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Antioxidant Vitamin C attenuates experimental abdominal aortic aneurysm development in an elastase-induced rat model

  • Tao Shang
    Affiliations
    Department of Vascular Surgery, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, People's Republic of China
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  • Author Footnotes
    1 Z.L. and C.L. equally contributed to this article.
    Zhao Liu
    Correspondence
    Corresponding author. Department of Vascular Surgery, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, 321 Zhongshan Road, Nanjing, 210008, People's Republic of China. Tel.: +86 025 83304616 60731; fax: +86 025 83308417.
    Footnotes
    1 Z.L. and C.L. equally contributed to this article.
    Affiliations
    Department of Vascular Surgery, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, People's Republic of China
    Search for articles by this author
  • Author Footnotes
    1 Z.L. and C.L. equally contributed to this article.
    Chang-jian Liu
    Correspondence
    Corresponding author. Department of Vascular Surgery, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, 321 Zhongshan Road, Nanjing, 210008, People's Republic of China Tel:+86 025 83304616 60731; Fax: +86 025 83308417.
    Footnotes
    1 Z.L. and C.L. equally contributed to this article.
    Affiliations
    Department of Vascular Surgery, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, People's Republic of China
    Search for articles by this author
  • Author Footnotes
    1 Z.L. and C.L. equally contributed to this article.
Published:December 09, 2013DOI:https://doi.org/10.1016/j.jss.2013.11.1105

      Abstract

      Background

      We investigated the hypothesis that an antioxidant, Vitamin C, could attenuate abdominal aortic aneurysm (AAA) development in a rat model.

      Methods

      An AAA model induced by intraluminal infusion was created in 36 male Sprague Dawley rats, which were randomly distributed into three groups: Sham (saline infused, placebo treated), Control (elastase infused, placebo treated), and Vitamin C (elastase infused, vitamin C treated). Vitamin C and placebo were intraperitoneally injected, initiating 1 wk before the infusion and continuing throughout the study. The aortic dilatation ratio was measured, and aortic tissues were further examined using biochemical and histologic techniques.

      Results

      Vitamin C attenuated the development of AAA, decreasing maximal aortic diameter by 25.8% (P < 0.05) and preserving elastin lamellae (P < 0.05). Vitamin C also decreased 8-hydroxyguanine (a marker of oxidative damage to DNA) and 8-isoprostane content (a marker of oxidative stress) in aortic tissues (P < 0.05, respectively). The proteins of matrix metalloproteinase (MMP)-2, MMP-9, and interleukin 6 were markedly downregulated (P < 0.05, respectively), accompanied with notably reduced messenger RNA expression of tumor necrosis factor-α, MMP-2/9, and interleukin 1β (P < 0.05, respectively). However, messenger RNA of tissue inhibitors of metalloproteinase-1 and tissue inhibitors of metalloproteinase-2 were both significantly upregulated in Vitamin C group. Vitamin C treatment had no significant effect on systolic blood pressure (P > 0.05).

      Conclusions

      Vitamin C attenuated AAA development in an elastase-induced rat model via crucial protective effect, which was mediated by an increased level of antioxidant in cooperation with preserving elastin lamellae, inhibiting matrix-degrading proteinases and suppressing inflammatory responses.

