Ang II-induced Abdominal Aortic Aneurysm Research Articles

Aldehyde dehydrogenase 2 protects against abdominal aortic aneurysm formation by reducing reactive oxygen species, vascular inflammation, and apoptosis of vascular smooth muscle cells

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is an enzyme that detoxifies aldehydes by converting them to carboxylic acids. ALDH2 deficiency is known to increase oxidative stress. Increased oxidative stress plays a pivotal role in abdominal aortic aneurysm (AAA) pathogenesis. Reactive oxygen species (ROS) promote degradation of the extracellular matrix (ECM) and vascular smooth muscle cell (VSMC) apoptosis. Reducing oxidative stress by an ALDH2 activator could have therapeutic potential for limiting AAA development. We hypothesized that ALDH2 deficiency could increase the risk for AAA by decreasing ROS elimination and that an ALDH2 activator could provide an alternative option for AAA treatment. The National Center for Biotechnology (NCBI) Gene Expression Omnibus (GEO) database was used. Human aortic smooth muscle cells (HASMCs) were used for the in vitro experiments. Gene-targeted ALDH2*2 KI knock-in mice on a C57BL/6J background and apolipoprotein E knockout (ApoE KO) mice were obtained. An animal model of AAA was constructed using osmotic minipumps to deliver 1000ng/kg/min angiotensin II (AngII) for 28days. Patients with AAA had significantly lower ALDH2 expression levels than normal subjects. ALDH2*2 KI mice were susceptible to AngII administration, exhibiting significantly increased AAA incidence rates and increased aortic diameters. Alda-1, an ALDH2 activator, reduced AngII-induced ROS production, NF-kB activation, and apoptosis in HASMCs. Alda-1 attenuated AngII-induced aneurysm formation and decreased aortic expansion in ApoE KO mice. We concluded that ALDH2 deficiency is associated with the development of AAAs in humans and a murine disease model. ALDH2 deficiency increases susceptibility to AngII-induced AAA formation by attenuating anti-ROS effects and increasing VSMC apoptosis and vascular inflammation. Alda-1 was shown to attenuate the progression of experimental AAA in a murine model.

The Role of a Selective P2Y6 Receptor Antagonist, MRS2578, on the Formation of Angiotensin II-Induced Abdominal Aortic Aneurysms.

Objective The P2Y6 receptor has been shown to be involved in many cardiovascular diseases, including hypertension and atherosclerosis. The study is aimed at exploring the role of the P2Y6 receptor in Ang II-induced abdominal aortic aneurysm (AAA) formation in apolipoprotein E-deficient (apoE−/−) mice by using its selective antagonist. Methods Male apoE−/− mice were fed with high-fat diet and infused with angiotensin (Ang) II (1000 ng/kg/min) for 4 weeks to induce AAA or saline as controls. Mice were divided into four groups: normal saline (NS, placebo control) group (n = 8), Ang II+vehicle (Ang II) group (n = 14), Ang II-low dose MRS2578 (Ang II+MRS-16 mg) group (n = 14), and Ang II-high dose MRS2578 (Ang II+MRS-32 mg) group (n = 14). Daily intraperitoneal injection with vehicle or MRS2578 was pretreated one week before Ang II infusion. On postoperative day 10, aorta imaging of each group was taken by ultrasonography. After 4 weeks of Ang II infusion, the excised aortas were processed for diameter measurement and quantification of aneurysm severity and tissue characteristics; the blood samples were collected for measurement of the lipid profile and levels of cytokines. Verhoeff's Van Gieson (EVG) staining and immunochemistry staining were performed to evaluate disruption of the extracellular matrix (ECM) and infiltration of macrophages. Expression and activity of matrix metalloproteinases (MMPs) was measured by gelatin zymography. Results Treatment with MRS2578 made no significant difference in AAA formation, and maximal aortic diameter yet caused higher AAA rupture-induced mortality from 7% (Ang II) to 21.4% (Ang II+MRS-16 mg) or 42.9% (Ang II+MRS-32 mg), respectively (p < 0.05). Consistently, the severity of aneurysm tended to be more deteriorated in MRS2578-treated groups, especially the high-dosage group. The ratios of type III and IV aneurysm were much higher in the MRS2578-coadministered groups (p < 0.05). Furthermore, histological analyses showed that administration of MRS2578 significantly increased infiltration of macrophages, expression of monocyte chemotactic protein 1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1), and activities of MMP-2 and MMP-9 followed by aggravating degradation elastin in vivo (p < 0.05). However, the multiple effects of MRS2578 on the development of AAA are independent of changes in systolic blood pressure and lipid profiles. Conclusions The present study demonstrated that administration of MRS2578 exacerbated the progression and rupture of experimental AAA through promoting proinflammatory response and MMP expression and activity, which indicated a crucial role of the P2Y6 receptor in AAA development. Clinical Relevance. Purinergic P2Y receptors have attracted much attention since the P2Y12 receptor antagonist had been successfully applied in clinical practice. Elucidating the underlying mechanisms of AAA and exploring potential therapeutic strategies are essential to prevent its progression and reduce the mortality rate.

