted image analysis software ImageJ. 2.2. Lentiviral RNA interference transfection in vivo Lentivirus encoding HIF-1a shRNA was generated by Genomeditech Company (Shanghai, China). The most effective sequences of murine HIF-1a shRNA were: sense, 50 -GCAGGAAUUGGAACAUUAU dTdT-30 ; antisense, 30 dTdTCGUCCUUAACCUUGUAAUA-50 . Lentivirus expressing scrambled shRNA used as control was also purchased from Genomeditech. Lentivirus was delivered by tail vein injection at 108 pfu per mouse and experiments were carried out 7 days after injection, as described previously [10]. 2.3. AAA model of different groups, monitoring of blood pressure, aortic diameter and tissue harvest Three groups of ApoE
/
mice were included: PBS infusion þ scramble shRNA lentivirus (Sham group n ¼ 20), AngII infusion þ scramble shRNA lentivirus [Con group (control-group), n ¼ 20], and AngII infusion þ HIF-1a shRNA lentivirus (HIF-1a group, n ¼ 20). PBS or AngII was infused by ALZET miniosmotic pumps (Model 1004) as described above. Maximal aortic diameter was measured in all mice by ultrasound examination at 40 MHz (Vevo770; Visualsonics, Toronto, Ontario, Canada) at 3d, 7d, 14d, 28d after pump implantation. At 3 days and 28 days after pump implantation, murine heart rate (HR) and blood pressure (BP) were measured by tail-cuff method, under conscious conditions. Ten mice in each group were sacrificed at day 3; suprarenal aortic tissue was harvest for PCR, Western blot and Gelatin zymography analysis. The remaining 10 mice in each group were sacrificed at day 28 for histological analysis (Supplemental Fig. 1). 2.4. Quantitative real-time reverse-transcription polymerase chain reaction Total RNA was extracted from suprarenal aorta using Trizol Reagent (Invitrogen) according to manufacturer's protocol. 1 mg

Proteins were isolated from frozen suprarenal aortas or cells using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitors. Protein extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrically transferred to polyvinylidene difluoride membranes, which were blocked with 10% nonfat dry milk in TBS0.05% Tween 20 for 1 h, then anti-HIF-1a antibody (Ab463, Abcam) and anti-b-actin antibody (sc-81178, Santa Cruz Biotechnology, Santa Cruz, Calif) were applied overnight at 4  C, followed by exposure to the secondary antibody. Blots were detected using an ECL Prime Western Blotting Detection reagent (Millipore). The signals were quantified by ImageJ software. 2.6. Gelatin zymography Proteins were isolated from frozen suprarenal aortas or cells using RIPA buffer. Equal amounts of 10 mg protein or equal amount of CM were electrophoresed on 10% polyacrylamide resolving gel containing 1% gelatin and 4% polyacrylamide stacking gel. After that, the gels were renatured in renaturing buffer: 50 mM Tris-HCl containing 100 mM NaCl and 2.5% Triton X-100; then they were incubated with developing buffer: 50 mM Tris-HCl containing 10 mM CaCl2 overnight. After that, they were stained with staining buffer (0.8% Brilliant Blue R) (Beyotime, Beijing, China), and distained in water. Band intensities were quantified with ImageJ software. 2.7. Histological study Mouse suprarenal aortas were fixed in 4% paraformaldehyde for at least 24 h, paraffin-embedded, cut into 5-mm slices, and deparaffinized. HE staining and elastic Van-Gieson (EVG) staining were performed by a standard protocol. Immunohistochemical studies were performed using the anti-CD68 (cluster of differentiation 68) antibody (Ab955), anti-CD31 (platelet/endothelial cell adhesion molecule 1) antibody (Ab 38364), anti-MMP-2 antibody (Ab 37150) and anti-MMP-9 antibody (Ab 38898) as described previously [11]. After primary incubation, slices were washed and incubated with the appropriate secondary antibody and stained with 3, 3diaminobenzidine. For immunofluorescent examination, anti-HIF1a antibody (Ab463) and anti- a-SMA (alpha-smooth muscle actin) antibody (Ab5694) were used. After incubation with the appropriate Alexa Fluor 488/546-conjugated secondary antibodies (Invitrogen, Carlsbad, Calif), the sections were observed by confocal laser scanning microscopy (Leica, Solms, Germany). Medial elastin density was calculated by dividing the elastinpositive area by the total media area. Number of CD68-positive macrophages and CD31-positive microvessels was measured by counting the total number in 5 grid fields composed of a 400  250mm rectangle (0.1 mm2) in each mouse (n ¼ 4 in each group). 2.8. Statistical analyses All data are presented as means ± S.E.M. or S.D. Student's t-test or c2 test was used to examine differences between the two groups. Comparisons among three or more groups were made