      Keywords

      1. Introduction

      Abdominal aortic aneurysm (AAA) is a silent and potentially life-threatening disease characterized by transmural aortic wall degeneration, leading to dilatation, progressive growth, and catastrophic rupture [
      • Kent K.C.
      • Zwolak R.M.
      • Egorova N.N.
      • et al.
      Analysis of risk factors for abdominal aortic aneurysm in a cohort of more than 3 million individuals.
      ,
      • Ailawadi G.
      • Eliason J.L.
      • Upchurch Jr., G.R.
      Current concepts in the pathogenesis of abdominal aortic aneurysm.
      ,
      • Lederle F.A.
      • Johnson G.R.
      • Wilson S.E.
      • et al.
      Veterans Affairs Cooperative Study #417 Investigators
      Rupture rate of large abdominal aortic aneurysms in patients refusing or unfit for elective repair.
      ]. Although open or endovascular surgical therapy of large AAA is recommended, repair of small abdominal aortic aneurysms (sAAA) does not provide a significant benefit [
      Mortality results for randomised controlled trial of early elective surgery or ultrasonographic surveillance for small abdominal aortic aneurysms. The UK Small Aneurysm Trial Participants.
      ,
      • Lederle F.A.
      • Wilson S.E.
      • Johnson G.R.
      • et al.
      Aneurysm Detection and Management Veterans Affairs Cooperative Study Group
      Immediate repair compared with surveillance of small abdominal aortic aneurysms.
      ,
      • Brewster D.C.
      • Cronenwett J.L.
      • Hallett Jr., J.W.
      • Johnston K.W.
      • Krupski W.C.
      • Matsumura J.S.
      Joint Council of the American Association for Vascular Surgery and Society for Vascular Surgery
      Guidelines for the treatment of abdominal aortic aneurysms. Report of a subcommittee of the Joint Council of the American Association for Vascular Surgery and Society for Vascular Surgery.
      ]. Although pharmacologic treatment for sAAA has not been established clinically, experimental studies have demonstrated that various inflammatory networks contribute to the AAA formation through certain signaling pathways to promote the degradation of extracellular matrix or impair the biosynthesis of extracellular matrix [
      • Brophy C.M.
      • Reilly J.M.
      • Smith G.J.
      • Tilson M.D.
      The role of inflammation in nonspecific abdominal aortic aneurysm disease.
      ,
      • Freestone T.
      • Turner R.J.
      • Coady A.
      • Higman D.J.
      • Greenhalgh R.M.
      • Powell J.T.
      Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm.
      ,
      • Shah P.K.
      Inflammation, metalloproteinases, and increased proteolysis: an emerging pathophysiological paradigm in aortic aneurysm.
      ,
      • Newman K.M.
      • Jean-Claude J.
      • Li H.
      • Ramey W.G.
      • Tilson M.D.
      Cytokines that activate proteolysis are increased in abdominal aortic aneurysms.
      ,
      • Koch A.E.
      • Haines G.K.
      • Rizzo R.J.
      • et al.
      Human abdominal aortic aneurysms. Immunophenotypic analysis suggesting an immune-mediated response.
      ]. Moreover, the enhanced oxidative stress, which occurs during the inflammatory response has been demonstrated to contribute to the formation of AAA [
      • Gavrila D.
      • Li W.G.
      • McCormick M.L.
      • et al.
      Vitamin E inhibits abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice.
      ].
      Oxidative stress results from an imbalance between free radical production and insufficient endogenous antioxidant defense mechanism [
      • Praticò D.
      • Reilly M.
      • Lawson J.A.
      • FitzGerald G.A.
      Novel indices of oxidant stress in cardiovascular disease: specific analysis of F2-isoprostanes.
      ]. As a potent water-soluble antioxidant, Vitamin C can scavenge reactive oxygen species and reactive nitrogen species by forming semidehydroascorbic acid and increasing endothelial nitric oxide synthase activity to prevent oxidative damage to important biological macromolecules [
      • Alleva R.
      • Di Donato F.
      • Strafella E.
      • et al.
      Effect of ascorbic acid-rich diet on in vivo-induced oxidative stress.
      ,
      • d'Uscio L.V.
      • Milstien S.
      • Richardson D.
      • Smith L.
      • Katusic Z.S.
      Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity.
      ]. Vitamin C has also been proven to enhance elastin and collagen production from aortic smooth muscle cells and these two proteins have been reported to be crucial in arterial structure according to the previous studies [
      • Qiao H.
      • Bell J.
      • Juliao S.
      • Li L.
      • May J.M.
      Ascorbic acid uptake and regulation of type I collagen synthesis in cultured vascular smooth muscle cells.
      ,
      • Hospelhorn A.C.
      • Martin B.M.
      • Franzblau C.
      Type IV collagen synthesis and accumulation in neonatal rat aortic smooth muscle cell cultures.
      ].
      In the present study, we hypothesized that Vitamin C could attenuate aneurysmal development in a rat model by elastase infusion and investigated the possible molecular mechanisms.