Runx2 (Runt-Related Transcription Factor 2)-Mediated Microcalcification Is a Novel Pathological Characteristic and Potential Mediator of Abdominal Aortic Aneurysm

Abdominal aortic aneurysms (AAAs) are highly lethal diseases without effective clinical predictors and therapeutic targets. Vascular microcalcification, as detected by fluorine-18-sodium fluoride, has recently been recognized as a valuable indicator in predicting atherosclerotic plaque rupture and AAA expansion. However, whether vascular microcalcification involved in the pathogenesis of AAA remains elusive. Approach and Results: Microcalcification was analyzed in human aneurysmal aortas histologically and in AngII (angiotensin II)-infused ApoE-/- mouse aortas by fluorine-18-sodium fluoride positron emission tomography and X-ray computed tomography scanning in chronological order in live animals. AAA patients' aortic tissue showed markedly enhanced microcalcification in the aortic media within the area proximal to elastic fiber degradation, compared with non-AAA patients. Enhanced fluorine-18-sodium fluoride uptake preceded significant aortic expansion in mice. Microcalcification-positive mice on day 7 of AngII infusion showed dramatic aortic expansion on subsequent days 14 to 28, whereas microcalcification-negative AngII-infused mice and saline-induced mice did not develop AAA. The application of hydroxyapatite, the main component of microcalcification, aggravated AngII-induced AAA formation in vivo. RNA-sequencing analysis of the suprarenal aortas of 4-day-AngII-infused ApoE-/- mice and bioinformatics analysis with ChIP-Atlas database identified the potential involvement of the osteogenic transcriptional factor Runx2 (runt-related transcription factor 2) in AAA. Consistently, vascular smooth muscle cell-specific Runx2 deficiency markedly repressed AngII-induced AAA formation in the ApoE-/- mice compared with the control littermates. Our studies have revealed microcalcification as a novel pathological characteristic and potential mediator of AAA, and targeting microcalcification may represent a promising strategy for AAA prevention and treatment.

AVE0991, a nonpeptide angiotensin-(1-7) mimic, inhibits angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E knockout mice.

AVE0991, a nonpeptide angiotensin-(1-7) mimic, has similar protective effects for cardiovascular system to Ang-(1-7). In this article, we aimed to explore the effects of AVE0991 and Ang-(1-7) on abdominal aortic aneurysm (AAA) induced by Ang II in apolipoprotein E knockout mice. The mice AAA model was established by Ang II infusion, and then mice received different treatment with saline, Ang II (1.44mg/kg/day), different dose AVE0991 (0.58 or 1.16μmol/kg/day), or Ang-(1-7) (400ng/kg/min). The incidence of AAA was 76%, 48%, 28%, and 24% in the vehicle, the low-dose AVE0991, high-dose AVE0991, and the Ang-(1-7) group, respectively. In comparison with control group, AVE0991 and Ang-(1-7) treatment significantly increased smooth muscle cells and decreased macrophage accumulation, the expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α), and the expression and activity of metalloproteinases 2 and 9 in mice AAA model or in human smooth muscle cells (hVSMCs). The therapeutic effects may be contributed to reduction of oxidative stress and downregulation of P38 and ERK1/2 signal pathways via Mas receptor activation, whereas the positive impacts were reversed by co-administration with the Mas antagonist A779 (400ng/kg/min) and AVE0991 in Ang II-infused mice or in hVSMCs. Therefore, AVE0991 and Ang-(1-7) might be novel and promising interventions in the prevention and treatment of AAA. KEY MESSAGES: • AVE0991 dose-dependently inhibited Ang II-induced AAA formation in Apoe-/- mice. • Ang-(1-7) played the same protective role as high-dose AVE0991. • Inhibition of Mas receptor with A779 could reverse the protective effect of AVE0991. • The therapeutic effects may be contributed to reduction of oxidative stress and downregulation of P38 and ERK1/2 signal pathways via Mas receptor activation.

Itaconate Prevents Abdominal Aortic Aneurysm Formation Through Inhibiting Inflammation Via Activation of Nrf2

Background: Identifying effective drugs to suppress vascular inflammation is a promising strategy to delay the progression of abdominal aortic aneurysm (AAA). Itaconate has a vital role in regulating inflammatory activation in various inflammatory diseases. However, the role of itaconate in the progression of AAA is unknown. In this study, we explored the inhibitory effect of itaconate on AAA formation and its underlying mechanisms. Methods: Quantitative PCR, western blotting and immunohistochemistry were used to determine Irg1 and downstream Nrf2 expression in human and mouse AAA samples. Liquid chromatograph-mass spectrometry (LC-MS) analysis was performed to measure the abundance of itaconate. OI treatment and Irg1 knockdown were performed to study the role of OI in AAA formation. Nrf2 intervention in vivo were performed to detect the critical role of Nrf2 in the beneficial effect of OI on AAA. Findings: We found that itaconate suppressed the formation of angiotensin II (Ang II)-induced AAA in apolipoprotein E-deficient (Apoe-/-) mice, while Irg1 deficiency exerted the opposite effects. Mechanistically, itaconate inhibited vascular inflammation by enabling Nrf2 to function as a transcriptional repressor of downstream inflammatory genes via alkylation of Keap1. Moreover, Nrf2 deficiency significantly aggravated inflammatory factor expression and promoted AAA formation. In addition, Keap1 overexpression significantly promoted Ang II-induced AAA formation, which were inhibited by itaconate. Interpretation: Itaconate inhibited AAA formation by suppressing vascular inflammation, and therapeutic approaches to increase itaconate are potentially beneficial for preventing AAA formation. Funding: National Natural Science Foundations of China and Guangzhou regenerative medicine and Health Laboratory of Guangdong. Declaration of Interest: None. Ethical Approval: Human AAA samples were obtained from patients undergoing open surgical repair according to protocols approved by the Research Ethics Committees of Nanfang Hospital (ethical approval number: NFEC-2019-086). All animal protocols were approved by the Institutional Animal Care and Use Committee at Southern Medical University.