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using one-way ANOVA and post hoc analysis with Bonferroni test. p < 0.05 was considered to be statistically significant. All data analyses were calculated by GraphPad Prism version 6 (GraphPad Software, Inc.).

the HIF-1a-group mice compared with the Con-group mice (maximal diameter 2.84 ± 1.96 mm vs. 1.41 ± 0.85 mm at day 28, p ¼ 0.0002, Fig. 2D and E). 3.4. HIF-1a knockdown preserved the ECM structure of AAA

3. Results 3.1. Sustained high levels of HIF-1a expression during AAA formation In the AngII infusion model, AAA develops over a period of 28 days, as defined by at least 50% increase in the original aortic diameter [12]. Although maximal aortic diameter of the PBS-group mice slightly increased during the 28-day period (Fig. 1A), a significant aortic dissection and dilation were observed 7 days after AngII infusion and with further expansion on day 28 (0.99 ± 0.04 mm vs. 1.36 ± 0.28 mm at day 7, p ¼ 0036, Fig. 1A). AAA was observed in 8 of 8 AngII-group mice on day 28but none in the PBS-group mice. These results are consistent with a previous study [13]. HIF-1 plays a central role in the transcriptional response to oxygen tension and HIF-1a is the major regulatory subunit of HIF-1 [14]. The suprarenal aortas harvested from different time points during AAA induction were analyzed for the time-course profiles of HIF-1a expression. In the PBS-group mice, the expression of HIF-1a mRNA did not show significant change during the 28-day period. In contrast, HIF-1a mRNA was remarkably increased in the AngIIgroup mice on day 1, much earlier than the timing of significant aortic dilation (1.09 ± 1.24 vs. 5.37 ± 2.73, p ¼ 0.0078). Moreover, HIF-1a mRNA expression persisted at a high level during the 28-day period of AAA induction (all p < 0.001 at day 3, 7, 14, and 28, Fig. 1B). Levels of HIF-1a protein were elevated at 3 days after AAA induction and remained at sustained high level over the period of AAA induction (3.32 ± 0.24 vs. 1.05 ± 0.24 at day 1, p ¼ 0.0015, all p < 0.001 at day 3, 7, 14, and 28). By contrast, in the PBS-group, the expression of HIF-1a protein did not change significantly during the 28-day period (Fig. 1C and D). Finally, in the aortic sample harvested at day 3 and day 28, HIF-1a was abundant in a-smooth muscle actin-positive VSMCs in the AngII-group, while in the PBSgroup, no signals of HIF-1a were detected (Fig. 1E). 3.2. Effects of HIF-1a shRNA on AngII-induced activation of HIF-1a Global HIF-1a deficient mice died at the embryonic day 10.5 due to cardiac and vascular malformation [15]. This embryonic lethality makes it difficult to examine the in vivo role of HIF-1a. To determine the pathophysiologic significance for AngII-induced HIF-1a expression, we conducted shRNA-induced in vivo knockdown of HIF-1a mRNA as described in the Methods section. Three days after AAA induction, HIF-1a protein levels of suprarenal aorta were successfully downregulated in the HIF-1a-group mice compared with Con-group mice (3.99 ± 0.39 vs. 2.22 ± 0.40, p ¼ 0.0082, Fig. 2A and B). We also confirmed the reduction of HIF-1a mRNA levels in the suprarenal aorta of the HIF-1a-group mice by qRT-PCR (Fig. 2C). These results confirmed successful knockdown of HIF-1a gene in the suprarenal aorta.