      2. Materials and methods

      2.1 Experimental groups and AAA model

      All the experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals, which is approved by the Ethical Committee of Researches of the Nanjing University. Six-wk-old male rats (SLAC Laboratory Animal Co, Ltd, Shanghai, China) weighing 180–220 g were used in our study. All rats (n = 12/each group) were randomly distributed into three groups: Vitamin C, Control, and Sham. Rats were housed under a 12-h light/dark cycle with standard diet and water. And they were anesthetized and underwent laparotomy mainly following previous methods [
      • Anidjar S.
      • Salzmann J.L.
      • Gentric D.
      • Lagneau P.
      • Camilleri J.P.
      • Michel J.B.
      Elastase-induced experimental aneurysms in rats.
      ]. Briefly, the abdominal aorta was isolated, and a tiny incision was made on the aorta bifurcation. Then, a PE-10 polyethylene (PE) tube (Smiths Medical International Ltd, Ashford, UK) was introduced through the incision into the abdominal aorta. The aorta was clamped below the renal artery and above the tip level of the PE tube, and then ligated with a 4-0 silk suture (Ethicon, Johnson-Johnson Company, New Brunswick) near the aortic bifurcation, followed by infusion with 40 μL (40 U) type I porcine pancreatic elastase (5.9 U/mg; Sigma-Aldrich, Co, St. Louis) in both Vitamin C and Control group for 10 min using a microinfusion pump at 100 mmHg and with isotonic saline in Sham group. After infusion, the clamp and ligatures were removed, and the PE tube was withdrawn. The incision was sutured with an 8-0 polypropylene suture (Prolene, Johnson-Johnson Company New Brunswick, USA). Aortic segments were harvested for further study on the 28th day after infusion. The systolic blood pressure of the rats was measured through tail-cuff technique before drug administration and sacrifice.

      2.2 Drug administration

      Initiating 1 wk before the aneurysm preparation, Vitamin C (100 mg/mL) was injected intraperitoneally to the Vitamin C group (n = 12) twice daily at a dose of 1 g/kg/d and continued throughout the duration of the study. Each rat received 180–200 mg (1.8–2.0 mL) Vitamin C, depending on its weight. The same volume of placebo was given to Sham (n = 12) and Control groups (n = 12) at the same time via the same method.

      2.3 Measurement of aortic size via ultrasound

      Ultrasound system (Sono Site Inc, Bothell), which contained a linear transducer (25 MHz), was used to demonstrate the dilation of the rats' aortas. The maximum inner luminal diameters of abdominal aortas were measured before drug administration, before laparotomy and on the 7th, 14th, 21st, and 28th day after the operation. Two experienced operators who were unaware of the protocols did the quantitative analysis of the ultrasound data.

      2.4 Histologic studies

      All rats were sacrificed 28 d after the operation. The excised aorta was fixed in 10% neutral-buffered formalin and processed using routine paraffin embedding. Aortic tissue cross-sections (5 μm) were stained with hematoxylin and eosin and Miller elastin-Van Gieson (EVG) following standard procedures. The percentage of the surface area occupied by the EVG-stained elastic fibers was quantified by morphometry system MacScope Ver. 2.2 (Mitani Corporation, Kanazawa Japan).

      2.5 Enzyme-linked immunosorbent assay

      8-Isoprostane content assay was performed following the manufacturer's instructions (#516351; Cayman Chemical, Ann Arbor), using abdominal aortic tissue homogenates. All samples were run in duplicates and at minimum of two dilutions. Results were expressed per milligram of protein as determined by Bradford assay (Bio-Rad, Hercules).

      2.6 Immunohistochemical staining

      Mouse monoclonal antibodies for matrix metalloproteinase (MMP)-2, MMP-9, and 8-hydroxyguanine (8-OHdG) (ab3158, ab58803, ab62623; Abcam Ltd, Cambridge) were used to analyze the local expression of matrix-degrading proteinases and oxidative stress. Immunohistochemical (IHC) staining was performed using an immunoperoxidase avidin-biotin complex system. After blocking the activity of endogenous peroxidase, the sections were incubated in the primary antibodies (1:100) overnight at 4°C. After that, according to the manufacturer's specifications (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA), we incubated the sections with biotinylated anti-mouse IgG antibody for 30 min (Vector Laboratories, Burlingame) and then avidin-biotinylated horseradish peroxidase complex in phosphate-buffered saline for 10 min. Immune complexes were visualized using 0.05% 3, 3′-diaminobenzidine (Vector Laboratories, Burlingame). The slides were counterstained using hematoxylin.