2009-P: Molecular Mechanisms of Obesity-Associated Vascular Vulnerability via Signals of Appetite Regulation

Background: Obesity is a risk factor of hypertension and abdominal aortic aneurysm (AAA). Melanocortin 4 receptor (MC4R) is activated by α-melanocyte-stimulating hormone (α-MSH) to mediate most of the anorectic effects of leptin. In humans, deficiency or mutation of MC4R is the most common monogenic form of obesity. However, it remains unknown whether MC4R signaling affects the development of obesity-associated hypertension, vascular vulnerability and aortic aneurysm. Methods and Results: MC4R-knockout (KO) mice fed a high-fat/high-sucrose diet (HF/HS) for 20 weeks displayed obesity associated with hyperleptinemia compared to HF/HS-fed wild type (WT) mice. MC4R-KO mice exhibited hypertension, increase of medial thickness and elastic lamina destruction, and elevation of inflammatory and fibrosis-related genes including TGF-β and osteopontin (OPN) in aortae. MC4R-KO mice showed higher incidence of angiotensin II (Ang II, 500 ng/kg/min)-induced AAA (73%) than WT mice (8%). MC4R-KO mice with ob/ob background exhibited lower incidence of Ang II-induced AAA (50%) than MC4R-KO mice, but higher than ob/ob mice (12.5%). Treatment of OPN-blocking antibody decreased Ang II-induced AAA incidence in HF/HS-fed MC4R-KO mice. In vascular smooth muscle cells (VSMCs), leptin (100 ng/ml) significantly upregulated OPN gene expression. α-MSH suppressed AngII-induced TGF-β gene expression in VSMCs, and LPS/IFN-γ-induced iNOS gene expression in peritoneal macrophages. However, these effects of α-MSH were abolished in primary macrophages from MC4R-KO mice and RAW264.7 cells pretreated with MC4R antagonist HS024. Conclusion: Our observation indicates that deficiency of MC4R promotes the vascular vulnerability in mice. It suggests that, not only hyperleptinemia, but also impairment of α-MSH-MC4R signaling in VSMCs and macrophages, may also promote the vascular vulnerability. Disclosure K. Mori: None. K. Tsuchiya: None. S. Nakamura: None. K. Shiba: None. Y. Miyachi: None. Y. Ogawa: None. K. Kitamura: None.

One amino acid change of Angiotensin II diminishes its effects on abdominal aortic aneurysm

Angiotensin (Ang) A is formed by the decarboxylation of the N terminal residue of AngII. The present study determined whether this one amino acid change impacted effects of AngII on abdominal aortic aneurysm (AAA) formation in mice. Computational analyses implicated that AngA had comparable binding affinity to both AngII type 1 and 2 receptors as AngII. To compare effects of these two octapeptides in vivo, male low-density lipoprotein receptor (Ldlr) or apolipoprotein E (Apoe) deficient mice were infused with either AngII or AngA (1 μg/kg/min) for 4 weeks. While AngII infusion induced AAA consistently in both mouse strains, the equivalent infusion rate of AngA did not lead to AAA formation. We also determined whether co-infusion of AngA would influence AngII-induced aortic aneurysm formation in male Apoe−/− mice. Co-infusion of the same infusion rate of AngII and AngA did not change AngII-induced AAA formation. Since it was reported that a 10-fold higher concentration of AngA elicited comparable vasoconstrictive responses as AngII, we compared a 10-fold higher rate (10 μg/kg/min) of AngA infusion into male Apoe−/− mice with AngII (1 μg/kg/min). This rate of AngA led to abdominal aortic dilation in three of ten mice, but no aortic rupture, whereas the 10-fold lower rate of AngII infusion led to abdominal aortic dilation or rupture in eight of ten mice. In conclusion, AngA, despite only being one amino acid different from AngII, has diminished effects on aortic aneurysmal formation, implicating that the first amino acid of AngII has important pathophysiological functions.