HE staining revealed marked enlargement of the luminal area, destruction of the media and marked thickening of the adventitia in the Con group after 28 days. However, these changes were attenuated by HIF-1a knockdown (Fig. 3A). EVG staining revealed the fractured of the elastic lamellae in the Con group, while in the Sham group, continuous and wavy elastic lamellae could be found. Knockdown of HIF-1a expression preserved the elastin structure of the aorta (Fig. 3B). 3.5. HIF-1a knockdown reduced the inflammatory response of AAA IHC staining for macrophages with an anti-CD68 antibody showed a marked infiltration of macrophages into the media and adventitia of the aorta in the Con group. Knockdown of HIF-1a decreased the number of infiltrated macrophages (38.8 ± 14.7 vs. 11.4 ± 4.4 macrophages/0.1 mm2, p < 0.0001, Fig. 4A and B). We also examined the expression of inflammatory cytokines in the suprarenal aorta, after 3 days of AngII infusion, when enlargement of the aorta is minimal. Expression of interleukin 1 beta (IL-1b), IL-6, monocyte chemotactic protein 1 (MCP-1) and TNF-a was up-regulated in the Con group compared with the Sham group. In the HIF-1a group, the expression of IL-1b, IL-6, MCP-1 and TNF-a was significantly lower than Con group (all p < 0.05, Fig. 4C). 3.6. HIF-1a knockdown attenuated neovascularity in AAA IHC staining showed CD31-positive vessels in periaortic tissue in the Con group compared with the Sham group (0.27 ± 0.44 vs. 5.56 ± 2.14 microvessels/0.1 mm2, p ¼ 0.0006), while HIF-1a knockdown attenuated the mural neovascularity compared with the Con group (5.56 ± 2.14 vs. 1.27 ± 1.05 microvessels/0.1 mm2, p ¼ 0.0008, Fig. 4D and E). The expression of vascular endothelial growth factor A (VEGF-A), fms related tyrosine kinase 1(Flt-1) and cluster of differentiation 31(CD31) was elevated in the suprarenal aorta after 3 days of AngⅡ infusion, when the enlargement of aorta was minimal. HIF-1a knockdown attenuated the secretion of these cytokines (all p < 0.05, Fig. 4F). 3.7. HIF-1a knockdown decreased MMPs activity IHC staining showed a reduced MMP-2 and MMP-9 positive area in the HIF-1a group compared with the Con group (Fig. 5A). After 3 days of AngⅡ infusion, The level of the zymographic active form of MMP-2 and MMP-9 was increased in the Con group compared with the Sham group and was attenuated by knockdown of HIF-1a (MMP-2, 1.36 ± 0.071 vs. 1.12 ± 0.052, p ¼ 0.032; MMP-9, 4.06 ± 0.217 vs. 1.67 ± 0.121, p ¼ 0.0007; Fig. 5B and C). PCR analysis showed reduced MMP-2 and MMP-9 mRNA and increased TIMP-1 mRNA in HIF-1a group compared with Con group, after 3 days of AngⅡ infusion (Fig. 5D).

3.3. HIF-1a knockdown prevented the development of AAA 4. Discussion AngII infusion modestly increased the HR and BP of mice after 3 and 28 days, while HIF-1a knockdown had no significantly effect on body mass (BM), HR, and BP (Supplemental Table 2), Although mean aortic diameters increased in both Con-group and HIF-1agroup after AAA induction, absolute growth was significantly less in the HIF-1a-group mice. Dilatation of the aorta was attenuated in

In the present study, we have shown that the expression of HIF1a remained high during the entire cause of AAA induction. HIF-1a knockdown attenuated the AngII induced aneurysms by reducing inflammatory cytokine expression, MMP activity, macrophage infiltration and neovascularity. Moreover, silencing of HIF-1a

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Fig. 1. Upregulation of HIF-1a during AAA formation. (A) Aortic diameter change at different time points after infusion of AngⅡ (AngⅡ group, n ¼ 5 for each time point) and PBS (PBS group, n ¼ 5 for each time point). The aortic diameter was larger in the AngⅡ group compared with the PBS group. (B) Time course of HIF-1a mRNA expression during AAA induction. (CeD) Western blot analysis of HIF-1a protein level in different groups. Expression levels on day 0 of different groups were set at 1. (E) Double immunofluorescent staining for HIF-1a and a-SMA are shown for aortas harvested at 3 days and 28 days after the infusion of AngⅡ and PBS. Results are means ± S.E.M.(n ¼ 5 per group) *p < 0.05, ** p < 0.01, ***p < 0.001 vs. PBS group.

suppressed inflammatory cytokine expression, and MMP activity at 3 days after induction, when dilatation of the aorta is minimal. These results indicated HIF-1a plays vital importance role in the formation of aneurysm. Previous studies focused on the effect of HIF-1a under the

hypoxia conditions, causing ILTs in the aneurysmal portion [6,16,17]. However, growing evidence suggested doses of HIF-1a protein could be modulated under normoxia conditions [18]. AngⅡ, which plays a vital role in the pathogenesis of the aneurysm, could stimulate HIF-1a expression in vascular smooth muscle cells under