      2.7 Quantitative (real-time) reverse transcriptase–polymerase chain reaction

      The expression of MMP-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α messenger RNA (mRNA) in aortic tissues was determined via quantitative (real-time) reverse transcriptase–polymerase chain reaction. Total mRNA was extracted from the aorta using TRIzol reagent (Invitrogen Life Technologies, Grand Island, USA) and complementary DNA produced via reverse transcription using oligo-(dT) primer 5.0 and M-MLV reverse transcriptase (Fermentas, Thermo Fisher Scientific Inc, Vilnius, Lithuania). The polymerase chain reactions (PCRs) were performed in quadruplicate with SYBR Green PCR Core Reagents (TOYOBO, CO., LTD, Osaka, Japan), and fluorescence signals were analyzed using the DA7600 Sequence Detection System (Zhong-shan Da-An Inc, Guangzhou, China). The results for each sample were normalized to the concentration of β-actin mRNA. PCR amplification was performed under the following conditions: denaturation for 15 s at 95°C, annealing for 30 s at 60°C, and extension for 30 s at 72°C.

      2.8 Western blotting

      Aortic tissues were obtained and homogenized 28 d after the operation, and total proteins were extracted from the frozen aorta tissues. The samples (70 μg) were electrophoresed in sodium dodecyl sulfate–polyacrylamide gel at 80 V, transferred onto polyvinylidene difluoride membranes at 300 mA, and incubated for 1 h in Tris Buffer Solution, 5% nonfat milk, and 0.2% Tween-20 at room temperature. The membranes were then incubated for 24 h at 4°C with different antibodies, including mouse monoclonal antibodies for MMP-2, MMP-9 (1:2000 dilution; Abcam Ltd), rabbit polyclonal antibody for IL-6 (1:500 dilution; Abcam Ltd), and 8-OHdG (1:2000 dilution; Santa Crus Bio, Dallas). They were washed in Tris Buffer Solution and 0.1% Tween-20 and then incubated for 2 h at room temperature with sheep anti-mouse IgG secondary antibody for MMP-2\MMP-9\IL-6 (1:5000; Amersham Biosciences) and goat anti-rabbit IgG for 8-OHdG (1:5000; Amersham Biosciences, Pittsburgh). The membranes were visualized using an ECL plus chemiluminescent kit (Amersham Biosciences) following the manufacturer's instructions and exposed to X-ray film (Kodak Co, Rochester). β-actin levels were used to standardize protein loading. To quantify and compare the levels of proteins, the density of each band was measured via densitometry (Shimadzu Co, Japan).

      2.9 Statistical analysis

      All values were expressed as the mean ± standard error of the mean. Statistical analysis was performed using SPSS ver. 16.0 (SPSS Inc, Chicago, IL). Differences between two groups were analyzed by Student t-test, and differences between multiple groups were analyzed by one-way analysis of variance followed by Bonferroni t-testing. Fisher exact test was used to analyze categorical data. P-values <0.05 were considered as significant.

      3. Results

      3.1 Vitamin C did not affect systolic blood pressure in this experimental model

      As hypertension is a documented risk factor for AAA enlargement, we monitored this parameter in all groups. Twenty-eight days after intra-aortic infusion, the values of systolic blood pressure in all three groups had no significant difference (Table). This result indicated that Vitamin C at a dose of 1 g/kg/d had not affected the blood pressure (P > 0.05).
      TableEffect of Vitamin C (1 g/kg/d) on systolic blood pressure (mmHg).
      Group1 wk Before infusion (n)Day 28 after (n)P value
      Sham113 ± 4 (12)108 ± 4 (12)0.322
      Control109 ± 3 (12)114 ± 4 (10)0.301
      Vit C111 ± 4 (12)110 ± 3 (9)0.253
      P value0.2270.289