Mas receptor deficiency augments angiotensin II-induced atherosclerosis and aortic aneurysm ruptures in hypercholesterolemic male mice

ObjectivePrevious studies demonstrated that deficiency of angiotensin-converting enzyme 2 (ACE2) augmented angiotensin II (AngII)-induced atherosclerosis and abdominal aortic aneurysm (AAA) formation in hypercholesterolemic mice. Effects of ACE2 deficiency could arise from increased concentrations of its substrate, AngII, or decreased concentrations of its product, angiotensin-(1-7) [Ang-(1-7)]. Infusion of Ang-(1-7), a Mas receptor (MasR) ligand, to hypercholesterolemic male mice reduced AngII-induced atherosclerosis, suggesting a protective role of the Ang-(1-7)/MasR axis. However, it is unclear whether endogenous Ang-(1-7) acts at MasR to influence AngII-induced vascular diseases. The purpose of this study was to define the role of MasR deficiency in AngII-induced atherosclerosis and AAA formation and severity in hypercholesterolemic male mice. MethodsMasR+/+ and MasR−/− male mice on a low-density lipoprotein receptor-deficient (Ldlr−/−) or apolipoprotein E-deficient (Apoe−/−) background were infused with AngII at either 600 or 1000 ng/kg/min by osmotic minipump for 28 days. Atherosclerosis was quantified at study end point as percentage lesion surface area of the aortic arch in Ldlr−/− mice. Abdominal aortic internal diameters were quantified by ultrasound, and maximal external AAA diameters were quantified at study end point. Blood pressure was quantified by radiotelemetry and a tail cuff-based technique. Serum cholesterol concentrations and vascular tissue characterization were examined at study end point. ResultsMasR deficiency did not influence body weight, systolic blood pressure at baseline and during AngII infusion, or serum cholesterol concentrations in either Apoe−/− or Ldlr−/− mice. MasR deficiency increased AngII-induced atherosclerosis in aortic arches of Ldlr−/− mice (P < .05), associated with increased oxidative stress and apoptosis in aortic root sections (P < .05). MasR deficiency also augmented internal and external AAA diameters and increased aortic ruptures of both Ldlr−/− and Apoe−/− mice (P < .05). These effects were associated with increased elastin breaks and T-lymphocyte and macrophage accumulation into abdominal aortas of AngII-infused MasR-deficient mice (P < .05). ConclusionsThese results demonstrate that MasR deficiency augmented AngII-induced atherosclerosis and AAA rupture through mechanisms involving increased oxidative stress, inflammation, and apoptosis, suggesting that MasR activation may provide therapeutic efficacy against vascular diseases.

Caloric Restriction Exacerbates Angiotensin II-Induced Abdominal Aortic Aneurysm in the Absence of p53.

p53-dependent vascular smooth muscle cell senescence is a key pathological process of abdominal aortic aneurysm (AAA). Caloric restriction (CR) is a nonpharmacological intervention that prevents AAA formation. However, whether p53 is indispensable to the protective role of CR remains unknown. In this study, we investigated the necessity of p53 in the beneficial role of CR in AAA formation and the underlying mechanisms. We subjected p53+/+ and p53-/- mice to 12 weeks of CR and then examined the incidence of Ang II (angiotensin II)-induced AAA formation. We found that both CR and p53 knockout reduced Ang II-induced AAA formation; however, CR markedly increased the incidence of AAA formation and exacerbated aortic elastin degradation in p53-/- mice, accompanied by increased vascular senescence, reactive oxygen species generation, and reduced energy production. Analysis of mitochondrial respiratory activity revealed that dysfunctional complex IV accounts for the abnormal mitochondrial respiration in p53-/- vascular smooth muscle cells treated by CR serum. Mechanistically, ablation of p53 almost totally blocked the protective role of CR by inhibiting SCO2 (cytochrome C oxidase assembly protein 2)-dependent mitochondrial complex IV activity. Overexpression of SCO2 restored the beneficial effect of CR on antagonizing Ang II-induced expression of AAA-related molecules and reactive oxygen species generation in p53-/- vascular smooth muscle cells. Together, our findings demonstrate that the existence of p53 in vascular smooth muscle cells is critical to the protective role of CR in Ang II-induced AAA formation by maintaining an appropriate mitochondrial function.

Knockdown of lncRNA PVT1 Inhibits Vascular Smooth Muscle Cell Apoptosis and Extracellular Matrix Disruption in a Murine Abdominal Aortic Aneurysm Model.