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Fig. 2. Effect of HIF-1a knockdown on AngⅡ Ⅱ infusion model. (AeB) Protein expression of HIF-1a in the aortic wall, 3 days after AAA induction of the Sham group (PBS þ Scram. shRNA), Con group (AngⅡþScram. shRNA) and HIF-1a group (AngⅡþ HIF-1a shRNA). (C) qRT-PCR analysis of HIF-1a mRNA levels of suprarenal aortas in Sham, Con and HIF-1a groups, 3 days after AAA induction. (D) Representative macroscopic appearance of the aorta in different groups, 28 days after AAA induction. (E) The diameter of the abdominal aorta is larger in the Con group than in the Sham group. The diameter is smaller in the HIF-1a group than in the Con group after 28 days. Results are means ± S.E.M. (n ¼ 10 per group) *p < 0.05, **p < 0.01, ***p < 0.001 vs. Sham group; #p < 0.05, ##p < 0.001, ###p < 0.001 vs. Con group.

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Fig. 3. HematoxylineEosin (HE) and elastic Van Gieson (EVG) staining of the mouse aortic wall. (A) Representative HE staining of the aortic wall in the Sham group, Con group and HIF-1a group. (B) Representative EVG staining of the aortic wall in the Sham group, Con group and HIF-1a group. Original magnification: 40and 200. Scale bars indicate 200 mm.

normoxia conditions [19]. Studies of HIF-1a expression under normoxia conditions at the early phase of AAA induction are lacking. Our study confirmed that the accumulation of HIF-1a protein occurred at the 3rd day of AAA induction, preceded the enlargement of the aorta and formation of ILT [20]. Van Vickle et al. [21] had reported similar results in the intra-aortic elastase infusion model. These results indicated that HIF-1a may be upregulated in a hypoxia independent manner at the early phase of AAA induction. As persistent upregulation of HIF-1a protein exists along the induction of the murine AAA model, we further investigated the role of HIF-1a in the pathogenesis of murine AAA. Right now, the effect of HIF-1a on inflammatory disease remains controversial. Several HIF-1 target genes, such as VEGFA, endothelin 1 (ET-1) and stromal cell-derived factor 1 (SDF-1), have been proven to exert a contributive effect on vascular inflammation and AAA development [22e24]. On the other hand, there are many examples in which stabilization of HIF-1a dampens the inflammatory response. Cobalt chloride (CoCl2), as a PHD inhibitor, which can stabilize HIF-1a, has shown an anti-inflammatory effect in the CaCl2 induced murine AAA model [9]. Besides, HIF-1 plays a vital role in reducing the tissue damage induced by ischemia, seen in peripheral arterial

disease and myocardial infarction [25e28]. Several recent articles provided evidence to reconcile these discrepant results. Toshihiro Ichiki et al. reported tat PHD inhibitor suppressed lipopolysaccharide induced TNF-a upregulation in HIF1a depleted cells [29]. CoCl2 was found to downregulate vascular angiotensin II type 1 receptor (AT1R) expression of VSMCs in a rat model, while HIF-1a does not contribute to AT1R expression in VSMCs [30]. These results indicated that PHD inhibitors may exert an anti-inflammatory effect in a HIF-1a independent manner. The stabilization of HIF-1a under hypoxia conditions may attenuate tissue damage caused by insufficient oxygen supply. However, nonhypoxic stimuli of HIF-1a activation, such as TNF-a and AngII, may promote inflammation and recruit macrophages [24,31,32]. Our study confirmed that knockdown of HIF-1a reduced inflammatory cytokines secretion and macrophage infiltration in the murine model of AAA. In this study, we confirmed that HIF-1a mediated VEGF upregulation is preceded by the aorta enlargement. VEGF-induced macrophage recruitment and neovascularity are a critical feature of AAA, and macrophages themselves secrete MMP-9 and VEGF [22,33]. Several recent articles revealed that angiogenesis inhibitors

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Fig. 4. HIF-1a knockdown attenuate inflammation and neovascularity in AngⅡ Ⅱ infusion model. (AeB) IHC staining for CD68 in the mouse aorta at day 28. (C) Expression of MCP-1, TNF-a, IL-1b, IL-6 in the aortic wall 3 days after AAA induction. (DeE) IHC staining for CD31 in the mouse aorta at day 28. (F) Expression of VEGFA, FLT-1, and CD31 in the aortic wall 3 days after AAA induction. Scale bars indicate 200 mm. Results are means ± S.E.M. (n ¼ 5 per group) *p < 0.05, **p < 0.01, ***p < 0.001 vs. Sham group; #p < 0.05, ##p < 0.001, ### p < 0.001 vs. Con group.