      3.2 Vitamin C decreased AAA diameter

      Measurement of aortic size by ultrasound was initiated 1 wk prior the operation and performed on every week afterward. Aneurysms were successfully established 1 week after the infusion, and the diameter of AAA was more than twice of the normal aorta at the end of 4 wk after the operation (Fig. 1, Fig. 2; Control versus Sham, 0.31 ± 0.038 versus 0.16 ± 0.012, t = 12.94, P < 0.001). Animals that received intraperitoneal Vitamin C showed a 25.8% reduction in maximal aortic diameter as compared with Control group (Fig. 1, Fig. 2; Vitamin C versus Control, 0.23 ± 0.023 versus 0.31 ± 0.038, t = 5.62, P < 0.001). Three rats in Control group died during the study due to aneurysms rupture, whereas two animals in Vitamin C group died during ultrasound measurement under anesthesia due to choke because food scraps were found in both windpipes.
      Figure thumbnail gr1
      Fig. 1The photographs show treated lesions of abdominal aorta before tissue harvest in each group. 0 d: the infusion day; n = 12 per group; Vit C, Vitamin C group. Sham, saline infused, placebo treated; Control, elastase infused, placebo treated; Vitamin C, elastase infused, Vitamin C treated. (Color version of figure is available online.)
      Figure thumbnail gr2
      Fig. 2Aortic diameters were detected by ultrasound 28 d after infusion (A). Development of aortic size after drug administration was assessed by ultrasound (B). All the data are expressed as the mean ± standard deviation; Vit C, Vitamin C group. Sham, saline infused, placebo treated; Control, elastase infused, placebo treated; Vitamin C, elastase infused, Vitamin C treated; *P < 0.05 versus Sham, †P < 0.05 versus Control.

      3.3 Vitamin C preserved elastin lamellae in aortic walls

      Hematoxylin and eosin staining showed that the Control group had weaker staining of smooth muscle cells and more mural thrombus than other groups (Fig. 3A1 and A2 ). In addition, we found observable degeneration of elastic lamellae in Control group by EVG staining (Fig. 3B1 and B2, Control versus Sham, 7.6 ± 2.6 versus 22.3 ± 3.7, t = 10.16, P < 0.001). The elastin content in Vitamin C group, however, was markedly preserved compared with Control group (Fig. 3B1 and B2, Vitamin C versus Control, 14.6 ± 3.3 versus 7.6 ± 2.6, t = 5.09, P < 0.001).
      Figure thumbnail gr3
      Fig. 3Histologic sections of rat aortas stained with hematoxylin and eosin staining (A1, ×40; A2, ×200). Histologic sections of rat aorta stained with Miller EVG staining (B1, ×100); elastin is stained dark purple. The quantification of elastin at 4 wk (B2). All the data are expressed as the mean ± standard deviation; Vit C, Vitamin C group. Sham, saline infused, placebo treated; Control, elastase infused, placebo treated; Vitamin C, elastase infused, Vitamin C treated; *P < 0.05 versus Sham, †P < 0.05 versus Control. (Color version of figure is available online.)

      3.4 Vitamin C decreased the expression of MMP-2 and MMP-9

      Activated MMP-2 and MMP-9 in the aortic walls were analyzed by IHC staining (Fig. 4A1 ). Strongest staining of activated MMP-2 or MMP-9 subunits was detected in the Control group (Fig. 4A1 and A2, Control versus Sham, MMP-2, 72.2 ± 11.8 versus 12.4 ± 2.8, t = 17.06, P < 0.001; MMP-9, 115.6 ± 12.9 versus 15.2 ± 4.3, t = 25.33, P < 0.001). The staining was weaker in Vitamin C group than Control group but deeper than Sham group (Fig. 4A1 and A2, Vitamin C versus Control, MMP-2, 20.6 ± 3.1 versus 72.2 ± 11.8, t = 13.36, P < 0.001; MMP-9, 30.1 ± 6.6 versus 115.6 ± 12.9, t = 18.48, P < 0.001). Western blot (WB) showed that both MMP-2 and MMP-9 within the aortic segment were overexpressed in the Control group (Fig. 4B1 and B2, Control versus Sham, MMP-2, 3.61 ± 0.31 versus 1.63 ± 0.17, t = 18.76, P < 0.001; MMP-9, 3.84 ± 0.28 versus 1.43 ± 0.18, t = 23.98, P < 0.001). Meanwhile, the expression of MMP-2 and MMP-9 were significantly downregulated in the Vitamin C group (Fig. 4B1 and B2, Vitamin C versus Control, MMP-2, 1.85 ± 0.27 versus 3.61 ± 0.31, t = 13.18, P < 0.001; MMP-9, 2.28 ± 0.23 versus 3.84 ± 0.28, t = 13.30, P < 0.001). The mRNA expression analysis by quantitative (real-time) reverse transcriptase–polymerase chain reaction had a similar result. (Fig. 4C1, MMP-2, Vitamin C versus Control, 1.52 ± 0.44 versus 2.14 ± 0.54 t = 2.76, P = 0.007; C2, MMP-9, 1.35 ± 0.44 versus 2.76 ± 0.77, t = 5.48, P < 0.001). Moreover, it was also found that the mRNA expression of TIMP-1 and TIMP-2 by Vitamin C treatment were notably upregulated compared with Control group (Fig. 4C3, TIMP-1, Vitamin C versus Control, 0.93 ± 0.26 versus 0.48 ± 0.18, t = 4.34, P < 0.001; C4, TIMP-2, 3.11 ± 0.58 versus 0.41 ± 0.17, t = 13.42, P < 0.001).
      Figure thumbnail gr4
      Fig. 4IHC staining for MMP-2 and MMP-9 in aortic tissue (A1, ×100); The quantification of staining (A2); WB results of MMP-2 and MMP-9 in the aneurysm wall (B1). Quantitative analysis of MMP-2 and MMP-9 expression by densitometry (B2); mRNA expression of MMP-2 (C1), MMP-9 (C2), TIMP-1 (C3), and TIMP-2 (C4) measured by real-time reverse transcriptase–polymerase chain reaction. All the data are expressed as the mean ± standard deviation; Vit C, Vitamin C group. Sham, saline infused, placebo treated; Control, elastase infused, placebo treated; Vitamin C, elastase infused, Vitamin C treated; *P < 0.05 versus Sham, †P < 0.05 versus Control. (Color version of figure is available online.)