This study was designed to determine the effects of the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) on vascular smooth muscle cell (VSMC) apoptosis and extracellular matrix (ECM) disruption in a murine abdominal aortic aneurysm (AAA) model. After injection of PVT1-silencing lentiviruses, AAA was induced in Apolipoprotein E-deficient (ApoE−/−) male mice by angiotensin II (Ang II) infusion for four weeks. After Ang II infusion, mouse serum levels of pro-inflammatory cytokines were analysed, and aortic tissues were isolated for histological, RNA, and protein analysis. Our results also showed that PVT1 expression was significantly upregulated in abdominal aortic tissues from AAA patients compared with that in controls. Additionally, Ang II treatment significantly increased PVT1 expression, both in cultured mouse VSMCs and in AAA murine abdominal aortic tissues. Of note, the effects of Ang II in facilitating cell apoptosis, increasing matrix metalloproteinase (MMP)-2 and MMP-9, reducing tissue inhibitor of MMP (TIMP)-1, and promoting switching from the contractile to synthetic phenotype in cultured VSMCs were enhanced by overexpression of PVT1 but attenuated by knockdown of PVT1. Furthermore, knockdown of PVT1 reversed Ang II-induced AAA-associated alterations in mice, as evidenced by attenuation of aortic diameter dilation, marked adventitial thickening, loss of elastin in the aorta, enhanced aortic cell apoptosis, elevated MMP-2 and MMP-9, reduced TIMP-1, and increased pro-inflammatory cytokines. In conclusion, our findings demonstrate that knockdown of lncRNA PVT1 suppresses VSMC apoptosis, ECM disruption, and serum pro-inflammatory cytokines in a murine Ang II-induced AAA model.

Chemokine (C-X-C motif) receptor 2 blockade by SB265610 inhibited angiotensin II-induced abdominal aortic aneurysm in Apo E-/- mice.

Inflammation plays a critical role in the development of abdominal aortic aneurysm (AAA). Chemokine receptor CXCR2 mediates inflammatory cell chemotaxis in several diseases. However, the role of CXCR2 in AAA and the underlying mechanisms remain unknown. In this study, we found that the CXCR2 expressions in AAA tissues from human and angiotensin II (Ang II)-infused apolipoprotein E knockout (Apo E-/-) mice were significantly increased. The pharmacological inhibition of CXCR2 (SB265610) markedly reduced Ang II-induced AAA formation. Furthermore, SB265610 treatment significantly reduced collagen deposition, elastin degradation, the metal matrix metalloprotease expression and accumulation of macrophage cells. In conclusion, these results showed CXCR2 plays a pathogenic role in AAA formation. Inhibition of CXCR2 pathway may represent a novel therapeutic approach to treat AAA.

Invited commentary

Precision medicine and molecular therapeutics offer significant potential in vascular patients because of the chronicity and heterogeneity of disease. Currently, there is no established, targeted molecular therapeutic intervention for abdominal aortic aneurysm (AAA) disease. The work on using metalloproteinase as a biomarker and targeting its activity as a therapeutic option continues on the basis of earlier work, in whole and in part, in murine models. Initial studies in humans have not come to fruition.1Golledge J. Norman P.E. Murphy M.P. Dalman R.L. Challenges and opportunities in limiting abdominal aortic aneurysm growth.J Vasc Surg. 2017; 65: 225-233Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Other potential biologic interventions have also proved unsuccessful. Thus, a greater understanding of AAA pathobiology and the identification of one or more ligand or intracellular pathways that can interrupt the pathobiologic process of aneurysm formation remain important areas of study. The current study sheds more light on receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclastogenic differentiation of macrophages in a phenotypically unique model of AAA formation. Several studies have demonstrated that there is a balanced mineralization process within the wall of diseased arteries mediated by monocyte-macrophage-derived osteoblast-like and osteoclast-like cells.2Yamanouchi D. Takei Y. Komori K. Balanced mineralization in the arterial system: possible role of osteoclasto-genesis/osteoblasto-genesis in abdominal aortic aneurysm and stenotic disease.Circ J. 2012; 76: 2732-2737Crossref PubMed Scopus (27) Google Scholar Osteoclastogenic activation of macrophages has been described both in human AAAs and in experimental aneurysm formation. Mature osteoclasts express matrix metalloproteinase 9, which provides an iterative link back to the original hypotheses that matrix metalloproteinases have a role in AAA formation. The current study builds on this background and shows that osteoclast-like macrophages in AAA are associated with increased expression of RANKL.2Yamanouchi D. Takei Y. Komori K. Balanced mineralization in the arterial system: possible role of osteoclasto-genesis/osteoblasto-genesis in abdominal aortic aneurysm and stenotic disease.Circ J. 2012; 76: 2732-2737Crossref PubMed Scopus (27) Google Scholar Administration of a RANKL-neutralizing antibody resulted in a significant decrease in the diameter of the experimental aneurysms. This would suggest that targeting RANKL may be a potential new therapeutic approach to inhibition of AAA growth. RANKL neutralization is currently used clinically to treat osteoporosis and other osteoclast-related diseases; thus, a medication and therapeutic profile in humans already exists. The importance of this paper is that it identifies a unique target within macrophages that reside within the wall of an AAA and this target has a known therapeutic antagonist in clinical practice. However, given the uniqueness of the model with a phenotype of a dissecting aortic aneurysm, validation in the other murine and nonmurine models of AAA will be necessary. Once validated, there will be a need to define the timing for RANKL intervention, to ensure precision in homing and targeting to an aneurysmal aortic wall, to estimate the duration of therapy (acute, pulsed, or chronic), and to ensure that there are no unintended cellular consequences in other arteries and organs. Irrespective of these issues, the current study reveals a novel and new pathway to interrupt AAA formation that has prospects to move to a clinical study and marks an advance in the understanding of the role of macrophages in AAA pathobiology. The opinions or views expressed in this commentary are those of the authors and do not necessarily reflect the opinions or recommendations of the Journal of Vascular Surgery or the Society for Vascular Surgery. RANKL-mediated osteoclastogenic differentiation of macrophages in the abdominal aorta of angiotensin II-infused apolipoprotein E knockout miceJournal of Vascular SurgeryVol. 68Issue 6PreviewOsteoclastogenic activation of macrophages (OCG) occurs in human abdominal aortic aneurysms (AAAs) and in calcium chloride-induced degenerative AAAs in mice, which have increased matrix metalloproteinase activity. As the activity of OCG in dissecting aneurysms is not clear, we tested the hypothesis that OCG contributes to angiotensin II (Ang II)-induced dissecting aneurysm (Ang II-induced AAA) in apolipoprotein E knockout mice. Full-Text PDF Open Archive