attenuate murine AAA progression [35]. These results suggest macrophage recruitment, neovascularity and MMP secretion are interdependent and of vital importance in the formation and progression of AAAs. Both of them appeared to be abolished by HIF-1a knockdown. MMP-2 and MMP-9 work in concert to produce aortic

aneurysms [34]. AngII can induce MMP-2 upregulation in VSMCs and this upregulation of MMP-2 plays a vital role in the early development of AAAs [35,36]. Several in vitro studies had revealed HIF-1a may participate in this progression. Previous studies showed HIF-1 can upregulate MMP-2 expression in fibroblasts [37] and adipocytes [38]. Both of them are the main source of MMP-2 in

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Fig. 5. HIF-1a knockdown suppressed MMP activity in the aortic wall. (A) IHC staining for MMP-2 and MMP-9 in the mouse aorta at day 28. (BeC) Gelatin zymography assay of suprarenal aorta, 3 days after AAA induction. (D) Expression of MMP-2, MMP-9, and TIMP-1 in the aortic wall, 3 days after AAA induction. Results are means ± S.E.M. (n ¼ 5 per group) *p < 0.05, **p < 0.01, ***p < 0.001 vs. Sham group; #p < 0.05, ##p < 0.001, ###p < 0.001 vs. Con group.

AAA. The reduced MMP-9 activity may be associated with reduced aortic mural macrophage infiltration considering macrophages are the major sources of MMP-9. Alternatively, the expression of HIF-1a may have direct effect on the expression of MMP-2 and MMP-9. HIF1a overexpression may be involved in the increased MMP-2 and MMP-9 production in U937 cells exposed to non-hypoxia stimuli [39]. Tissue inhibitors of metalloproteinase 1 (TIMP-1), which binds pro-MMP-9 forming a complex, was decreased in aortic aneurysms [40]. Knockdown of HIF-1a maintained the expression of TIMP-1.

Therefore, silencing of HIF-1a maintained the balance of MMPs and TIMPs under AngⅡ stimulation and prevented the development of aneurysms. It is worth mentioning that chronic renal injury caused by AngII infusion may participate in the formation of aneurysm as renal function plays a vital role in the renin-angiotensin system. In the study by Zhu et al. [31], renal injury caused by AngⅡ infusion could be reversed by silencing the HIF-1a pathways in the kidney, which implies HIF-1a knockdown could also attenuate aneurysmal

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formation by indirect pathways. The in vitro experiment using isolated aorta or VSMCs may be need in the future because it would set aside the influence of renal function. A growing number of agents aiming to inhibit HIF-1a are under investigation for cancer therapy [41]. Manipulation of HIF-1a activity by genetic or pharmacological means has marked effects on tumor growth because of effects on angiogenesis, glucose metabolism and/or cell survival. Our study showed that some of these agents may have a retardation effect on the development of AAA. However, considering HIF-1a plays vital role in the cell survival under ischemia and hypoxia conditions, treating AAA with HIF-1a inhibitors needs to be done with great vigilance. There are several limitations in the present studies. First, silencing of HIF-1a was achieved by RNA interference. Further investigation using conditional HIF-1a knockout mice needs to be carried out to evaluate HIF-1a in different cells. Second, upregulation of HIF-1a in the early phase of AAA was elastase-induced and AngII-induced in mouse models, in a previous study [21] and the present study. However, considering the patho-morphological differences between animal models and human AAA [12,42], any conclusion on human AAA formation would be premature. Further studies are needed to address these issues. In conclusion, activation of HIF-1 signaling pathway participates in the Ang Ⅱ-induced AAA formation in mice. Silencing HIF-1a attenuates angiotensin Ⅱ-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice.

[6]

[7]

[8]

[9]

[10]

[11]

[12] [13]

[14] [15]

[16]

Funding information [17]

This work was supported, in part, by the National Natural Science Foundation of China [grant number: 81470575], and the National Natural Science Foundation of China [grant number: 8157020626].

[18]

[19]

Declaration of interests [20]

There were no declarations of pecuniary interests. [21]

Acknowledgements We thank Dr Shang Jin for helping with statistical analysis.

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Appendix A. Supplementary data

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Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2016.07.010.

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