      3.5 Vitamin C decreased the oxidative stress in abdominal aorta

      To examine whether Vitamin C exerted an antioxidant effect in the abdominal aorta during AAA formation, we determined the concentration of 8-isoprostane and expression of 8-OHdG in aortic tissue through IHC and WB. IHC staining demonstrated stronger 8-OHdG–positive cells in Control group. However, Vitamin C successfully decreased the expression of 8-OHdG 4 wk after the infusion (Fig. 5A1 and A2 , Vitamin C versus Control, 18.4 ± 3.5 versus 96.9 ± 10.3, t = 22.75, P < 0.001; Fig. 5C1 and C2, 1.07 ± 0.18 versus 1.91 ± 0.21, t = 9.36, P < 0.001). The enzyme-linked immunosorbent assay also showed a similar result of concentration of 8-isoprostane in aortic tissue homogenates, which proved that Vitamin C could downregulate the oxidative stress (Fig. 5B, Vitamin C versus Control, 13.38 ± 3.27 versus 28.84 ± 5.53, t = 7.51, P < 0.001).
      Figure thumbnail gr5
      Fig. 5IHC staining for 8-OHdG and IL-6 in aortic tissue (A1, ×100); The quantification of staining (A2); enzyme-linked immunosorbent assay result of 8-isoprostane in abdominal aortic tissue homogenates (B); Western blot results of 8-OHdG and IL-6 in the aneurysm wall (C1). Quantitative analysis of 8-OHdG and IL-6 expression by densitometry (C2); mRNA expression of IL-1β (D1) and TNF-α (D2) measured by real-time reverse transcriptase–polymerase chain reaction. All the data are expressed as the mean ± standard deviation; Vit C, Vitamin C group. Sham, saline infused, placebo treated; Control, elastase infused, placebo treated; Vitamin C, elastase infused, Vitamin C treated; *P < 0.05 versus Sham, †P < 0.05 versus Control. (Color version of figure is available online.)

      3.6 Vitamin C decreased the inflammatory response in aneurysmal tissue

      Inflammation is believed to contribute to the etiology of MMP-regulated and TIMP-regulated AAA formation. We examined the expression of IL-6, an inflammatory cytokine, by IHC and WB, accompanied with the mRNA expression of IL-1β and TNF-α. As illustrated in Figure 5, the outcome of IHC and WB had similar suggestions that the overexpressed IL-6 could be suppressed by Vitamin C (Fig. 5A1 and A2, Vitamin C versus Control, 48.3 ± 9.7 versus 144.2 ± 13.9, t = 17.59, P < 0.001; Fig. 5C1 and C2, 1.60 ± 0.21 versus 2.46 ± 0.27, t = 7.83, P < 0.001). In addition, the mRNA expression levels of inflammatory cytokines, IL-1β and TNF-α, were both downregulated in Vitamin C group (Fig. 5D1 and D2, Vitamin C versus control, 0.73 ± 0.19 versus 1.35 ± 0.22, t = 6.59, P < 0.001; 0.68 ± 0.19 versus 2.07 ± 0.36, t = 10.69, P < 0.001).