Licochalcone A attenuates abdominal aortic aneurysm induced by angiotensin II via regulating the miR-181b/SIRT1/HO-1 signaling.

Licochalcone A (LA), a chalcone derived from liquorice, exhibits multiple biological activities, including anti-oxidation and anti-inflammation. This study aimed to investigate the role and underlying mechanism of LA in the abdominal aortic aneurysm (AAA). AAA model was established by continuous infusion of 1000 ng/kg/min of angiotensin II (AngII) in ApoE -/- mice for 4 weeks. At 7 days before AngII administration, 5 mg/kg/day or 10 mg/kg/day of LA was intraperitoneally administered to mice and continued for 4 weeks. The characteristics and quantification of AAAs were determined in situ. Real-time PCR or western blot was used to measure mRNA or protein levels of matrix metalloproteinase 2 and matrix metalloproteinase 9; pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6; apoptosis-related proteins Bax, Bcl-2, and active caspase-3; miR-181b; Sirtuin 1 (SIRT1); and heme oxygenase-1 (HO-1). Mouse-aorta-origin vascular smooth muscle (MOVAS) cells were used to confirm the involved pathways in vitro. We found LA administration dose-dependently reduced the incidence of AngII-induced AAA, aneurysm diameter enlargement, elastin degradation, matrix metalloproteinase production, pro-inflammatory cytokines and miR-181b expression, and vascular smooth muscle cell apoptosis. It elevated SIRT1 and HO-1 expression that was suppressed by AngII. AngII enhanced miR-181b but reduced SIRT1 and HO-1 expression in MOVAS cells. In AngII-stimulated MOVAS cells, downregulation of miR-181b significantly upregulated the expression of SIRT1 and HO-1, the effect of which was abrogated by SIRT1 siRNA. Collectively, LA could attenuate AngII-induced AAA by modulating the miR-181b/SIRT1/HO-1 signaling. LA might be a potential medical therapy for small AAA.

Recombinant leptin attenuates abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice

Vascular disease can manifest as stenotic plaques or ectatic aneurysms. Human abdominal aortic aneurysms (AAA) comprise an inflammatory disease characterized by the predominance of T helper type 2 (Th2) cytokine expression. Leptin has been clearly demonstrated to play an important role in regulating Th0 cell to Th1. So, we hypothesize that leptin has a protective effect on aneurysm formation. In this study, we demonstrated that intraperitoneal injection of leptin attenuated Ang II-induced AAA formation in ApoE−/− mice with no effect on serum lipids and systolic blood pressure. To investigate the mechanisms involved, we found that leptin pretreatment exhibited decreased protein expression of matrix metalloproteinase 2 (MMP-2) and MMP-9 and increased transforming growth factor-β1 (TGF-β1). We also examined potential mechanism of leptin as a modulator of the immune response. Our results proved that pretreatment with leptin downregulated protein expression of Th2 cytokine IL-4 and mRNA levels of GATA-3, the key transcription factor for Th2 polarization, and upregulated Th1 cytokine INF-γ and T-bet, the key transcription factor for Th1 polarization. Taken together, leptin, with the effect of regulation of Th1/Th2 cytokines, may have therapeutic potential for the treatment of AAA. Leptin may constitute a novel therapeutic strategy to prevent AAA formation.

Inhibition of endoplasmic reticulum stress by intermedin1-53 attenuates angiotensin II-induced abdominal aortic aneurysm in ApoE KO Mice.

Endoplasmic reticulum stress (ERS) is involved in the development of abdominal aortic aneurysm (AAA). Since bioactive peptide intermedin (IMD)1-53 protects against AAA formation, here we investigated whether IMD1-53 attenuates AAA by inhibiting ERS. AAA model was induced by angiotensin II (AngII) in ApoE KO mouse background. AngII-treated mouse aortas showed increased ERS gene transcription of caspase12, eukaryotic translation initiation factor 2a (eIf2a) and activating transcription factor 4(ATF4).The protein level of ERS marker glucose regulated protein 94(GRP94), ATF4 and C/EBP homologous protein 10(CHOP) was also up-regulated by AngII. Increased ERS levels were accompanied by severe VSMC apoptosis in human AAA aorta. In vivo administration of IMD1-53 greatly reduced AngII-induced AAA and abrogated the activation of ERS. To determine whether IMD inhibited AAA by ameliorating ERS, we used 2 non-selective ERS inhibitors phenyl butyrate (4-PBA) and taurine (TAU). Similar to IMD, PBA, and TAU significantly reduced the incidence of AAA and AAA-related pathological disorders. In vitro, AngII infusion up-regulated CHOP, caspase12 expression and led to VSMC apoptosis. IMD siRNA aggravated the CHOP, caspase12-mediated VSMC apoptosis, which was abolished by ATF4 silencing. IMD infusion promoted the phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) in aortas in ApoE KO mice, and the AMPK inhibitor compound C abolished the protective effect of IMD on VSMC ERS and apoptosis induced by AngII. In conclusion, IMD may protect against AAA formation by inhibiting ERS via activating AMPK phosphorylation.

Deficiency of Melanocortin 4 Receptor Promotes Vascular Vulnerability in Mice

Background: Obesity is known as a risk factor of hypertension and abdominal aortic aneurysm (AAA) in humans. Melanocortin 4 receptor (MC4R) is primarily expressed in brain, and mediates most of the anorectic effects of leptin; in humans, deficiency or mutation of MC4R is the most common monogenic form of obesity. However, it remains unknown whether MC4R signaling affects the development of obesity-associated hypertension, vascular vulnerability and aortic aneurysm. Methods and Results: MC4R-knockout (KO) mice fed a high-fat/high-sucrose diet (HF/HS) for 20 weeks displayed obesity and insulin resistance associated with hyperleptinemia compared to HF/HS-fed wild type mice. HF/HS-fed MC4R-KO mice exhibited hypertension, and showed increase of medial thickness and elastic lamina destruction in aortae. In aortae of HF/HS-fed MC4R-KO mice, genes of MCP-1, TNF-α, and F4/80 were markedly upregulated relative to HF/HS-fed wild type mice. Following the administration of angiotensin II (Ang II, 500 ng/kg/min), HF/HS-fed MC4R-KO mice showed significantly lower survival rate and higher incidence of AAA than HF/HS-fed wild type mice, with increased expression of F4/80- and osteopontin-positive cells in aortae. Furthermore, HF/HS-fed ob/ob mice exhibited significantly lower incidence of Ang II-induced AAA with a trend of higher survival rate than HF/HS-fed MC4R-KO mice. Leptin (100 ng/ml) significantly upregulated osteopontin gene expression in primary cultured vascular smooth muscle cells (VSMCs), but not in primary cultured macrophages. Conclusion: Our observation clearly demonstrates that deficiency of melanocortin 4 receptor promotes the development of hypertension, vascular vulnerability, and Ang II-induced AAA in mice. It also suggests that hyperleptinemia caused by central leptin resistance could contribute to the development of these vascular phenotypes in MC4R-KO mice, at least partly via induction of osteopontin in VSMCs. Disclosure K. Mori: None. K. Tsuchiya: None. S. Nakamura: None. Y. Miyachi: None. K. Shiba: None. Y. Ogawa: Research Support; Self; AstraZeneca. Advisory Panel; Self; Gilead Sciences, Inc.. Research Support; Self; Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science, Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd.. K. Kitamura: None.

Inhibition of interleukin-1 suppresses angiotensin II-induced aortic inflammation and aneurysm formation

BackgroundAngiotensin II (Ang II) activates components of the inflammatory cascade, which promotes hypertension and development of abdominal aortic aneurysm (AAA). This study aimed to elucidate the effects of an IL-1 receptor antagonist (IL-1Ra) and an anti-IL-1β antibody (01BSUR) on Ang II-induced AAA. Methods and resultsMale wild-type (WT) and IL-1Ra-deficient (IL-1Ra−/−) mice were infused with Ang II (1000 ng/kg/min) using subcutaneous osmotic pumps for 28 days. Fourteen days post-infusion, both systolic blood pressure (SBP) (Ang II-treated IL-1Ra−/−:149 ± 2 vs. Ang II-treated WT:126 ± 3 mm Hg, p < 0.001) and abdominal aortic width (0.94 ± 0.09 vs. 0.49 ± 0.03 mm, p < 0.001) were significantly higher in IL-1Ra−/− mice than in WT mice. Because 28-day infusion with Ang II in IL-1Ra−/− mice significantly increased the occurrence of fatal aortic rupture (89% vs. 6%, p < 0.0001), both types of mice were infused with Ang II for only 14 days, and histological analyses were performed at 28 days. Interestingly, AAA increased more significantly in IL-1Ra−/− mice than in WT mice (p < 0.001), although SBP did not differ at 28 days in IL-1Ra−/− and WT mice (117 ± 4 vs. 115 ± 3 mm Hg, p = 0.71 (after cessation of Ang II infusion)). Histological analyses showed numerous inflammatory cells around the abdominal aorta in IL-1Ra−/− mice, but not in WT mice. Finally, compared with IgG2a treatment, treatment with 01BSUR decreased Ang II-induced AAA in IL-1Ra−/− mice. ConclusionsThe present study demonstrates that inhibition of IL-1β significantly suppresses AAA formation after Ang II infusion, suggesting that suppression of IL-1β may provide an additional strategy to protect against AAA in hypertensive patients.