      4. Discussion

      The present study demonstrated for the first time that Vitamin C exerted crucial protective effect against AAA development in an elastase-induced rat model. This effect was mediated by an increased level of antioxidant in cooperation with preserving elastin lamellae, inhibiting proteolysis of extracellular matrix proteins and suppressing inflammatory response.
      The elastase-induced AAA model is a standard aneurysm model for in vivo research in small animals [
      • Miwa K.
      • Nakashima H.
      • Aoki M.
      • et al.
      Inhibition of ets, an essential transcription factor for angiogenesis, to prevent the development of abdominal aortic aneurysm in a rat model.
      ]. Elastase breaks down elastin, which determines the structural and mechanical properties of aortic extracellular matrix [
      • Nakashima H.
      • Aoki M.
      • Miyake T.
      • et al.
      Inhibition of experimental abdominal aortic aneurysm in the rat by use of decoy oligodeoxynucleotides suppressing activity of nuclear factor kappaB and ets transcription factors.
      ]. The main involvement of elastase in the aneurysm formation is through enhanced elastolytic activity and a loss of elastin in the aortic walls. This model has attracted considerable interest for its potential relevance to human AAAs due to the similar histologic features including leukocyte infiltration, medial degeneration, and excessive production of various matrix-degrading proteinases [
      • Aoki H.
      • Yoshimura K.
      • Matsuzaki M.
      Turning back the clock: regression of abdominal aortic aneurysms via pharmacotherapy.
      ]. We successfully established AAA according to the instruction, although three rats died within 2 wk after the elastase infusion because of aneurysms rupture. It should be emphasized that only animals surviving to the end of the study were included in the analysis.
      Elastin and collagens are the major structural components of the aortic wall. Collagens are responsible for tensile strength and prevent aneurysm rupture. Elastic fibers maintain the structure of the aortic wall against hemodynamic stress, resulting in the prevention of aortic dilatation [
      • Liapis C.D.
      • Paraskevas K.I.
      The pivotal role of matrix metalloproteinases in the development of human abdominal aortic aneurysms.
      ,
      • Longo G.M.
      • Xiong W.
      • Greiner T.C.
      • Zhao Y.
      • Fiotti N.
      • Baxter B.T.
      Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms.
      ]. Aneurysm development involves a complex remodeling process with an imbalance between the synthesis and degradation of elastin and collagens. MMP-2 and MMP-9 have attracted interest and been considered as the predominant proteinases in the process of AAA development [
      • Longo G.M.
      • Xiong W.
      • Greiner T.C.
      • Zhao Y.
      • Fiotti N.
      • Baxter B.T.
      Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms.
      ]. The activation of MMPs is tightly regulated by TIMPs, and mRNA levels of TIMPs are decreased in AAA tissue [
      • Tsarouhas K.
      • Soufla G.
      • Apostolakis S.
      • et al.
      Transcriptional regulation of TIMPs in ascending aorta aneurysms.
      ]. Our experiment revealed that MMP-2 and MMP-9 were highly expressed in the aortic walls of AAA rats, whereas animals received intraperitoneal Vitamin C showed a significant downexpression of the two proteinases at mRNA and protein levels. In addition, we also found the mRNA of TIMP-1 and TIMP-2 were notably increased by treatment with Vitamin C, confirming that Vitamin C effectively inhibited the proteolysis of extracellular matrix proteins in this animal model.
      Oxidative stress has been demonstrated to play a significant role in human AAA formation and progression [
      • Morimoto K.
      • Hasegawa T.
      • Tanaka A.
      • et al.
      Free-radical scavenger edaravone inhibits both formation and development of abdominal aortic aneurysm in rats.
      ,
      • Yajima N.
      • Masuda M.
      • Miyazaki M.
      • Nakajima N.
      • Chien S.
      • Shyy J.Y.
      Oxidative stress is involved in the development of experimental abdominal aortic aneurysm: a study of the transcription profile with complementary DNA microarray.
      ]. Oxidative damage to vascular smooth muscle cells sharply decreased the synthesis capability of elastin and collagens, resulting in the degeneration of aortic walls and rupture in the end [
      • Morimoto K.
      • Hasegawa T.
      • Tanaka A.
      • et al.
      Free-radical scavenger edaravone inhibits both formation and development of abdominal aortic aneurysm in rats.
      ,
      • Yajima N.
      • Masuda M.
      • Miyazaki M.
      • Nakajima N.
      • Chien S.