PM2.5 promotes abdominal aortic aneurysm formation in angiotensin Ⅱ-infused apoe-/- mice

BackgroundParticulate matter 2.5 (PM2.5) has proven to be associated with morbidity and mortality from cardiovascular diseases. However, whether PM2.5 could promote the formation of abdominal aortic aneurysm (AAA) is unclear. Present study aimed to explore the relationship between PM2.5 exposure and AAA development. MethodsAng Ⅱ-infused apoe−/− mice were treated with PM2.5 or saline by intranasal instillation. Four weeks later, histological and immunohistological analyses were used to evaluate the effect of PM2.5 on AAA formation. Human aortic smooth muscle cells (HASMCs) were also employed to further analyze the adverse effect of PM2.5 in vitro. ResultsWe found that PM2.5 could significantly increase the AAA incidence, the maximal abdominal aortic diameter and could promote the degradation of elastin. Additionally, the expression of senescence markers, P21 and P16 were also enhanced after PM2.5 exposure. We also found that PM2.5 significantly increased the AAA related pathological changes, MMP2 and MCP-1 expression in HASMCs. Meanwhile, PM2.5 could increase the expression of senescence markers P21, P16 and SA-β-gal activity, also the reactive oxygen species levels in vitro. ConclusionsPM2.5 promoted the formation of AAA in an Ang Ⅱ-induced AAA model. The underlying mechanism might be cellular senescence after PM2.5 exposure.

Ginkgo biloba extracts prevent aortic rupture in angiotensin II-infused hypercholesterolemic mice.

Abdominal aortic aneurysms (AAAs) are a chronic vascular disease characterized by pathological luminal dilation. Aortic rupture is the fatal consequence of AAAs. Ginkgo biloba extracts (GBEs), a natural herb extract widely used as food supplements, drugs, and cosmetics, has been reported to suppress development of calcium chloride-induced AAAs in mice. Calcium chloride-induced AAAs do not rupture, while angiotensin II (AngII)-induced AAAs in mice have high rate of aortic rupture, implicating potentially different mechanisms from calcium chloride-induced AAAs. This study aimed to determine whether GBE would improve aortic dilation and rupture rate of AngII-induced AAAs. Male apolipoprotein E (apoE) -/- mice were infused with AngII and administered either GBE or its major active ingredients, flavonoids and ginkgolides, individually or in combination. To determine the effects of GBE in mice with established AAAs, male apoE-/- mice were firstly infused with AngII for 28 days to develop AAAs, and then administered either GBE or vehicle in mice with established AAAs, which were continuously infused with AngII for another 56 days. GBE, but not the two major active components separately or synergistically, prevented aortic rupture, but not aortic dilation. The protection of GBE from aortic rupture was independent of systolic blood pressure, lipid, and inflammation. GBE also did not attenuate either aortic rupture or progressive aortic dilation in mice with established AAAs. GBE did not reduce the atherosclerotic lesion areas, either. In conclusion, GBE prevents aortic rupture in AngII-infused hypercholesterolemic mice, but only in the early phase of the disease development.

Abstract 108: Lipopolysaccharide Fails to Augment Development of Angiotensin II-induced Abdominal Aortic Aneurysms in Mice

Background and objective: Our previous study demonstrated that deficiency of toll-like receptor 4 (TLR4) reduced angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Lipopolysaccharide (LPS) is a well-known agonist of TLR4. Interaction of LPS and TLR4 promotes inflammation and augments hypercholesterolemia-induced atherosclerosis. The purpose of this study was to determine whether LPS augments AngII-induced AAAs. Methods and Results: Low-density lipoprotein (LDL) receptor -/- mice fed a Western diet develop hypercholesterolemia and have augmentation of AngII-induced AAAs. We found that LDL receptor -/- mice fed Western diet had increased LPS concentrations, as quantified by a Limulus Amebocyte lysate chromogenic endotoxin quantitation kit, compared to the same mice prior to Western diet feeding. To determine whether LPS plays a role on AngII-induced AAAs, 4 groups of male C57BL/6J mice, 10-week-old, were infused subcutaneously with: (1) Vehicle (N=5), (2) LPS (250 μ g/kg/day; N=10), (3) AngII (1,000 ng/kg/min; N=10), or (4) both AngII and LPS (N=10) through Alzet mini osmotic pumps for 28 days. Abdominal aortic expansion was monitored serially at 2 and 4 weeks of infusion using high frequency ultrasonography (Vevo 3100 system; FUJIFILM). Continuous luminal expansion was detected in both AngII-infused mice and mice co-infused with both AngII and LPS. These results were confirmed by ex vivo measurements of maximal outer diameters of suprarenal aortas after termination showing AngII infusion led to significant increase of abdominal aortic dilation, compared to vehicle or LPS infused mice. However, co-infusion of LPS with AngII did not augment AngII-induced AAAs. Conclusion: LPS has no effects on AngII-induced AAAs in normolipidemic mice.