      • Shyy J.Y.
      Oxidative stress is involved in the development of experimental abdominal aortic aneurysm: a study of the transcription profile with complementary DNA microarray.
      ,
      • Shang T.
      • Liu Z.
      • Zhou M.
      • Zarins C.K.
      • Xu C.
      • Liu C.J.
      Inhibition of experimental abdominal aortic aneurysm in a rat model by way of tanshinone ⅡA.
      ]. Our team recently reported that Tanshinone IIA could inhibit the development of AAA through multiple effects, and one of them was reducing oxidative stress [
      • Shang T.
      • Liu Z.
      • Zhou M.
      • Zarins C.K.
      • Xu C.
      • Liu C.J.
      Inhibition of experimental abdominal aortic aneurysm in a rat model by way of tanshinone ⅡA.
      ]. In the present study, 8-OHdG–positive cells, a marker of oxidative damage to DNA, were strongly illustrated in the aortic walls of Control group. Vitamin C, as a well-known antioxidant, significantly suppressed the oxidative stress and the content of 8-OHdG were obviously reduced in Vitamin C group. Moreover, 8-isoprostanes are stable products of membrane lipid peroxidation, and their tissue concentration correlates with the level of oxidative stress. We demonstrated that elastase infusion markedly increased tissue concentration of aortic 8-isoprostanes, which was significantly lowered by treatment with Vitamin C, confirming that Vitamin C effectively reduced aortic oxidative stress.
      Chronic inflammation of the aortic walls plays an important role in the pathogenesis of AAA, which could lead macrophages and lymphocytes to infiltrate into adventitia [
      • Rentschler M.
      • Baxter B.T.
      Pharmacological approaches to prevent abdominal aortic aneurysm enlargement and rupture.
      ,
      • Thompson R.W.
      • Curci J.A.
      • Ennis T.L.
      • Mao D.
      • Pagano M.B.
      • Pham C.T.N.
      Pathophysiology of abdominal aortic aneurysms: insights from the elastase-induced model in mice with different genetic backgrounds.
      ]. Various proinflammatory cytokines are secreted from the recruited macrophages to induce more inflammatory cells. Both smooth muscle cells and infiltrating inflammatory cells produce MMPs, resulting in contribution to AAA development [
      • Shiraya S.
      • Miyake T.
      • Aoki M.
      • et al.
      Inhibition of development of experimental aortic abdominal aneurysm in rat model by atorvastatin through inhibition of macrophage migration.
      ]. In the present study, staining and WB of IL-6 and mRNA expressions of IL-1β and TNF-α in the aortic walls showed Vitamin C successfully suppressed recruitment of inflammatory cells and inflammatory responses.
      An interesting finding in the present study was that dietary Vitamin C did not exert much protective effects on AAA. We preformed a preliminary experiment (n = 3) with oral Vitamin C treatment of the same volume as the present study. However, it could not prevent dilatation (187%, 204%, and 216% dilatation at 4 wk). One reasonable explanation was that the intraperitoneal injection in the present study delivered Vitamin C more directly to the target aorta. However, the number of experiment animals was still not enough, thus the conclusion needed more solid evidences to support.
      The limitation of this study was that we did not perform experiments to evaluate the effect of Vitamin C on existing AAA because human AAAs are always pre-existing when they are found. Our results strongly suggested Vitamin C as an effective preventive medicine to many patients with sAAA, but it was important to demonstrate whether a pharmacologic treatment could induce AAA regression before it was established as a clinical therapy. No side effects were observed with Vitamin C at 1 g/kg/d in this study. We believed that Vitamin C had beneficial antioxidative actions as long as the dose was optimal. Further study with other doses of Vitamin C would be necessary to evaluate its dose-dependent effects on AAA prevention.

      5. Conclusion

      We demonstrated for the first time that intraperitoneally injected Vitamin C exerted crucial inhibition against AAA development in an elastase-induced rat model. The protective effect was mediated by an increased level of antioxidative stress in cooperation with the preserving elastin lamellae, inhibiting matrix-degrading proteinases and suppressing inflammatory response.

      Acknowledgment

      This work was supported by grants from National Nature Science Foundation of China (81270396) and Nature Science Foundation of Jiangsu Province, China (BK2009035). The authors thank Dr Wei Wang and Dr Cheng-yan Zhu for assistance in ultrasound measurement.
      Conflict of Interest: None declared.

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